Dis3745 PDF

Identification, characterization and high-resolution
mapping of resistance genes to Phytophthora

infestans in potato

Promotors:
Prof. dr. Richard G.F. Visser, Hoogleraar in de Plantenveredeling
Prof. dr. ir. Evert J acobsen, Hoogleraar in de Plantenveredeling

Promotiecommissie:
Prof. dr.ir. P.J .G.M. de Wit, Wageningen Universiteit
Dr. J .H. de J ong, Wageningen Universiteit
Dr. A. Pereira, Plant Research International
Prof. dr. W.J . Stiekema, Plant Research International

Dit onderzoek is uitgevoerd binnen de onderzoekschool Experimental Plant Sciences

Identification, characterization and high-resolution
mapping of resistance genes to Phytophthora
infestans in potato

Tae-Ho Park

Proefschrift
ter verkrijging van de graad van doctor
op gezag van de rector magnificus
van Wageningen Universiteit
Prof. dr. ir. L. Speelman
in het openbaar te verdedigen
op woensdag 11 mei 2005
des namiddags te vier uur in de Aula.
3

Identification, characterization and high-resolution mapping of resistance genes to
Phytophthora infestans in potato

Tae-Ho Park

PhD thesis, Wageningen University, The Netherlands, 2005
With references With summaries in English, Dutch and Korean

ISBN 90-8504-189-9

Contents

Chapter 1 General introduction 7
Chapter 2 Dissection of foliage and tuber late blight resistance in mapping
populations of potato 19
Chapter 3 High-resolution mapping and analysis of the resistance locus
Rpi-abpt against Phytophthora infestans in potato 33
Chapter 4 Characterization and high-resolution mapping of a late blight
resistance locus similar to R2 49
Chapter 5 The late blight resistance locus Rpi-blb3 from Solanum
bulbocastanum belongs to a major late blight R gene cluster on
chromosome 4 of potato 63
Chapter 6 Genetic positioning of centromeres using half-tetrad analysis
in a 4x-2x cross population of potato 81
Chapter 7 General discussion 101
Summary 119
Samenvatting 123
Summary in Korean 127
Acknowledgements 131
Curriculum vitae 134

5

Chapter 1

General introduction

Chapter 1

Potato

The cultivated potato Solanum tuberosum L. that originated from the Andean region
of South America is one of the most important food crops in the world. Potato was
first domesticated and eaten by man in South America particularly in the region of the
Andes about 8,000 years ago. It was probably introduced into Europe from South
America more than four centuries ago when the Spanish invaded the Inca Empire.
The native potato cultivars that were introduced were day-length sensitive, which
means that tuber formation was inhibited by a day-length longer than 12 hours. After
many generations of natural selection in Western Europe, the potato became adapted
to the long days of the western hemisphere (Hawkes, 1978 and 1994; Brown, 1990).
Potato is ranked at the fourth place in the world food production after wheat,
corn and rice. Of the root crops, it is on the top of the list, followed by cassava, sweet
potato and yams. Potato is very productive and nutritious. Compared with wheat and
rice, the potato has an approximately five times higher crop yield per hectare and one
and half times more energy production per ha and day (Struik and Wiersema, 1999).
Potato is also an important source of starch and contains high quality protein and
vitamin C. Moreover, potato can be grown widely under various climatic conditions
such as tropical, sub-tropical and temperate environments and in various altitudes
because of its high adaptation ability (Doehlonan and Sleper, 1995). Purposes for
growing potato are also very diverse such as for fresh potato consumption, starch
production, chips and French-fries.
Potato belongs to the Solanaceae family, which includes eggplant (S.
melongena), pepper (Capsicum annum), tomato (Lycopersicum esculentum) and
tobacco (Nicotiana tabacum). Many alkaloid drug plants also belong to this family
such as Atropa, Hyoscyamus, Scopolia and Mandragora and ornamentals in the
genera Petunia, Nicotiana and Schizanthus (Hawkes, 1990; Spooner et al., 1993).
Potato breeding is performed to improve resistance for diseases, viruses and
pests and to improve taste, cooking qualities, skin colour, shape and the yield as the
most important criteria according to demands of growers and consumers. There are
several ploidy levels from monoploid (2n=x=12) to hexaploid (2n=6x=72) with
tetraploid being common in cultivated potatoes. The triploids and the pentaploids are
often sterile, however, they can be maintained because of their vegetative
propagation (Howard, 1970; Hawkes, 1990). The high degree of ploidy not only
makes it genetically diverse, but also makes it difficult to create new cultivars.

Phytophthora infestans

Phytophthora infestans is the casual agent of late blight, which is the most
devastating disease in potato worldwide. It caused the great Irish famine in the 1840s
8

Figure 1. Pictures of infected potatoes in resistance assays performed on different tissues and organs
using different methods. A: Plants inoculated by pipetting inoculum on the leaves in vitro (in vitro
assay). B: Leaf inoculated by pipetting inoculum on the abaxial side (detached leaf assay). C: Plants
inoculated by spraying inoculum in the field (field assay). D: Tuber inoculated by pipetting inoculum on
the wound site (wounded tuber assay). E: Tuber after cutting the infected tuber (D) in the wounded
tuber assay. F: Tuber kept for three additional days after cutting the infected tuber (D) in the wounded
tuber assay. G: Tuber slice inoculated by pipetting inoculum on the upper side. H: Tuber inoculated by
spraying with a perfume spray (whole tuber assay). I: Tuber after cutting the infected tuber (H) in the
whole tuber assay.
9
Chapter 1

resulting in famine-related diseases, which made over 1 million people die and over
1.5 million people immigrate.
The pathogen belongs taxonomically to the oomycetes, a phylum that was
traditionally classified as a fungus due to their filamentous growth habit. However,
based on biochemical analyses and phylogenetic analyses, oomycetes are closer to
heterokont algae than to fungi belonging to basidiomycete fungi and ascomycete
fungi (van de Peer and de Vachter, 1997). Chesnick et al. (1996) also showed that
Phytophthora is related to two other stramenopiles (Chrysodidymus and
Ochromonas), but not to fungi using the mitochondrial nad4L genes protein
sequences. The oomycetes consist of 60 species of the genus Phytophthora, several
genera of the biotrophic downy mildew and more than 100 species of the genus
Pythium (referred to from http://www.oardc.ohio-state.edu/phytophthora). The host
plant range of P. infestans encompasses at least 90 plant species. Most of them are
members of the plant family Solanaceae (Erwin and Ribeiro, 1996). Late blight
diseased potatoes are shown in Figure 1. P. infestans can infect any tissue and organ
of potato. The figure includes pictures of infected tubers and leaves in different
resistance assays.
The life of P. infestans has been well-studied (Figure 2). It follows three basic
steps, formation of mycelium in the host plant, spatial expansion of the affected area
lesion in the host plant and formation and dispersal of spores. P. infestans has a
complex life cycle with distinctly different spore forms like zoospores produced by
self-reproduction and oospores produced by sexual reproduction. In the 1940s or
1950s, an A2 type, the sexual reproduced type, was discovered in Mexico where the
pathogen originated and was spreading through the USA, Europe, Asia and North
Africa in the 1970s and 1980s (Fry et al., 1992). Since the appearance of A2 types,
the chance of genetic variation of the pathogen is getting higher and it causes the
problem that new strains can develop and overcome resistance genes.

Late blight resistance

Potato late blight causes 10 to 15 percent reduction of the global annual production
(CIP, 1995). The economic value of this loss and the costs of crop protection have
been estimated at 5 billion US dollar annually (Duncan, 1999). Late blight is mainly
controlled by application of enormous amounts of chemicals. In spite of the
application of the chemical, late blight is increasingly more difficult to control and in
most countries chemical control is being restricted. There is thus an urgent need for
durable resistant potato cultivars. Introduction of resistance genes from wild Solanum
species into potato cultivars is considered as the most promising and environment-
safe approach to achieve late blight resistance.
10

Figure 2. Disease cycle of potato late blight caused by Phytophthora infestans

In early breeding programs, eleven R genes from S. demissum were
introduced into cultivated potatoes. Eight R genes, R3 (now known to exist of R3a
and R3b), R5, R6, R7, R8, R9, R10 and R11, were found to be located close to each
other on the short arm of chromosome 11 (El Kharbotly, 1994 and 1996; Huang et al.,
2004; Huang, 2005). Other mapped R genes include R2 on chromosome 4 (Li et al.,
1998) and R1 on chromosome 5 (Leonards-Schippers et al., 1992; El-Kharbotly,
1994). Unfortunately all of these genes have already been broken because new
pathogens quickly evolve that are virulent to the previously resistant hosts (Umaerus
and Umaerus, 1994). Besides S. demissum, other wild Solanum species have been
recognized as new sources of the resistance against P. infestans. For instance, R
ber
from S. berthaultii has been mapped on chromosome 10 (Ewing et al., 2000), RB or
Rpi-blb1 and Rpi-blb2 from S. bulbocastanum on chromosome 8 and chromosome 6,
respectively (Nasess et al., 2000; van der Vossen et al., 2003 and 2004), Rpi1 from S.
pinnatisectum on chromosome 7 (Kuhl et al., 2001) and quantitative trait loci (QTL)
involved in resistance from S. microdontum on chromosome 4, 5 and 10 (Sandbrink
et al., 2000).
Until now, research for late blight has been focussed mainly on foliage blight
resistance. However, since infected seed tubers have been recognised as highly
11
Chapter 1

important in the spread of the disease, more emphasis should be placed on
understanding the genetic basis of tuber blight resistance.
Tuber blight resistance is largely dependent on the testing conditions. It can be
effective in various different tissues and the character of tuber resistance can be
diverse in different plant genotypes (Flier, 2001; wieyski and Zimnoch-Guzowska,
2001). Detailed studies on foliar blight resistance to P. infestans have shown that
plant R genes specifically recognize avirulence gene products or elicitors of P.
infestans. Strong R genes act qualitatively, but weaker R genes act quantitatively and
reveal a partial resistance phenotype (Vleeshouwers et al., 2000). Similarly, R gene-
based resistance mechanisms are also thought to operate at tuber level (Hohl and
Stssel, 1976; Shimony and Friend, 1976, wieyski and Zimnoch-Guzowska,
2001). However, insight on the correlation between leaf and tuber blight resistance
phenotypes is limited, most likely because of the difficulty to separate tissue specific
genetic effects from the variation obtained due to testing conditions (wieyski and
Zimnoch-Guzowska, 2001). As mentioned above, the expression of tuber resistance
is affected by several factors such as testing conditions, storage conditions, tuber
tissues, plant genotypes, harvest date, interval between harvest and inoculation,
testing location, year etc. Tubers grown in the glasshouse, for instance, gave more
consistent phenotypes then tubers grown in the field (Stewart et al., 1996). More
reliable disease responses were observed on post-harvest inoculated tubers than
field tubers (Platt and Tai, 1998). With regard to the testing location and year, Darsow
(1987) presented results of testing varieties for 12 years and he found that Adretta
was very resistant and Mariella was very susceptible in 1975 and vice versa in 1979.
This is an extreme example. wieyski et al. (2001) reported the consistency of
tuber blight resistance, which was assayed in five different countries. They found 25
varieties giving consistency and eight varieties with a discrepancy. Furthermore, the
effect of harvest date and the interval between harvest and inoculation for tuber blight
resistance was examined by Stewart et al. (1983). They concluded that the optimum
date for the test would probably vary with the aggressiveness of P. infestans used
and other conditions influencing infection. The rate of suberization of the lenticels and
differences in the relative importance of eyes and lenticels as sites of infection are
important factors in determining tuber blight resistance.
So far, any resistance genes, which are only active in foliage or tuber, have not
been identified. There were the first contemporary reports on mapping QTL for tuber
blight resistance (Collins et al., 1999; Oberhagemann et al., 1999). However, it was
related to the major QTL for foliage blight resistance, foliage maturity and vigor. That
a relation exists between tuber and foliage blight resistance was confirmed by Collins
et al. (1999). A strong influence of foliar R1, R2 and R3 genes on tuber blight
resistance was verified (Roer et al., 1961; Toxopeus, 1961; Stewart and Bradshaw,
2001).
12

Resistance gene cloning

Genes that confer race-specific resistance often control disease resistance in plants.
Those resistance genes interact with specific avirulence genes in the pathogen in a
gene-for-gene interaction (Flor, 1977). During the last decade, plant disease
resistance genes controlling monogenic resistance have been cloned (reviewed in
Gebhardt and Vlkonen, 2001). About 40 disease resistance genes have now been
isolated from a variety of plant species. Nine of those resistance genes have been
cloned from potato (Table 1).

Table 1. Potato disease resistance genes cloned.
Gene Pathogen Chr.* Reference
Rx1 Potato virus X 12 van der Vossen et al. (2000)
Rx2 Potato virus X 5 Bendahmane et al. (2000)
Gpa2 Globodera pallida 12 van der Vossen et al. (2000)
Gro1 Globodera rostochiensis 7 Paal et al. (2004)
R1 Phytophthora infestans 5 Ballvora et al. (2002)
R3a Phytophthora infestans 11 Huang et al. (2005)
R3b Phytophthora infestans 11 Huang S. (in preparation)
Rpi-blb1 or RB Phytophthora infestans 8 van der Vossen et al. (2003); Song et al. (2003)
Rpi-blb2 Phytophthora infestans 6 van der Vossen et al. (2004)
* Chr. indicates the chromosomal location where the genes belong to.

Importance of the centromere

From the viewpoint of plant breeding, the centromere is one of the most important
functional elements of eukaryotic chromosomes, because it facilitates proper cell
division in meiosis and stable transmission of the genetic material. During meiosis,
each chromosome replicates, inducing homologous chromosome and crossover
events generate new allelic combinations.
In potato, breeding is not easy because the cultivated potato is tetraploid.
However, the introgression of new genes from diverse 2x relatives of potato for the
improvement of traits, especially disease resistance, is often possible via 4x-2x
crosses because 2n gametes can be produced (reviewed by J ansky, 2000; Buso et
al., 2003). These 2n gametes lead to various breeding schemes to synthesize highly
heterozygous 4x clones. Douches and Maas (1998) investigated the relationship
between yield production and heterozygosity transmission in 4x-2x populations. The
heterozygosity of genetic traits and marker loci in the 4x-2x cross is dependent on the
distance between genetic loci and centromere and the genetic consequences
associated with different cytological mechanisms that are related to the functional
importance of the centromere. Therefore, the identification of the centromeric position
13
Chapter 1

in potato chromosomes could be of great value in potato breeding (reviewed by
Carputo et al., 2000).

Objectives and outline of this thesis

The aim of this research was to identify foliage and tuber resistance genes against
Phytophthora infestans in potato.
In Chapter 2, we investigated the relationship between foliage and tuber blight
resistance to identify foliage or tuber specific resistance in four different populations of
which indications were obtained that three of them contained both types of
resistances and one did not. We found successfully two foliage specific resistance
genes, but unfortunately no tuber specific resistance genes. Additionally, we found
more resistance genes in one of the populations by comparing resistance specificity
with molecular marker data.
In Chapter 3, a new resistance gene was identified in a tetraploid population
derived from an ABPT breeding line. The gene was expected to be originated from
the wild species S. bulbocastanum. A high-resolution map of the resistance locus was
constructed towards resistance gene cloning and the resistance locus was analyzed
by using the homology between the sequence of markers and sequences available in
databases.
In Chapter 4, a resistance locus, which is phenotypically indistinguishable from
the S. demissum derived-R2 locus, was identified in a diploid population. A marker-
saturated high-resolution map was constructed using bulk segregant analysis
combined with AFLP marker technology towards resistance gene cloning and also
here additional genes were found in the population.
In Chapter 5, another gene derived from the wild species S. bulbocastanum
was identified. Four different methods were used to construct a marker-saturated
high-resolution map towards resistance gene isolation: PCR-based marker
development from published RFLP markers, the RGA candidate gene approach, BAC
chromosome walking and bulk segregant analysis combined with AFLP marker
technology. Additionally comparative genetics and R gene clusters on potato
chromosome 4 were studied.
In Chapter 6, we obtained results that are important for potato breeding in
general. A population, already described in Chapter 2, was created by a cross
between tetraploid female and diploid male parents. In this case, the male parent
produces unreduced 2n pollen. The analysis of markers derived from the male parent
allowed us to localize the position of centromeres using half-tetrad analysis.
Additionally we proved that crossovers occur only once per chromosome arm.
The genes we identified for foliage blight resistance in this research were all
located on chromosome 4. They could be very closely related or allelic to each other.
14

To make full use of this fact, resistance gene clusters and comparative genetics for
the isolation of these genes are discussed in Chapter 5 and in the general discussion.

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Umaerus, V. and Umaerus, M. 1994. Inheritance of resistance to late blight. In: Bradshaw,
J.E. and Mackay, G.R. Potato genetics. CAB International. Wallingford. UK. pp 365-401
van de Peer, Y. and de Wachter, R. 1997. Evolutionary relationships among crown taxa
taken into account site-to-site rate variation in 185 rRNA. J Mol Evol 45: 619-630
van der Vossen, E.A.G., Rouppe van der Voort, J.N.A.M., Kanyuka, K., Bendahmane, A.,
Sandbrink, H., Baulcombe, D.C., Bakker, J., Stiekema, W.J. and Klein-Lankhorst,
R.M. 2000. Homologues of a single resistance-gene cluster in potato confer resistance
to distinct pathogens: a virus and a nematode. Plant J 23: 567-576
van der Vossen, E., Sikkema, A., Hekkert, B.L., Gros, J., Stevens, P., Muskens, M.,
Wouters, D., Pereira, A., Stiekema, W. and Allefs, S. 2003. An ancient R gene from
the wild potato species Solanum bulbocastanum confers broad-spectrum resistance to
Phytophthora infestans in cultivated potato and tomato. Plant J 36: 867-882
van der Vossen, E.A., Muskens, M., Sikkema, A., Gros, J., Wouters, D., Wolters, P.,
Pereira, A. and Allefs, S. 2004. The Rpi-blb2 gene from Solanum bulbocastanum is
allelic to the Mi-1 gene from tomato and confers broad spectrum resistance to
Phytophthora infestans both in cultivated potato and tomato. 1
st
Solanaceae Genome
Workshop 2004, September 19-21, 2004, Wageningen, The Netherlands. p 121
Vleeshouwers, V.G.A.A., van Dooijeweert, W., Govers, F., Kamoun, S. and Colon, L.T.
2000. The hypersensitive response is associated with host and nonhost resistance to
Phytophthora infestans. Planta 210: 853-864

18
Chapter 2

Dissection of foliage and tuber late blight resistance
in mapping populations of potato

Tae-Ho Park, Vivianne G.A.A. Vleeshouwers, J ong-Bo Kim,
Ronald C.B. Hutten and Richard G.F. Visser

Accepted by Euphytica

Chapter 2

Abstract

The devastating late blight pathogen Phytophthora infestans infects foliage as
well as tubers of potato. To date, resistance breeding has often focused on
foliage blight resistance, but tuber blight resistance is becoming more and
more important in cultivated potatoes. In this study, a reliable tuber assay for
resistance assessment was developed and foliage and tuber blight resistance
(R) was compared in four mapping populations. In the RH4X-103 population,
tuber blight resistance inherited independently from foliage blight resistance.
Three specific R genes against P. infestans were segregating. The Rpi-abpt and
R3a genes function as foliage-specific R genes, whereas the R1 gene acts on
both foliage and tuber. In the segregating populations SHRH and RH94-076,
tuber and foliage blight resistance correlated significantly, which suggests that
resistance in foliage and tuber is conferred by the same gene (could be R3b)
and QTL, respectively. In the CE population neither tuber nor foliage resistance
was observed.

Introduction

Late blight caused by the oomycete Phytophthora infestans is a devastating disease
in potato (Solanum tuberosum L.). The economic value of the crop protection and
losses by the disease has reached several billion US dollars annually (Kamoun,
2001). Although numerous chemicals are applied to control late blight, introduction of
resistance (R) genes from wild Solanum species into potato cultivars is considered
the most promising and environmentally safe approach to achieve late blight
resistance.
For late blight resistance in foliage, eleven R genes from S. demissum have
been introduced into cultivated potatoes. Five genes, R1, R2, R3, R6 and R7 were
mapped and shown to confer race-specific resistance (Leonards-Schippers et al.,
1992; Li et al., 1998; El-Kharbotly, 1994 and 1996). Also in other wild species such as
S. microdontum, S. berthaultii, S. pinnatisectum and S. bulbocastanum, R genes and
quantitative trait loci (QTL) have been localized as new sources for foliage resistance
against P. infestans (Sandbrink et al., 2000; Ewing et al., 2000; Kuhl et al., 2001;
Naess et al., 2000; van der Vossen et al., 2003 and 2004).
The genetics of tuber blight resistance has not extensively been studied. The
inheritance of tuber blight resistance was proven by three different testcrosses in
potato: resistant x resistant, resistant x susceptible and susceptible x susceptible
(Toxopeus, 1961; Wastie et al., 1987), as the mean percentage of susceptible tubers
was significantly different among the three testcrosses. The inheritance of tuber blight
20
Tuber blight resistance

resistance was then explored using combining ability analysis by Stewart et al. (1992).
In 1999, there were the first contemporary reports on mapping QTL for tuber blight
resistance. Oberhagemann et al. (1999) analyzed five populations derived from
crosses between heterozygous potato clones devoid of any known major R genes in
foliage. One major QTL for tuber blight resistance was observed near marker GP179
on chromosome 5 in three populations. This QTL was closely linked to foliage blight
resistance, foliage maturity and vigour. In general, cultivars with a good level of
foliage blight resistance show a good level of tuber blight resistance (Collins et al.,
1999), but this is depending on the plant genotypes (wieyski and Zimnoch-
Guzowska, 2001). To date, no R genes against late blight were identified to
specifically act in tuber or foliage.
Tuber resistance against P. infestans is an agriculturally important trait. For
instance, when cut potato seeds are planted, one or two cycles of late blight
transmission may occur between cutting and emergence (Lambert et al., 1998). A
higher degree of resistance in seed tubers is expected to lower the infection pressure
in the field (Toxopeus, 1958; Wastie, 1991). Toxopeus (1958 and 1961) reported that
important barriers to prevent the pathogen from penetrating the tuber are the tuber
skin and the cambial layer just below the tuber skin. Also the periderm, the cortex and
the medulla are thought to be involved in tuber blight resistance (Flier et al., 2001).
Therefore the expression of tuber blight resistance is largely dependent on inoculated
tissues (wieyski and Zimnoch-Guzowska, 2001).
In this study, four mapping populations bearing various R genes and QTL from
different sources were used to investigate the inheritance of, and the relation between
tuber and foliage blight resistance. Most R genes and QTL appeared to be active in
both foliage and tuber. In one mapping population, foliage specific R genes were
identified, including a novel R gene derived from S. bulbocastanum.

Materials and methods

Plant material
The parents and progeny of four mapping populations, SHRH, RH4X-103, RH94-076
and CE were used in this study (Table 1). The diploid SHRH population is widely
used in potato genetic mapping at the Laboratory of Plant Breeding and Nematology,
Wageningen University, The Netherlands and used for an ultra high density (UHD)
map with more than 10,000 AFLP (Amplified Fragment Length Polymorphism)
markers (Rouppe van der Voort et al., 1997a, 1997b and 1999; Huang et al., 2004;
van Os et al., submitted). The UHD map is available in the web
(http://www.dpw.wageningen-ur.nl/uhd/). For this population, 14 resistant and 16
susceptible genotypes were chosen based on the results of inoculation with isolate
IPO-0 in foliage (Huang et al., 2004). The tetraploid RH4X-103 population is derived
21
Chapter 2

from a cross between 707TG11-1 and RH89-039-16. 707TG11-1 was obtained from
ABPT material, which was produced via bridge crosses as quadruple hybrids with
four Solanum species: S. acaule, S. bulbocastanum, S. phureja and S. tuberosum
(Hermsen and Ramanna, 1973). For this population, 15 resistant and 15 susceptible
genotypes were chosen based on the results of inoculation with P. infestans isolate
90128 in foliage (Chapter 3). The diploid RH94-076 population is derived from a
cross between RH90-038-21, an interspecific hybrid between S. microdontum and S.
tuberosum and RH88-025-50, an interspecific hybrid between S. phureja and S.
tuberosum. For this population, 15 genotypes with the highest and 15 genotypes with
the lowest AUDPC (Area Under Disease Progress Curve) value, which were
evaluated with a complex P. infestans isolate IPO-82001 in a field experiment (Celis
et al., unpublished), were selected. The diploid CE population is derived from a cross
between C (USW5337.3) and E (77.2102.37). Clone C is an interspecific hybrid
between S. phureja and S. tuberosum. Clone E was obtained from a cross between
the clone C and S. vernei S. tuberosum backcross clone (J acobs et al., 1995).
Thirty genotypes were randomly selected. Potato cultivars Nicola and Bintje were
used as resistant and susceptible controls, respectively, in all experiments.
Table 1. Mapping populations used in this study. Parents with known R genes or QTL in foliage, wild
Solanum species origin of resistance and genetic location of the R gene or QTL are presented.
Population Maternal clone
(R gene)
Paternal clone Resistance source R gene / QTLs
and location
Reference
SHRH SH83-92-488
(R3)
RH89-039-16 S. demissum R3 on
chromosom 11
Huang et al.
(2004)
RH4x103 707TG11-1
(Rpi-abpt)
RH89-039-16 S. bulbocastanum Rpi-abpt on
chromosome 4
Chapter 3
RH94-076 RH90-038-21
(QTL)
RH88-025-50 S. microdontum QTLs on
chromosome 4
Celis et al.
(unpublished)
CE USW5337.3 77.2102.37 None None J acobs et al.
(1995)

Table 2. Isolates of Phytophthora infestans and virulence factors.
Isolate Race Source
IPO-0 0 W. Flier, Plant Research International, The Netherlands
90128 1.3.4.7.8.11 F. Govers, Wageningen University, The Netherlands
99018 1.4 F. Govers, Wageningen University, The Netherlands
H30P04 3.7 F. Govers, Wageningen University, The Netherlands

Isolates of P. infestans and preparation of inoculum
The P. infestans isolates used in this study are presented in Table 2. The isolates
were cultured on rye sucrose agar medium in the dark at 15
o
C for 1-2 weeks (Caten
and J inks, 1968). Sporulating mycelium was flooded with cold water and the
sporangiaspore suspension was incubated at 4
o
C for three hours. After the release of
22

the zoospores, the inoculum was adjusted to a concentration of 5 x 10
5
zoospores/ml
(Vleeshouwers et al., 1999).

Resistance assays
The wounded tuber assay was developed based on the method described by Roer
and Toxopeus (1961). Intact tubers of similar size were taken from cold storage. They
were washed in tab water, sterilized in 5 % sodium hypochlorite for 5 minutes and
rinsed in tap water twice. Holes of 5 mm depth were made on the surface of the
tubers and a 10 droplet of inoculum was applied in the wound. The tubers were
incubated in a closed tray with 100 % relative humidity in the dark at 15
o
C and
resistance was evaluated at 5-14 days after inoculation. After the evaluation, the
tubers were cut and kept in the same condition for three additional days.
Two types of tuber slice methods were performed, i.e. the medulla slice assay
and the cortex slice assay as described by Dorrance & Inglis (1998). Tuber slices of
about 7 mm thickness were taken from the medulla and the cortex, i.e. the central
and the outer part of tubers, respectively. The slices were placed individually in 9 cm
petri-dishes and spot-inoculated with a 10 l droplet of inoculum on the middle upper
side of the slices. They were incubated in the dark at 15
o
C and evaluated at 3-7 days
after inoculation.
Foliage blight resistance was determined by a detached leaf assay. Fully
expanded and healthy leaves collected from greenhouse plants were placed
randomly on wet paper in trays with high relative humidity. The leaflets were
inoculated by pipetting 10 l droplet of inoculum on the abaxial side and incubated in
the climate chamber at 15
o
C. The symptoms were evaluated at 3-14 days after
inoculation.

DNA isolation and molecular marker analysis
DNA isolation was performed with fresh leaf tissue using the Retsch machine (Retsch
Inc., Haan, Germany) in which DNA of 96 samples can be isolated simultaneously.
The fresh leaf tissue was grinded in the presence of a 2 % CTAB buffer composed of
Tris (pH 7.5), 5 M NaCl and 0.5 M EDTA (pH 8.0).
Markers linked to the resistance locus were identified by a bulked segregant
analysis (BSA) as described by Michelmore et al. (1991) using the AFLP method (Vos
et al., 1995). AFLP bands were separated on a 6 % polyacryl amide gel in a Li-cor
sequencer (Li-cor, Lincoln, NB, U.S.A.). From pre-amplified products, the bulks for
resistant and susceptible genotypes were composed according to the phenotypic data
obtained from the resistance assay. Each bulk consisted of pre-amplified products of
eight genotypes. In the BSA analysis, the resistant parent, the susceptible parent, the
resistant bulk and the susceptible bulk were screened with 256 primer combinations.
Candidate markers, which seemed to be linked to the resistance gene, were selected
23
Chapter 2

on the basis of absence/presence of bands. Candidate markers were tested for
confirmation of linkage on 16 individual genotypes used for resistant and susceptible
bulk composition.
For linkage analysis and characterization of resistances in the RH4X-103
population, PCR-based markers were used to detect different known R genes
(Chapter 3; Ballvora et al., 2002; Meksum et al., 1999; Huang et al., 2005). The
markers are listed in Table 3.

Table 3. List of primers of PCR based-markers used in this study.
Marker PCR primer (5`-3`)* Tm
Enz.
Reference
GP179 GGTTTTAGTGATTGTGCTGC
AATTTCAGACGAGTAGGCACT
55 BssKI Meksem et al. (1995)
Th2 ATTCATCGTCATCGCCTTTA
CCTTTGTATCATTCGCAGTT
56 a.s. Chapter 3
76_2S CACTCGTGACATATCCTCACTA
CAACCCTGGCATGCCACG
50-55 a.s. Ballvora et al. (2002)
SH23_2 ATCGTTGTCATGCTATGAGATTGTT
CTTCAAGGTAGTGGGCAGTATGCTT
64-50 a.s. Huang et al. (2005)
*Nucleotide sequence of primers. Both forward and reverse primers are shown.
Annealing temperature. For 76_2S, the annealing temperature was 50

o
C for the first 7 cycles and 55
o
C for the last 30 cycles.
For SH23_2, a touch-down program was applied. For the first 12 cycles, the annealing temperature was gradually decreasing
from 64
o
C to 50
o
C and it was kept at 50
o
Restriction enzyme that reveals polymorphism between resistant and susceptible linked alleles of the marker. a.s. means allele
specific marker showing polymorphism without digestion.

Results

Tuber blight assessment
To optimize the method for tuber blight resistance assessment and to compare late
blight resistance between foliage and tuber, the parents of four mapping populations
(Table 1) were inoculated with the P. infestans isolates IPO-0, H30P04 and 90128
(Table 2) on tuber medulla slices, cortex slices and wounded tubers. No difference in
resistance outcome was found between the different methods. However, the
wounded tuber assay was selected for further experiments, because it showed
systematically clear phenotypes and more reliable patterns. At five days after
inoculation, resistant genotypes did not show any disease symptom (Figure 1A), but
massive mycelium was visible at the inoculation site on the wound of susceptible
genotypes (Figure 1B). The difference between resistant and susceptible became
clearer when the tubers were cut. Resistant genotypes showed a sharp border, which
looked like a hypersensitive response (HR; Figure 1A), whereas in susceptible
genotypes the tissue was browning that expanded from the inoculation site (Figure
1B). After re-incubation at high humidity for three additional days, resistant genotypes
remained devoid of any disease symptoms (Figure 1C), whereas abundant
24

sporulating mycelium was visible on the cut tuber surface in the susceptible
genotypes (Figure 1D). Occasionally, an intermediate phenotype was noted. In such
case, the HR-like border was less clear, but no sporulation could be noted on the cut
tubers. This phenotype was designated moderately resistant.

Figure 1. The wounded tuber assay with P. infestans
isolate IPO-0. Tubers of 707TG11-1 (A, C) and RH89-039-
16 (B, D) were cut 5 days after inoculation (A, B) and re-
incubated for 3 additional days (C, D).

Table 4. Tuber late blight resistance data of the parents of the mapping populations (Table 1) to the P.
infestans isolates IPO-0, H30P04 and 90128.
IPO-0 H30P04 90128
SH83-92-488 R* M S
RH89-039-16 S S S
RH90-038-21 M S S
RH88-025-50 S S S
707TG11-1 R M M
USW5537.3 S S S
77.2102.37 S S S
* Tuber blight resistance is classified into three different classes. R, S and M indicate resistant, susceptible and moderately
resistant respectively.

The resistances found in tubers of parents (Table 4) were similar to the
resistances in foliages (Table 1). RH89-039-16, RH88-025-50, USW5537.3 and
77.2102.37 were susceptible to all three P. infestans isolates in tubers as they were in
foliage to identical or different isolates (Table 1, data not shown). All genotypes were
susceptible to isolate 90128 except 707TG11-1, which contains a novel R gene
termed Rpi-abpt (Chapter 3). R3 race-specific resistance was found in tubers of
SH83-92-488 that also exists in foliage (Huang et al., 2004). Quantitative differences
in resistance to different isolates were also noted. SH83-92-488 and RH90-038-21
tubers were moderately resistant to H30P04 and IPO-0, respectively. RH90-038-21
contains a major QTL for foliage blight resistance (Celis et al., unpublished), which is
apparently strong enough to confer tuber resistance to P. infestans isolate IPO-0, but
25
Chapter 2

too weak for the more complex isolates H30P04 and 90128. 707TG11-1 was fully
resistant to IPO-0, but moderately resistant to H30P04 and 90128.

Inheritance of foliage and tuber blight resistance
In the resistance assay with seven parental genotypes, we found that tuber blight
resistance coincided with foliage blight resistance. To investigate the inheritance of
tuber blight resistance in comparison with foliage blight resistance, 30 genotypes
were selected for each mapping population and inoculated with isolate IPO-0 using
the wounded tuber assay. All 30 genotypes of the CE population were susceptible in
tubers, indicating the absence of any late blight R gene. Tuber blight resistance to
isolate IPO-0 segregated in the other three mapping populations. In SHRH and
RH94-076, the resistance in tubers correlated with the resistance in foliage. This
indicates that R3 in the SHRH population (Huang et al., 2004) and the QTL in the
RH94-076 population (Celis et al., unpublished) are expressed in foliage as well as in
tuber. It also would suggest that the QTL of RH94-076 is effective against isolate IPO-
0 (race 0) as well as isolate IPO-82001 (race 1.2.4.5.10.11). In RH4X-103, tuber and
foliage blight resistance were not correlated. Eleven and seven genotypes were
resistant and susceptible for both tuber and foliage resistance, respectively, but
conflicting results were found in the other 12 genotypes (Table 5).

Table 5. Comparison between foliage and tuber blight resistance in 30 genotypes of the RH4X-103
mapping population. Correlation between foliage and tuber resistance was determined by
2
test (
2
=
1.29; P <0.5).
Foliage\Tuber Resistant Susceptible Total
Resistant 11 4 15
Susceptible 8 7 15
Total 19 11 30

Localization of tuber resistance in RH4X-103
To further investigate the tuber blight resistance in RH4X-103, the population was
expanded to 233 offspring. The resistance of these genotypes was examined by
inoculation with the isolates 90128 and IPO-0 using the wounded tuber assay method.
All genotypes were susceptible to the isolate 90128. Race-specific resistance was
noted to isolate IPO-0, as 79 and 104 genotypes were clearly resistant and clearly
susceptible, respectively. The remaining 50 genotypes showing unclear
hypersensitive response were excluded from analysis. The resistance data indicates
a 1:1 segregation (
2
=3.48, 0.9<P<0.95) for a race-specific R gene to P. infestans.
The segregation of a part of the population is included in Figure 2A.
Based on the results of tuber blight resistance assay with isolate IPO-0, a
resistant and a susceptible bulk were composed for molecular marker analysis.
26

707TG11-1, RH89-039-16, the resistance bulk and the susceptible bulk were
screened for BSA (Michelmore et al., 1991), and ten AFLP markers were identified to
be linked to the locus for tuber blight resistance (data not shown). Blasting of the
sequence of the AFLP markers via the website (http://www.ncbi.nlm.nih.gov/BLAST)
showed that EATC/MCGA_332 was identical to the sequence of S. tuberosum strain
P6/210 clone BAC BA87d17, S. tuberosum strain P6/210 clone BAC BA213c14 and
S. demissum chromosome 5 clone PGEC472P22. A part of the AFLP marker
sequence (63 nucleotides) was 82 % similar to the sequence of Lycopersicon
pimpinellifolium Rio Grande 76R Pto locus (Chang et al., 2002), which is located on
tomato chromosome 5. These data suggest that the resistance gene that is active in
tuber is located on chromosome 5.
The tentative location of the R gene was further examined with known PCR
markers (Table 3). The RFLP marker-derived locus GP179 (Meksem et al., 1995),
which is located on chromosome 5, was mapped in the population of 233 genotypes
and found to be linked to the tuber blight R locus at 8.1 cM genetic distance. Also the
race-specific R1 gene is located near GP179 (Leonards-Schippers et al., 1992).
Based on the sequence of R1, an R1 specific marker 76_2s was developed (Table 3;
Ballvora et al., 2002) and applied to the 233 genotypes. The 76_2s marker was fully
co-segregating with tuber blight resistance locus. In combination with the race-
specific resistance data to isolates IPO-0 and 90128, these data indicate that the R1
or an R1-like gene is present in RH4X-103 and active in tubers.

Figure 2. Graphical genotyping and phenotyping of the parents 707TG11-1 (TG) and RH89-039-16
(RH) and 52 offspring and of RH4X-103. Segregation for resistance to each P. infestans isolate and
molecular marker is shown. A: The phenotypic results with isolate IPO-0 in tuber. B: The phenotypic
results with P. infestans isolates IPO-0, 90128, 99018 and H30P04 in foliage. C: The genotypic results
detected by the Rpi-abpt-specific Th2, the R1-specific 76_2S and the R3-specific SH23_2 markers
(Table 3). D: Classes determined by phenotypic and genotypic data. The number of genotypes within
each group is indicated. R, S, nd, ab and aa indicate resistant, susceptible, not determined, presence
and absence of the markers, respectively.
27
Chapter 2

Characterization of the foliage resistance genes in RH4X-103
To identify foliage blight resistance genes in the population RH4X-103, a subset of 52
genotypes and both parents were subjected to detached leaf assays with the P.
infestans isolates IPO-0 and 90128. A smaller subset of 28 progeny plants was
inoculated with the isolates 99018 and H30P04 (Table 2). All isolates showed
different segregation patterns in the population, which suggests that at least three R
genes for foliage resistance are segregating in the population (Figure 2B). The results
for resistance to isolate 90128 were adopted from the results of Chapter 3. In
addition, the population of 52 genotypes was analyzed with molecular markers (Table
3). The 76_2s marker (Ballvora et al., 2002) segregated 1:1 (Figure 2C), and exactly
matched to the R1 specific resistance in tuber (Figure 2A). Also the SH23_2 marker
for R3a race-specific resistance (Huang et al., 2005) appeared to segregate in
RH4x103 (Figure 2C). An additional marker Th2 was developed (Table 3, Chapter 3),
which appeared to be 1:1. By combining genotypic and phenotypic data, the 52
genotypes were assigned to eight different groups (Figure 2D). Genotypes that
contain any of the three R gene-linked markers, i.e. group I-VII, were resistant to
isolate IPO-0 in foliage. All genotypes that carry the Th2 marker, i.e. group I-IV, were
resistant to all isolates, which is explained by the broad-spectrum resistance of Rpi-
abpt (Chapter 3). The genotypes containing only 76_2s or SH23_2, i.e. group VI or
VII, show race-specific resistance to isolates 99018, H30P04 and IPO-0, as expected
based on virulence data to R1 and R3a. In summary, in the RH4X-103 population, the
three R genes Rpi-abpt, R1 and R3a segregate for foliage resistance, from which only
R1 is functional in tuber.

Discussion

Tuber resistance to P. infestans is one of the most important traits in cultivated
potatoes. Although it is considered as an essential component for potato breeding,
comparatively less efforts are made in breeding for resistance in tuber than in foliage
(wieyski and Zimnoch-Guzowska, 2001). There is rather limited knowledge on
inheritance of tuber blight resistance, but also occurrence of tissue-specific
expression of resistance and the difficulty of evaluation complicate the breeding
research. To develop a reliable method for tuber blight resistance, seven parental
genotypes of four mapping populations of which foliage blight resistances were
previously identified were tested using different inoculation methods. The different
tuber blight assay methods gave identical results in this genetic material.
The relationship between tuber and foliage blight resistance is not clear and
often contradicting. Platt and Tai (1998) and Stewart et al. (1994) found a correlation
between tuber and foliage blight resistances, whereas Stewart et al. (1992) and Kirk
28

et al. (2001) did not. R1, R2 and R3 and some QTL could confer both foliage and
tuber blight resistance (Toxopeus, 1961; Roer and Toxopeus, 1961; Stewart and
Bradshaw, 2001; Collins et al., 1999; Oberhagemann et al., 1999). However, other
genes may be differentially expressed in different tiss ues or organs. In this study,
therefore, we examined the inheritance of tuber blight resistance in four mapping
populations and compared the results with foliage blight resistance. The proportion of
resistant and susceptible tubers were accepted as a 1:1 ratio in the SHRH, RH94-076
and RH4X-103 populations indicating that the resistance was transmitted as a
monogenic trait from the parents to their progenies. Tuber blight resistance inherited
independently of the foliage resistance in the RH4X-103 population (Table 5). Tuber
blight resistance was conferred by one major gene, which was localized on
chromosome 5. This resistance co-segregated with the R1-specific marker 76_2s,
suggesting that tuber blight resistance in this population was caused by R1 or R1-like
as one cultivar containing S. demissum-derived fragment was present in the pedigree
of the population. In foliage, however, two additional R genes were identified to
cause foliage-specific resistance. R3a (Huang et al., 2005) and Rpi-abpt (Chapter 3)
were segregating in the population and specifically acting in foliage. However,
707TG11-1 was moderately resistant to isolate 90128 which could be due to the
presence of several unknown minor QTL, which do not have detectable effects on
resistance in the progeny after segregation. The moderate resistance to isolate
H30P04 could be explained by its high aggressiveness (Table 4). In the SHRH and
the RH94-076 populations, tuber blight resistance correlated with the presence of R3
and a major QTL for foliage blight resistance, respectively.
The R3 locus in the SHRH population consists of two functionally distinct R
genes R3a and R3b that are only 0.4 cM separated from each other (Huang et al.,
2004). R3-specific resistance occurred in both tuber and foliage in the SHRH
population, but the R3a gene alone was foliage-specific in the RH4x103 population. It
remains to be determined whether the R3 specific resistance for tuber blight in the
SHRH population is caused by R3b or another R3-like gene or differences in
expression patterns.
In conclusion, we found that the expression of resistance for tuber or foliage
late blight depends on the R genes. In the RH4X-103 population, Rpi-abpt and R3a
are foliage-specific whereas R1 or R1-like and the QTL from RH94-076 act both in
tuber and foliage.

Reference

29
Chapter 2

Caten, C.E. and Jinks, J.L. 1968. Spontaneous variability of single isolates of Phytophthora
infestans I. Cultural variation. Can J Bot 46: 329-347
Chang, J.H., Tai, Y.S., Bernal, A.J., Lavelle, D.T., Staskawicz, B.J. and Michelmore, R.W.
2002. Functional analysis of the Pto resistance gene family in tomato and the
identification of a minor resistance determinant in a susceptible haplotype. Mol Plant
Microb Interact 15: 281-291
P., De Jong, W., Gebhardt, C., Bonnel, E. and Waugh, R. 1999. QTL for field
Breeding 5: 387-398
Dorrance, A.E. and Inglis, D.A. 1998. Assesment of laboratory methods for evaluating
potato tubers for resistance to late blight. Plant Dis 82: 442-446
infestans in progeny of dihaploid potato parents. Mol Gen Genet 242: 749-754
El-Kharbotly, A., Palomino-Snchez, C., Salamini, F., Jacobsen, E. and Gebhardt, C.
infestans (Mont.) de Bary identified genetic loci clustering with the R3 locus on
Flier, W.G., Turkensteen, L.J., van den Bosch, G.B.M., Vereijken, P.F.G. and Mulder, A.
2001. Differential interaction of Phytophthora infestans of potato cultivars with different
levels of blight resistance. Plant Pathol 50: 292-301
Hermsen, J.G.T.H. and Ramanna, M.S. 1973. Double-bridge hybrids of Solanum
bulbocastanum and cultivars of Solanum tuberosum. Euphytica 22: 457-466
Huang, S., Vleeshouwers, V.G.A.A, Werij, J.S., Hutten, R.C.B., van Eck, H.J., Visser,
Huang, S., van der Vossen, E., Kuang, H., Vleeshouwers, V., Zhang, N., Borm, T.J.A.,
van Eck, H.J., Baker, B., Jacobsen, E. and Visser, R.G.F. 2005. Comparative
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Kirk, W.W., Felcher, K.J., Douches, D.S., Niemira, B.A. and Hammerschmidt, R. 2001.
Susceptibility of potato (Solanum tuberosum L.) foliage and tubers to the US8 genotype
of Phytophthora infestrans. Amer J Potato Res 78: 319-322
Lambert, D.H., Currier, A.I. and Olanya, M.O. 1998. Transmission of Phythphthora
infestans in Cut Potato Seed. Amer J Potato Res 75: 257-263
potato chromosome V. Mol Gen Genet 233: 278-283
Meksem, K. Leister, D., Peleman, J., Zabeau, M., Salamini, F. and Gebhardt, C. 1995. A
high-resolution map of the vicinity of the R1 locus on chromosome V of potato based on
RFLP and AFLP markers. Mol Gen Genet 249:74-81
Michelmore, R.W., Paran, I. and Kesseli, R.V. 1991. Identification of markers linked to
disease-resistance genes by bulked segregant analysis: A rapid method to detect
markers in specific genomic regions by using segregating populations. Proc Natl Acad
Sci USA 88: 9828-9832
Oberhagemann, P., Chatot-Balandras, C., Schfer-Pregl, R., Wegener, D., Palomino, C.,
resistance to late blight in potato: towards marker-assisted selection. Mol Breeding 5:
399-415
Platt, H.W. and Tai, G. 1998. Relationship between resistance to late blight in potato foliage
and tubers of cultivars and breeding selections with different resistance levels. Amer J
Roer, L. and Toxopeus, H.J. 1961. The effect of R-genes for hypersensitivity in potato-
leaves on tuber resistance to Phytophthora infestans. Euphytica 10: 35-42
Rouppe van der Voort, J., van Zandvoort, P., van Eck, H.J., Folkertsma, R.T., Hutten,
R.C.B., Draaistra, J., Gommers, F.J., Jacobsen, E., Helder, J. and Bakker, J. 1997a.
Use of allele specificity of comigrating AFLP markers to align genetic maps from
different potato genotypes. Mol Gen Genet 255: 438-447
Rouppe van der Voort, J., Wolters, P., Folkertsma, R., Hutten, R., van Zandvoort, P.,
Vinke, H., Kanyuke, K., Bendahmane, A., Jacobsen, E., Janssen, R. and Bakker, J.
1997b. Mapping of the cyst nematode resistance locus Gpa2 in potato using a strategy
based on comigrating AFLP markers. Theor Appl Genet 95: 874-880
Rouppe van der Voort, J., Kanyuka, K., van der Vossen, E., Bendahmane, A., Klein-
Lankhorst, R., Stiekema, W., Baulcombe, D. and Bakker, J. 1999. Tight physical
linkage of the nematode resistance gene Gpa2 and the virus resistance gene Rx on a
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single introgression segment from the wild potato species Solanum tuberosum ssp.
andigena CPC 1673. Mol Plant Microb Interact 12:197-206
Stewart, H.E. and Bradshaw, J.E. 2001. Assessment of the field resistance of potato
genotypes with major gene resistance to late blight (Phytophthora infestans (Mont.) de
Bary) using inoculum comprised of two complementary races of the fungus. Potato Res
44: 41-52
Stewart, H.E., Bradshaw, J.E. and Wastie, R.L. 1994. Correlation between resistance to
late blight in foliage and tubers in potato clones from parents of contrasting resistance.
Stewart, H.E., Wastie, R.L., Bradshaw, J.E. and Brown, J. 1992. Inheritance of resistance
to late blight in foliage and tubers of progenies from parents differing in resistance.
wieyski, K.M. and Zimnoch-Guzowska, E. 2001. Breeding potato cultivars with tuber
resistant to Phytophthora infestans. Potato Res 44: 97-117
Toxopeus, H.J. 1958. Some notes of the relations between field resistance to Phytophthora
infestans in leaves and tubers and ripening time in Solanum tuberosum subsp.
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Toxopeus, H.J. 1961. On the inheritance of tuber resistance of Solanum tubersosum to
Phytophthora infestans in the field. Euphytica 10: 307-314
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Solanaceae Genome
Vleeshouwers, V.G.A.A., van Dooijweert, W., Paul Keizer, L.C., Sijpkes, L., Govers, F.,
Colon, L.T. 1999. A laboratory assay for Phytophthora infestans resistance in various
Solanum species reflects the field situation. Eur J Plant Pathol 105: 241-250
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J., Peleman, J., Kuiper, M. and Zabeau, M. 1995. AFLP: a new technique for DNA
fingerprinting. Nucleic Acids Res 23: 4407-4414
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test for resistance to tuber blight (Phytophthora infestans). Potato Res 30: 533-538

32
Chapter 3

High-resolution mapping and analysis of the resistance
locus Rpi-abpt against Phytophthora infestans
in potato

Tae-Ho Park, Vivianne G.A.A. Vleeshouwers, Ronald C.B. Hutten,
Herman J . van Eck, Edwin van der Vossen,
Evert J acobsen and Richard G.F. Visser

Accepted by Molecular Breeding

Chapter 3

Abstract

Introduction of more durable resistance against Phytophthora infestans
causing late blight into the cultivated potato is of importance for sustainable
agriculture. We identified a new monogenically inherited resistance locus that
is localized on chromosome 4. The resistance is derived from an ABPT clone,
which is originally a complex quadruple hybrid in which Solanum acaule, S.
bulbocastanum, S. phureja and S. tuberosum were involved. Resistance data of
the original resistant accessions of the wild species and analysis of mobility of
AFLP markers linked to the resistance locus suggest that the resistance locus
is originating from S. bulbocastanum. A population of 1383 genotypes was
screened with two AFLP markers flanking the Rpi-abpt locus and 98
recombinants were identified. An accurate high-resolution map was
constructed and the Rpi-abpt locus was localized in a 0.5 cM interval. One
AFLP marker was co-segregating with the Rpi-abpt locus. Its DNA sequence
was highly similar with sequences found on a tomato BAC containing several
resistance gene analogues of chromosome 4 and its translated protein
sequence appeared to be homologous to several disease resistance related
proteins. The results indicated that it is clear that the Rpi-abpt gene is a
member of an R gene cluster.

Introduction

Late blight caused by Phytophthora infestans is one of the most serious threats to
potato cultivation. Controlling late blight usually involves the application of a lot of
chemicals. Nevertheless, restrictions of chemical control, environmental safety and
the spread of more virulent forms of the pathogen to these chemicals indicate that the
most promising approach to achieve late blight resistance is the introduction of
resistance genes from wild Solanum species into potato cultivars (Kato et al., 1997).
In the last century, 11 resistance genes against Phytophthora infestans were
introgressed from the wild Solanum species S. demissum into potato (Black et al.,
1953; Malcolmson and Black, 1966). However, these resistance genes confer race-
specific resistance controlled by single resistance factors and these genes are not
durable (Mastenbroek, 1953; Malcolmson and Black, 1966). In the meantime, not only
asexual but also sexual forms (A1 and A2) of P. infestans have been introduced into
Europe, enabling more flexibility of the pathogen both against chemicals and
resistance genes. Therefore, new sources are required for the creation of durable and
race-non-specific resistance. Instead of the genes from S. demissum, resistance loci
and QTLs originating from wild species like S. berthaultii, S. pinnatisectum and S.
microdontum have been localized on the chromosomes 10, 7 and 4, respectively, as
34
High-resolution mapping of Rpi-abpt

new sources of resistance (Ewing et al., 2000; Kuhl et al., 2001; Sandbrink et al.,
2000). S. bulbocastanum has also been considered as another new source of
resistance to P. infestans (Niederhausen and Mills, 1953). S. bulbocastanum is a self-
incompatible diploid wild species from Mexico which could not directly be crossed to
the common potato S. tuberosum (Hermsen and De Boer, 1971). Recently two
resistance genes Rpi-blb1/RB and Rpi-blb2 derived from S. bulbocastanum were
cloned (van der Vossen et al., 2003; Song et al., 2003; van der Vossen et al., 2004).
The RB gene was localized on chromosome 8 (Naess et al., 2000). Because of the
impossibility of direct sexual crosses between S. tuberosum and S. bulbocastanum,
BC
2
populations derived from somatic hybrids were used (Helgeson et al., 1998). Due
to the same reason, the Rpi-blb1 gene was cloned using an intraspecific hybrid
between resistant and susceptible S. bulbocastanum genotypes (van der Vossen et
al., 2003). To clone Rpi-blb2, which is located on chromosome 6, both an intraspecific
hybrid and a complex interspecific hybrid, which was designated ABPT, were used
(van der Vossen et al., 2004). ABPT is a quadruple species hybrid created through
bridge crosses using S. acaule (A), S. bulbocastanum (B), S. phureja (P) and S.
tuberosum (T) (Figure 1a; Hermsen and Ramanna, 1973).
Map-based cloning is one of the most challenging methods for gene cloning in
plant species with a relatively large genome (Lukowitz et al., 2000). In addition to Rpi-
blb1/RB and Rpi-blb2, the S. demissum derived R1 (Ballvora et al, 2002) and R3a
(Huang, 2005) have also been isolated using the map-based cloning approach. The
first essential step for map-based gene cloning is to determine the accurate position
of the target gene.
In this paper, we identify another new resistance gene named Rpi-abpt in
ABPT material, which differs from that used by van der Vossen et al. (2004). We
construct a high-resolution map of the Rpi-abpt locus and analyze this region by
investigating the sequence of closely linked markers. We also unraveled the predicted
wild species origin of the resistance gene.


Plant materials
The pedigree of the mapping population RH4X-103 is shown in Figure 1b. The
tetraploid resistant female parent 707TG11-1 was derived from a quadruple hybrid
ABPT clone (Hermsen and Ramanna, 1973). The diploid susceptible male parent
RH89-039-16, producing unreduced 2n pollen, is one of the advanced clones widely
used for mapping research at the Laboratory of Plant Breeding and the Laboratory of
Nematology, Wageningen University (Rouppe van der Voort et al., 1997a, 1997b,
1998, 1999; Huang et al., 2004; van Os et al., submitted).
35
Chapter 3

Seeds of RH4X-103 progeny were sterilized in three different steps: 70 %
alcohol for 1 minute, 1.5 % sodium hypochlorite for 5 minutes and three times rinsing
with sterilized water. Plants were generated from seeds sown in vitro. When the size
of the in vitro plants was approximately five centimeters, they were multiplied for DNA
isolation, resistance assay and genotype maintenance. Recombinants were
maintained in vitro and transferred to the greenhouse for generation of leaf material
for the detached leaf assays.

Figure 1. Plant materials used for creation of the tetraploid mapping population RH4X-103. A: Pedigree
of the tetraploid ABPT breeding clone. A, B, P and T indicate S. acaule, S. bulbocastanum, S. phureja
and S. tuberosum, respectively. B: Pedigree of RH4X-103 derived from two quadruple hybrid ABPTs.
The genotypes carrying resistance against P. infestans are indicated in bold.

Accession of S. acaule CGN17843 and CGN20620, S. bulbocastanum
CGN21306 and CGN17693, S. phureja CGN17667 and CGN18301, S. demissum
CGN17810 were obtained from the Center of Genetic Resources in The Netherlands
(CGN) and maintained in vitro. Potato clone Ceb44-158-4, which has S. demissum in
its pedigree and S. tuberosum cultivars Titana, Alcmaria, Van Gogh, Agria, Bintje and
Nicola were planted in the greenhouse, and leaves were collected for DNA isolation.

Resistance assay
The mapping population of 233 genotypes was subjected to a detached leaf assay in
four replications using leaf material from the maintenance field. Fully expanded and
healthy leaflets were collected and incubated in moist trays. Each leaflet was
inoculated with a 10 l droplet of inoculum (Vleeshouwers et al, 1999). Leaves of
plants were evaluated three and five days after inoculation.
P. infestans isolates 90128 (race 1.3.4.7.8.11), which was kindly provided by
Drs. F. Govers of Wageningen University, Wageningen, the Netherlands, USA618
(race 1.2.3.6.7.11) by W. Fry of Cornell University, Ithaca, USA and IPO-82001 (race
1.2.4.5.10.11) by W. Flier of Plant Research International, Wagenigen, The
Netherlands were used to determine the resistance. The isolates were cultured on rye
36

sucrose agar medium (Caten and J inks, 1968) in the dark at 15 for two weeks.
When mycelium was growing on the medium, it was rinsed with 5 ml ice-cold water
for the release of the zoospores from sporangia. After incubating the suspension at
4 for three hours, it was re-suspended to make an inoculum concentration of 5 x
10
5
zoospores/ml.

DNA isolation and AFLP analysis
DNA isolation was performed using a Retch protocol. Fresh leaf tissue was ground
with two steel balls, nuclear lysis buffer (0.2 M Tris-HCl, 0.05 M EDTA, 2 M NaCl, 2 %
CTAB, with an end pH of 7.5), DNA extraction buffer (100 mM Tris-HCl, 5 mM EDTA,
0.35 M Sorbitol, 20 mM NaBisulfite, with an end pH of 7.5) and 5 % sarcosyl in 96
wells Costar plates (Corning Inc., Corning, NY, U.S.A.) using a Retch machine
(Retsch Inc., Haan, Germany). The ground leaf tissue was incubated at 65 C for an
hour followed by adding ice-cold chloroform isoamyl alcohol (24:1). After
centrifugation, the supernatant was transferred to new tubes. Adding isopropanol and
another centrifugation allowed the precipitated DNA to be extracted. After the DNA
pellet was dried, the DNA was dissolved in T
0.1
E-buffer (+0.5 g RNAse).
AFLP analysis was performed as described by Vos et al. (1995). Primary
template DNA of 233 genotypes was prepared using the restriction enzymes
EcoRI/MseI and PstI/MseI combinations, ligated with adaptors fitting to the EcoRI,
MseI and PstI sites, and 10 times diluted prior to the selective pre-amplification with
single nucleotide extended primers. For the selective amplification, 11 E+3/M+3
primer combinations were selected based on their appearance in the Ultra High
Density (UHD) Map (http://www.dpw.wau.nl/uhd/). Extra primer combinations were
selected according to the results of a bulked segregant analysis (BSA) with 204
primer combinations as described by Michelmore et al. (1991) and six primer
combinations from the R2 map (Li et al., 1998).
In addition to this, AFLP marker analysis was performed to detect the wild
species origin of the resistance in five different Solanum species as described by Li et
al. (1998). The mobility of the AFLP markers, which are linked to the resistance locus,
was compared among them.

Map construction
The AFLP markers inherited from the female parent were scored. Band intensity was
discarded to use AFLP markers as dominant markers and only simplex-inherited
markers [single-dose restriction fragments, (SDRFs); Wu et al., 1992] with a simplex
trait allele for the detection of linkage in the coupling phase were included in the data
analysis. The linkage group containing the resistance locus was determined by
J oinMap 2.0 (Stam, 1993) and marker order by Record (van Os et al., 2000). The
37
Chapter 3

map distance was calculated based on the frequency of the recombination between
AFLP markers.

AFLP marker and BAC clone analysis
Blasting of the sequences was performed on the web site http://www.ncbi.nlm.nih.
gov/BLAST/. Genescan (http://bioweb.pasteur.fr/seqanal/interfaces/genscan.html;
Burge and Karlin, 1997) and Genemark (http://www.ebi.ac.uk/genemark/; Lukashin
and Borodovsky, 1998) were used to analyze the sequences of some BAC clones.
ClustalX (J eanmougin et al., 1998) was used to align sequences. For physical
alignment of the resistance gene, the BAC library constructed of the wild species S.
bulbocastanum, BGRC8005-8 (van der Vossen et al., 2003) was used and BAC-end
sequences were performed at Greenomics (Wageningen, The Netherlands).

Marker nomenclature
AFLP markers were named with acronyms of enzyme combinations EcoRI/MseI and
PstI/MseI, which were used to prepare the template DNA, followed by three selective
nucleotides and the size of each marker as described in reference autoradiograms
created by Keygene NV, Wageningen, The Netherlands. Developed PCR markers,
which were based on the sequence of a tomato BAC clone, were named with the
original accession number followed by a defined part of the sequence, for instance
right (R), left (L) and resistance gene analogue (RGA). Markers from potato BAC-end
sequences were named with the plate, row and column number of the BAC library.

Results

Localization of the resistance locus
ABPT-derived clones were routinely inoculated with P. infestans during the breeding
process and resistance was specifically noted in ABPT-30, ABPT-33, (ABPT30x33)-3,
ABNA1-2, RH87-707-1 and 707TG11-1 (Figure 1). A tetraploid mapping population of
233 tetraploid genotypes, which was generated by a cross between 707TG11-1 and
RH89-039-16, was assessed for resistance to P. infestans isolate 90128. 707TG11-1
was assigned as resistant, RH89-039-16 as susceptible (Figure 2) and 83 and 90
offspring were unambiguously classified as resistant and susceptible, respectively.
This 1:1 segregation ratio indicates a simplex inheritance of the resistance gene. The
remaining 60 genotypes were tentatively classified, but excluded from the analysis for
localization of the resistance locus because of less accurate observations and
insufficient humidity during the incubation period.
To localize the resistance locus on the genetic linkage map of potato, 11
primer combinations were selected from the UHD map. In total 1187 AFLP bands
were observed in 707TG11-1 ranging between 79 and 164 per primer combination.
38

243 bands (20.5 %) of these showed a polymorphism that was scorable with
accuracy in the offspring. A subset of 191 marker loci showed a 1:1 segregation
(simplex inherited markers) with the
2
-test (P <0.05). Those markers should be
considered as SDRFs. In the first round of the J oinMap analysis, two of the 191
markers, i.e. EAAC/MCAG_101 and PAC/MAAT_498 were grouped with the Rpi-abpt
locus.

Figure 2. Detached leaf assay on resistant
and susceptible parents of RH4X-103, five
days after inoculation with P. infestans
isolate 90128. A: The resistant parent
707TG11-1 shows the hypersensitivity
response. B: The susceptible parent RH89-
039-16 is heavily sporulating.

To obtain additional AFLP markers with linkage to the Rpi-abpt locus, BSA was
applied with 128 Eco/Mse and 76 Pst/Mse primer combinations using four samples:
resistant parent (Pr), susceptible parent (Ps), resistant bulk (Br) and susceptible bulk
(Bs). The bulks were composed of equal amounts of pre-amplified templates of eight
resistant and eight susceptible genotypes as determined by the resistance assay.
AFLP markers that were present in Pr and Br and absent in Ps and Bs were selected
as candidate markers with putative linkage to the Rpi-abpt locus. Three and five
markers from Eco/Mse and Pst/Mse primer combinations, respectively, were
confirmed progressively to have more definite linkage to the Rpi-abpt locus by
analyzing the 16 genotypes of Br and Bs individuals. An example is shown in Figure 3.
This analysis resulted in an efficiency of determining one marker with linkage to the
resistance locus per 25 AFLP primer combinations. In the second round of J oinMap
analysis, two AFLP markers from 11 primer combinations and eight AFLP markers
from the BSA were grouped with the Rpi-abpt locus.

Figure 3. Part of an AFLP fingerprint showing a bulk-specific band associated with resistance using the
PCG/MAGA primer combination. Pr, Ps, Br and Bs indicate the resistant parent, susceptible parent,
resistant bulk and susceptible bulk, respectively. R and S represent resistant and susceptible individual
genotypes of Br and Bs.
39
Chapter 3

One of the AFLP markers, PAT/MAGA_307, was converted into a CAPS
marker Th2 (Foward: AGGATTTCAGTATGTCTCG and Reverse: TCCATTGTTGATT
GCCCCT) to determine the chromosomal location of the Rpi-abpt locus. In the UHD
mapping population, Th2 was localized on chromosome 4. R2, one of the S.
demissum-derived R genes against P. infestans, was also mapped on chromosome 4
(Li et al., 1998). To confirm the chromosome number, six primer combinations
selected from the R2 map were tested on our mapping population of 233 genotypes
and four R2-linked markers were grouped with the Rpi-abpt locus in the third round of
J oinMap analysis. The marker order was determined by Record software (van Os et
al., 2000) and genetic map distances were determined by the recombination
frequencies between flanking markers. The linkage map containing 15 loci, i.e. 14
markers and the Rpi-abpt locus was constructed. Subsequently the map was merged
with the high-resolution map in Figure 4.

Figure 4. High-resolution map of Rpi-abpt on chromosome 4. The
constructed map was based on 98 recombinants selected from the
extended population of 1383 genotypes between the markers
EAGT/MCTT_247 and EAGA/MCAC_202. The rest was based on the
original map with 233 genotypes. The AFLP markers, which are also
present in the map of R2, are indicated in italic. In the population of 86
genotypes, R2 was co-segregating with EACT/MCAC_189 and was
located 1 cM below EATC/MCGA_186 (Li et al., 1998). Genetic
distance and the number of recombinants between markers are
shown on the left side of the map.

Construction of a high-resolution map
From the genetic linkage map of Rpi-abpt, two flanking AFLP markers were selected
for a large-scale recombinant analysis. The first one is EAGT/MCTT_247 mapping
3.4 cM telomeric from the Rpi-abpt locus and the other one is EAGA/MCAC_202
mapping 1.3 cM centromeric from the Rpi-abpt locus in the population with 233
genotypes. An additional 1383 offspring of the population was screened with the two
AFLP markers, and 98 recombinants were selected. This resulted in 7.1 cM genetic
40

distance between the two AFLP markers. The 98 recombinants were screened with
the AFLP markers located on and between EAGT/MCTT_247 and EAGA/MCAC_202
and tested for resistance to P. infestans isolate 90128. The high-resolution map
containing the resistance locus and six AFLP markers was constructed (Figure 4).
The closest markers on both sides of the Rpi-abpt locus, EATC/MCGA_186 and
EATG/MCAG_215, are separated with one recombinant in 0.1 cM and six
recombinants in 0.4 cM, respectively. One AFLP marker PAT/MAGA_307 was co-
segregating with the Rpi-abpt locus.

Figure 5. A physical alignment of the resistance gene Rpi-abpt and its homologues. A: Graphical
illustration of sequence homology among the ABPT-derived AFLP marker PAT/MAGA_307, tomato
derived NBS-LRR sequences of gene4, gene5 and gene7 and Solanum bulbocastanum derived BAC-
end sequences. GENESCAN predicted NBS-LRR genes in the tomato BAC clone with high homology
to the Rpi-abpt candidate tagged by PAT/MAGA_307 marker. B: Sequence alignment among the
sequences of resistance gene analogues and PAT/MAGA_307 created by using CLUSTAL-X software.

Analysis of the Rpi-abpt locus
AFLP marker PAT/MAGA_307 co-segregating with the Rpi-abpt locus was
sequenced. A blast search revealed that the protein sequence of the marker showed
a high degree of similarity to several well-known plant resistance proteins that belong
to the NBS-LRR class. In addition, the DNA sequence of the marker showed high
similarity with sequences of Lycopersicon esculentum BAC clone 127E11 (Gene back
number: AF411807) derived from the tomato cultivar Heinz 1709 (van der Hoeven et
al., 2002). This BAC has been localized between CT229A and TG370 at the short
arm of the tomato chromosome 4 with a total length of 95,845 bp. By using Genescan
and Genemark, 19 resistance gene analogues (RGA) were predicted in this clone.
41
Chapter 3

Three RGAs, i.e. Gene4, 5 and 7 out of the 19 RGAs are homologous to disease
resistance protein RPP13 against Peronospora parasitica (Downy Mildew) in
Arabidopsis (Bittner-Eddy et al., 1999). Marker PAT/MAGA_307 is highly similar to
the 5 end of these three RGAs of the tomato BAC clone (Figure 5B). The markers
AF411807L and AF411807R of the tomato BAC clone were localized at 1.2 cM and
0.8 cM distance from the Rpi-abpt locus, respectively. Based on the consensus
sequence of the three RGAs of the tomato BAC clone, a RGA marker was designed
(Forward: CCTTTGTATCATTTGCAGTT and Reverse: ACATCCTCCATTCTTTCTT).
Analysis of the RGA marker in the mapping population revealed that the RGA marker
was also co-segregating with the Rpi-abpt locus and PAT/MAGA_307 (Figure 5A).
For physical alignment of resistance gene homologues, we screened the BAC
library from S. bulbocastanum (van der Vossen et al., 2003) with PAT/MAGA_307
and the RGA marker. Several BACs contained these two markers. BAC clones
139K15 and 54I8 were selected to form a small contig (Figure 5A). The BAC-end
sequence marker from 139K15L is also coding for a resistance gene homologue.

Identification of the wild species origin of the resistance
The wild Solanum accessions CGN17843, CGN20620, CGN21306, CGN17693,
CGN17667 and CGN18301 that were involved in the pedigree of RH4X-103 were
inoculated with P. infestans isolate 90128, using an in vitro inoculation assay (Huang,
2005). All genotypes from S. acaule and S. phureja were heavily sporulating whereas
all genotypes from S. bulbocastanum showed a hypersensitive response. These
results suggest that S. bulbocastanum is the most likely the donor species of the Rpi-
abpt resistance.
To identify the wild species origin of the resistance gene Rpi-abpt at the
molecular level, the 14 AFLP markers linked to the Rpi-abpt locus (Figure 4) were
examined in the five Solanum species used in this study, i.e. S. acaule, S.
bulbocastanum, S. phureja, S. demissum and S. tuberosum (Figure 6). Four to six
genotypes of each accession or species were included in this experiment.
PAT/MAGA_307 generated AFLP fragments with the same mobility in S.
bulbocastanum as in 707TG11-1, but not in the other species. The AFLP markers
PCT/MACA_146, PCG/MAGA_275, EAGA/MCAC_202 and PAC/MAAG_97 were
identified in the fingerprints of both S. acaule and S. bulbocastanum. The AFLP
markers EAAC/MCTG_132, EAAC/MCAG_101, PAC/MAAT_498 and
EAGT/MCAA_473 that were more distant from Rpi-abpt were only identified in S.
acaule, S. tuberosum or both. Also within species, variation between genotypes was
found, which can be explained by the enormous genetic variation in outbreeding
Solanum spp. (Hawkes, 1991). Marker EATG/MCAG_215 was not identified in any of
the Solanum species we tested possibly because of the same reason. None of the
AFLP markers was detected in S. phureja. Two of the four R2-linked markers from S.
42

demissum were also observed in S. bulbocastanum and another one in S. acaule as
well. The pattern of the 14 Rpi-abpt-linked AFLP markers distributed over the
Solanum species revealed that the closest markers are present in S. bulbocastanum
that supports our previous indication that the introgression fragment containing Rpi-
abpt is likely derived from this species.
The ABPTderived Rpi-abpt is located at a similar position as R2 in S.
demissum (Figure 4). As described above, the P. infestans isolate 90128, which lacks
virulence to R2, was unable to infect any plant carrying Rpi-abpt. To test whether the
Rpi-abpt shares the same race-specificity as R2, the virulent P. infestans isolates
USA618 and IPO82001 were inoculated on 55 genotypes of the segregating
population RH4X-103. Both isolates showed an identical segregation for resistance
as P. infestans isolate 90128 (data not shown). We conclude that Rpi-abpt
functionally differs from R2, and thus represents a novel R gene to late blight.

Figure 6. A representation of mobility of AFLP markers in various Solanum species. The symbol, ,
indicates marker presence with the same mobility on the AFLP gels in one or more genotypes within
presented Solanum species and the symbol of the dotted line indicates the expectation of the Rpi-abpt
locus presence. Marker order and genetic distance are the same as those indicated in the high-
resolution map. The AFLP markers, which are also present in the map of R2, are indicated in italic. a,
b, p, t and d represent S. acaule, S. bulbocastanum, S. phureja, S. tuberosum and S. demissum,
respectively.

Discussion

The mapping population RH4X-103 was derived from ABPT clones in which the wild
species S. acaule, S. bulbocastanum and S. phureja were involved (Hermsen and
Ramanna, 1973). S. bulbocastanum is a well-known source of resistance to
43
Chapter 3

Phytophthora infestans (Umearus and Umearus, 1994; Helgeson et al., 1998; van der
Vossen et al., 2003 and 2004) but unfortunately is not directly crossable with potato.
In this study S. bulbocastanum was introgressed via ABPT bridge crosses (Hermsen
and Ramanna, 1973) and in the ABPT-derived mapping population RH4X-103,
monogenic inheritance of Rpi-abpt was identified. Inoculation studies with a panel of
P. infestans isolates revealed that the Rpi-abpt is a novel resistance gene with broad-
spectrum resistance to all tested isolates. The definite origin of the R gene cannot
unambiguously be determined, because the original breeding material has not been
maintained during the entire long-lasting process of ABPT breeding. However,
several lines of research suggest that the resistance was obtained from S.
bulbocastanum. First, the Rpi-abpt resistance can be traced back in the pedigree to
ABPT material, and only S. bulbocastanum accessions were identified to contain
resistance to late blight. Second, by applying a method previously described by Li et
al. (1998), the mobility of AFLP markers linked to Rpi-abpt was examined in S. acaule,
S. bulbocastanum, S. phureja, S. tuberosum and S. demissum. Except one marker
which could not be amplified from any wild species, the six markers that were closest
to Rpi-abpt showed an identical mobility in S. bulbocastanum accessions. The closest
co-segregating marker was specific to S. bulbocastanum only. Other markers were
less specific and appeared to be conserved also on S. acaule or S. demissum.
Although this indicates that this method is not providing ultimate proof about descent,
the introgression fragment is most likely of S. bulbocastanum origin.
In the BSA analysis, eight markers linked to the Rpi-abpt locus in coupling
phase were detected from 204 primer combinations. This observation is similar to
other results in tetraploid potato. Li et al. (1998) detected 11 AFLP markers linked to
R2 from 205 primer combinations. BSA in diploid potato is more efficient than in
tetraploid potato as Meksem et al. (1995) identified 29 bulk specific loci linked to R1
from 108 primer combinations. The difference of the results between diploid and
tetraploid levels can be explained by the fact that markers with linkage in repulsion
cannot be used in tetraploids and that the composed bulks were based on the
genotypes with RFLP markers linked to R1 in the diploid.
Since the molecular isolation of genes based on their map location could be
applicable to any gene whose phenotypic effect can be followed in a segregating
population (Ballvora et al., 2001), a reliable map position of the Rpi-abpt locus was
successfully determined using 1383 offspring. The Rpi-abpt locus was localized in a
0.5 cM interval between the closest flanking AFLP markers and was co-segregating
with one AFLP marker. Although the relationship between genetic and physical
distances is known to vary considerably in potato (Marano et al., 2002), we expect
that the physical distance between AFLP markers at the region of the Rpi-abpt locus
is relatively small because of the resistance gene homology of the co-segregating
AFLP marker and the RGA marker.
44

Comparative genomics between potato and tomato can facilitate the cloning of
R genes (Gebhardt et al., 1991; Tanksley et al., 1992; van der Vossen et al., 2004;
Huang, 2005). The sequence of the co-segregating AFLP marker is highly similar with
the sequence of one of the tomato BAC clones on chromosome 4 (van der Hoeven et
al., 2002), which contains 19 predicted genes, some of which belong to the NBS-LRR
gene family. The translated protein of the sequence of the co-segregating AFLP
marker is homologous to the resistance gene protein RPP13 which belongs to the
CC-NBS-LRR class in Arabidopsis (Bittner-Eddy et al., 1999) and is highly similar to
RGA open reading frames of the tomato BAC clone. These results suggest that Rpi-
abpt is a member of a resistance gene cluster. For future work, the construction of a
BAC library of the resistant parent 707TG11-1 is in progress. The co-segregating and
flanking AFLP markers are expected to facilitate the creation of a physical contig to
span the Rpi-abpt locus in a few BAC walking steps. The results described in this
paper predict that the novel resistance gene Rpi-abpt will be cloned soon.

Reference

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van der Hoeven, R., Ronning, C., Giovannoni, J., Martin, G. and Tanksley, S. 2002.
Deductions about the number, organization, and evolution of genes in the tomato
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Wouters, D., Pereira, A., Stiekema, W.J. and Allefs, S. 2003. An ancient R gene from
the wild species Solanum bulbocastanum confers broad-spectrum resistance to
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The detection and estimation of linkage in polyploids using single-dose restriction
fragments. Theor Appl Genet 83: 294-300

48
Chapter 4

Characterization and high-resolution mapping of
a late blight resistance locus similar to R2

Tae-Ho Park, Vivianne G.A.A. Vleeshouwers, Dirk J . Huigen,
Edwin van der Vossen, Herman J . van Eck
and Richard G.F. Visser

Accepted by Theoretical Applied and Genetics

Chapter 4

Abstract

Identification of resistance (R) genes to Phytophthora infestans is an essential
step in molecular breeding of potato. We identified three specific R genes
segregating in a diploid mapping population. One of the R genes is located on
chromosome 4 and proved phenotypically indistinguishable from the Solanum
demissum-derived R2 although S. demissum is not directly involved in the
pedigree of the population. By bulked segregant analysis combined with a
resistance assay, a genetic linkage map of the R2-like locus was constructed
with 30 coupling and 23 repulsion phase AFLP markers. Two markers flanking
the R2-like locus were applied to screen an extended population of 1586
offspring. 103 recombinants were selected and an accurate high-resolution
map was constructed. The R2-like resistance was localized in a 0.4 cM interval
and was found co-segregating with four AFLP markers, which can be used to
isolate the R2-like gene by map-based gene cloning. By analyzing race-
specificity and R gene specific molecular markers, we also found that an R1-
like gene and an additional unknown R gene are segregating in the population.

Introduction

Phytophthora infestans is the causal agent of late blight and one of the most
important pathogens in cultivated potato (Solanum tuberosum L.). The economic
value of the loss of global annual production and the cost of crop protection are
estimated to be 5 billion US dollar annually (Duncan, 1999). In early breeding
programs for late blight resistance, 11 resistance (R) genes originating from the
hexaploid wild species S. demissum were introduced into S. tuberosum (Black et al.,
1953; Malcolmson and Black, 1966). These R genes confer race-specific resistance
and they are controlled by major single dominant factors (Mastenbroek, 1953;
Malcolmson and Black, 1966). The potato - P. infestans interaction is following the
gene-for-gene model as proposed by Flor (1971). Although the R genes do not give
durable resistance, the isolation of these resistance genes is of importance to
understand the mechanism of the specific plant defense. Several R genes have been
localized, e.g. R1 on chromosome 5 (Leonards-Schippers et al., 1992) and R3, R6
and R7 clustered on chromosome 11 (El-Kharbotly et al., 1994 and 1996). R1 and R3
have been isolated (Ballvora et al., 2002; Huang, 2005). The R2 locus has been
localized on chromosome 4 using a tetraploid mapping population EJ 96-4061
comprising of 86 offspring (Li et al., 1997). Eleven AFLP markers linked to the R2
locus have been identified using bulked segregant analysis (BSA; Michelmore et al.,
1991) and the R2 linkage group has been assigned using the reference map of
50
High-resolution mapping of the R2-like gene

AM3778-16. Three AFLP markers that are tightly linked to R2 are present in the
diploid mapping population RHAM026, which is used in this study.
Recently many different haplotypes of R3-like genes were discovered at the
R3 locus in S. demissum and ample variation for functional R genes to late blight
appears to exist in Solanum species (Huang et al., 2004 and Huang, 2005). In this
research we identified resistances by assaying race-specificities and focused on a
locus that is indistinguishable from R2 in the diploid mapping population RHAM026.
Since the resistance was introgressed from different wild species, we designate the
gene as R2-like. From the R2-like locus, a high-resolution map was constructed.


Plant materials
The diploid mapping population RHAM026 was obtained from a cross between
AM3778-16 (AM) and RH89-039-16 (RH). The dihaploid female parent AM is derived
from the tetraploid breeding clone AM78-3778, which has various wild Solanum
species in its pedigree, including S. tuberosum spp. andigena, S. vernei, S. vernei
spp. ballsii, S. oplocense and S. edinense. Detailed pedigree of the mapping
population is shown in Figure 1.
The mapping population was generated from seeds that were sown in vitro
after they were sterilized. When the seeds germinated and the seedlings reached
approximately 3 to 5 centimeters, they were duplicated. One was used for
maintenance and another for DNA isolation.

Figure 1. Pedigree of the diploid mapping population RHAM026 used in this study.

Inoculum preparation and resistance assay
Four different isolates were used to identify race-specific resistance (Table 1). For
experiments, a plug of mycelium was transferred to a fresh plate with rye sucrose
51
Chapter 4

agar medium (Caten and J inks, 1968). One to two weeks later, ice-cold water was
added to the mycelium that covered the agar plate. The sporangiospore suspension
was pipetted into a tube and incubated at 4
o
C for 3 hours. After the release of
zoospores, the concentration of inoculum was adjusted to 5 x 10
4
spores/ml.
A resistance assay was performed by a detached leaf assay. Fully expanded
and healthy leaves were collected from greenhouse plants and inoculated on wet
paper in humid trays. Droplets of 10 l inoculum were applied to the leaves, which
were subsequently incubated at 15
o
C (Vleeshouwers et al., 1999). The symptoms on
the leaves were evaluated three and five days after inoculation.

Table 1. Isolates of Phytophthora infestans and virulence factors.
Isolate Race-specificity Source
IPO-0 0 W. Flier, Plant Research International, The Netherlands
99018 1.4 F. Govers, Wageningen University, The Netherlands
90128 1.3.4.7.8.11 F. Govers, Wageningen University, The Netherlands
USA618 1.2.3.6.7.11 W.E. Fry, Cornell University, USA

DNA isolation
A high throughput DNA isolation procedure was followed using a Retsch machine
(Retsch Inc., Haan, Germany) and 96-deep-well COSTER microtiter plates (Corning
Inc., Corning, Newyork, U.S.A.). Fresh leaf tissue was obtained by harvesting in vitro
plants and grinding with two steel balls in the presence of the Nuclear lysis buffer (0.2
M Tris-HCl, 0.05 M EDTA, 2 M NaCl, 2 % CTAB, with an end pH of 7.5), DNA
extraction buffer (100 mM Tris-HCl, 5 mM EDTA, 0.35 M Sorbitol, 20 mM NaBisulfite,
with an end pH of 7.5) and 5 % sarcosyl followed by incubation at 65C in a water
bath for one hour. Ice-cold chloroform isoamyl alcohol (24:1) was added and the
samples were mixed and centrifuged. The supernatant was transferred to new tubes
and an equal volume of isopropanol was added. Another centrifugation step allowed
the precipitation of DNA. The DNA pellet was dried and dissolved in T
0.1
E-buffer (+
0.5 g RNAse).

AFLP analysis
The AFLP analysis was essentially carried out as described by Vos et al. (1995).
Primary template DNA was prepared using EcoRI and MseI and adaptors fitting to the
restriction enzyme sites. Template DNA was diluted 10 times prior to the selective
pre-amplification with single nucleotide extended primers, which decreases marker
density. For the selective amplification, AFLP reactions with three nucleotides
extended primers (E+3/M+3 primers) were performed to find markers linked to the
resistance locus. AFLP bands were separated on a 6 % polyacryl amide gel in a Li-
cor sequencer (Li-cor, Lincoln, NB, U.S.A.).
52

AFLP markers were named with the enzyme, the primer combination and the
mobility of the fragment as described in reference autoradiograms created by
Keygene NV, Wageningen, The Netherlands.

Genetic analysis of resistance
AFLP markers linked to a resistance locus were identified by bulked segregant
analysis (BSA; Michelmore et al., 1991). The data were analyzed using J oinMap
software (Stam, 1993). Marker order was determined by Record (van Os et al., 2000)
and the map distance was calculated based on the frequency of the recombination
between markers. A marker saturated genetic linkage map was produced by
MapChart (Voorrips, 2002).
To identify race-specificity of the resistances, R1 specific marker 76_2s
(forward primer: 5-CACTCGTGACATATCCTCACTA-3 and reverse primer: 5-
CAACCCTGGCATGCCACG-3; Ballvora et al., 2002) and R2-linked AFLP markers
EATC/MCGA_186 and EACT/MCAC_189 (Li et al., 1998) were used.

Results

Characterization of resistance
A subset of the population of 28 genotypes and both parents AM and RH were
evaluated for resistance to four different isolates, which differ in race specificity (Table
1). All 28 genotypes were susceptible to the isolate USA618, but segregated for
resistance to the isolates IPO-0, 99018 and 90128 (Figure 2A). The differential
segregation for resistance to the three isolates suggests that three specific R genes
are segregating in the population.

Figure 2. Graphical genotyping of 28 offspring and parents AM and RH. A: Phenotypic results with the
P. infestans isolates IPO-0, 99018, 90128 and USA618. R, S and nd indicate resistant, susceptible and
undetermined, respectively. B: Genotypic results obtained with R1-specific marker 76_2s and the
AFLP markers EATC/MCGA_186 and EACT/MCAC_189 linked to R2. aa and ab indicate the absence
and presence of the marker. C: Classes distinguished based on the phenotypic and genotypic data.

To examine whether known or unknown R genes segregate in RHAM026,
specific molecular markers were designed. The presence of R1 gene specific
sequences was tested by applying the 76_2s marker (Ballvora et al., 2002) on
53
Chapter 4

parents and progenies and a clear segregation was found (Figure 2B). The R2-linked
AFLP markers EATC/MCGA_186 and EACT/MCAC_189 (Li et al., 1998) were also
tested. Both markers showed an identical segregation pattern in the progenies
(Figure 2B).
By comparing the genotypic results with the phenotypic results, we divided the
28 genotypes into five groups, which could have different candidate resistance genes
(Figure 2C). Presence of the 76_2s marker matched with the R1-specific resistance
to P. infestans isolate IPO-0 (group I and IV) and the R2-linked AFLP markers
EATC/MCGA_186 and EACT/MCAC_189 perfectly matched with the R2 specific
resistance to isolate 90128 (group I and II). Some genotypes that do not contain the
R1 and R2 markers were susceptible to the isolate 90128, but resistant to isolates
IPO-0 or 99018 (group III). This might be caused by another resistance gene that we
designated R
unknown
. In conclusion, the combination between race-specificity and
molecular marker data suggests that R
unknown
, R1- and R2-like genes are segregating
in the population.

Figure 3. Genetic linkage map of the
R2-like locus. The resistance locus is
underlined. Two flanking markers used
for screening the extended population
are dot-underlined. All 30 coupling
phase markers, but only four repulsion
phase markers and the resistance
locus are included in the map.

Marker-saturated genetic linkage map
To determine the location of the R2-like locus, a genetic linkage study was initiated
with the RHAM026 population comprising of 78 genotypes. A detached leaf assay
with P. infestans isolate 90128 revealed 24 resistant and 45 susceptible genotypes,
which fits to a 1 to 2 segregation according to the
2
-test. This segregation is skewed
to susceptible, which might be caused by the relatively small population size or by
postzygotic selection against unfavourable allelic combinations (Gebhardt et al.,
1991; El-Kharbotly et al., 1994). Nine genotypes were not assigned due to unclear
54

observation. A resistant and a susceptible bulk were composed of eight genotypes
each. 256 Eco+3/Mse+3 primer combinations were examined in the resistant parent,
the susceptible parent, the resistant bulk and the susceptible bulk by bulked
segregant analysis, and 33 coupling and 31 repulsion phase candidate markers were
revealed. After selecting the candidate markers, the 16 genotypes used to compose
the resistant and susceptible bulks were separately checked for linkage to the
resistance locus. Finally 30 coupling phase markers and 23 repulsion phase markers
were identified to be linked to the R2-like locus (Figure 3). Seven AFLP markers co-
segregate with the resistance locus. The linkage group was determined with two
AFLP markers, EATC/MCGA_186 and EACT/MCAC_189, which were bridged to the
genetic map of chromosome 4 where the R2 locus is located (Li et al., 1998).

Figure 4. Schematic representation for recombinant screening towards high-resolution mapping. AFLP
gel images of the two flanking AFLP markers EATC/MCGA_204 and EATG/MCAG_215, which were
used to select recombinants in 1582 genotypes. One example of a recombinant is indicated by X.

High-resolution map
To construct a high-resolution map, we selected two flanking AFLP markers (Figure
3) to screen an extended population for selecting recombinants around the resistance
locus. One is EATC/MCGA_204, which was mapped 1.3 cM telomeric from the
resistance locus. Another is EATG/MCAG_215, which was mapped 2.5 cM
centromeric from the resistance locus in the 78 offspring (Figure 4). By screening the
expanded population of 1582 genotypes, 103 recombinants were selected resulting in
a larger interval of 6.4 cM genetic distance between these two AFLP markers. We
screened the 103 recombinants with 14 AFLP markers located between
EATC/MCGA_204 and EATG/MCAG_215 (Figure 3) and tested them for their
55
Chapter 4

resistance to P. infestans isolate 90128. Subsequently a high-resolution genetic map
of the 6.4 cM interval containing the resistance locus was constructed (Figure 5), in
which the AFLP markers are precisely separated and mapped. Closest markers on
both sides of the resistance locus are EACA/MCAC_105 (three recombinants in 0.2
cM) and a group containing five markers (three recombinants in 0.2 cM). Four AFLP
markers are still co-segregating with the resistance locus.

Figure 5. High-resolution map of the R2-like locus. 1582
genotypes were screened with two AFLP markers
EATC/MCGA_204 and EATG/MCAG_215, which are
dot-underlined, and 103 recombinants were selected.
Four markers are co-segregating with the R2-like locus.

Comparative genetics
The R2 and R2-like loci derived from two different mapping populations were
compared. The bridged markers EATC/MCGA_186 and EACT/MCAC_189 are 0.2
cM apart in the R2-like diploid population (3 recombinants in 1582 genotypes) and 1.2
cM in the R2 tetraploid population (1 recombinant in 86 genotypes) (Li et al., 1998).
One AFLP marker EATC/MCGA_186 co-segregates with the R2-like locus, but is 1.2
cM separated from the R2 locus. Another AFLP marker EACT/MCAC_189 is 0.2 cM
separated from the R2-like locus, but co-segregates with the R2 locus. The position of
flanking markers that connect the two genetic linkage maps confirmed that the R2
and R2-like loci are located at the same region of chromosome 4.
56

Discussion

In this research, we identified three resistance loci in the diploid segregating
population RHAM026. One locus was localized on chromosome 4 where the R2 locus
from S. demissum was previously mapped using a tetraploid mapping population
EJ 96-4601 (Li et al., 1998). Accurate specificity studies with a panel of four different P.
infestans isolates revealed that R2-like resistance of this population is phenotypically
indistinguishable from R2.
The origin of the AFLP markers EACT/MCAC_189 and EATC/MCGA_186 was
previously thought to be S. demissum-specific because the AFLP markers were
identified in the fingerprints of three accessions of S. demissum (Li et al., 1998).
However, we observed that EACT/MCAC_189 is also present in S. acaule accessions
and EATC/MCGA_186 in S. bulbocastanum accessions (data not shown) and
therefore conclude that these markers are present in a broader range of Solanum
germplasm. Although S. demissum is not directly present in the ancestors of the R2-
like diploid population, the introgression fragment could be of wild species origin, e.g.
S. edinense which is a natural hybrid between S. tuberosum and S. demissum
(Hawkes, 1990). It remains unknown what the origin of the R2-like locus is. Even if it
is derived from S. demissum, definite identity with R2 (Li et al., 1998) can only be
determined after cloning both genes.
To construct a marker saturation and high-resolution map at the R2-like locus,
we used a diploid population. Compared to the tetraploid level, mapping studies at the
diploid level can avoid the complexities of tetrasomic inheritance and makes the study
of potato genetics more feasible (J acobs et al., 1995; Li et al., 1998). The number of
markers is at least fourfold higher in a diploid population than in a tetraploid
population, because marker alleles with linkage in repulsion can be used at the
diploid level (Wu et al., 1992). We identified 69 AFLP markers with linkage to R2-like
using 256 primer combinations. A similar high efficiency was achieved in another
study in which 29 AFLP markers with linkage to R1 using 108 primer combinations
were identified at the diploid level (Meksem et al., 1995). At the tetraploid level, much
lower efficiency was achieved. Li et al. (1998) detected only 11 AFLP loci with linkage
to R2 using 205 primer combinations.
Many resistance genes and resistance gene homologues are clustered in the
genome (Meyers et al., 1998; Michelmore and Meyers, 1998). In the potato genome,
for instance, at least five resistance genes against diverse pathogens are located at
the gene cluster on chromosome 5 (Leister et al., 1996; Marano et al., 2002), i.e. Gpa
conferring resistance to the potato cyst nematode Globodera pallida (Kreike et al.,
1994), Grp1 conferring resistance to the potato cyst nematode Globodera
rostochiensis (Rouppe van der Voort et al., 1998), Nb conferring resistance to potato
virus X (de J ong et al., 1997), Rx2 (Ritter et al., 1991) conferring resistance to potato
57
Chapter 4

virus X and R1 conferring resistance to Phytophthora infestans (Leonards-Schippers
et al., 1992). Also near the R2-like locus on chromosome 4, various known R genes
are located. The Hero gene, which confers broad-spectrum resistance against
Globodera rostochiensis in tomato, is located between CT229 and TG370 (Ganal et
al., 1995), Gpa4 conferring resistance to Globodera pallida (Bradshaw et al., 1998)
and R2 (Li et al., 1998), Rpi-abpt (Chapter 3) and Rpi-blb3 (Chapter 5) conferring
resistance to Phytophthora infestans are mapped at the same region on chromosome
4.
One of the most challenging methods for gene cloning in a plant species with a
large genome is map-based cloning, which can be considered routine in model
species such as Arabidopsis with a small genome (Lukowitz et al., 2000). Accurate
mapping of the target gene and identification of recombinants in the population are
essential steps for map-based cloning and here we report a reliable high-resolution
map of the R2-like locus within a 0.4 cM interval on chromosome 4 in the potato
breeding line RHAM026. The initial research for tightly linked AFLP markers was
facilitated using AFLP technology (Vos et al., 1995) in combination with bulked
segregant analysis (Michelmore et al., 1991). The R2-like locus is flanked by one
AFLP marker on the telomeric side, a group of five AFLP markers on the centromeric
side and co-segregating with four AFLP markers (Figure 5). The flanking and co-
segregating markers will facilitate isolation of the R2-like gene by BAC walking. A 0.2
cM (4/2109 recombinants) interval was covered by one BAC clone of 100 kb in the
cloning of the potato late blight resistance gene Rpi-blb1 (van der Vossen et al.,
2003) and in the cloning of R1, a 0.2 cM (2/1049 recombinants) interval was
estimated at 250-300 kb (Ballvora et al., 2002). We estimate that the 0.4 cM interval
spanning the R2-like gene could be covered by a single BAC clone or a few BAC
walking steps.
Currently a BAC library of diploid genotype AM is being constructed to
establish a physical contig with the co-segregating and flanking markers and isolation
of R2-like gene in near future is quite feasible. After that, physical analysis of the
gene cluster containing the R2-like gene might accelerate isolation of the homologous
genes conferring resistance to Phytophthora infestans in different genetic
backgrounds.

Reference

58

Bradshaw, J.E., Hackett, C.A., Meyer, R.C., Milbourne, D., McNicol, J.W., Phillips, M.S.
and Waugh, R. 1998. Identification of AFLP and SSR markers associated with
quantitative resistance to Globodera pallida (Stone) in tetraploid potato (Solanum
tuberosum subsp. tuberosum) with a view to marker-assisted selection. Theor Appl
Genet 97: 202-210
de Jong, W., Forsyth, A., Leister, D., Gebhardt, C. and Baulcombe, D.C. 1997. A potato
hypersensitive resistance gene against potato virus X maps to a resistance gene cluster
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Duncan, J.M. 1999. Phytophthora an abiding threat to our crops. Microbiol Today 26: 114-
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infestans in progeny of dihaploid potato parents. Mol Gen Genet 242:749-754
El-Kharbotly, A., Palomino-Sanchez, C., Salamini, F., Jacobsen, E. and Gebhardt, C.
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Ganal, M.W., Simon, R., Brommonschenkel, S., Arndt, M., Phillips, M.S., Tanksley, S.D.
and Kumar, A. 1995. Genetic mapping of a wide spectrum nematode resistance gene
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Gebhardt, C., Ritter, E., Barone, A., Debener, T., Walkemeier, B., Schachtschabel, U.,
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and Salamini, F. 1991. RFLP maps of potato and their alignment with the homologous
tomato genome. Theor Appl Genet 83:49-57
Hawkes, J.G. 1990. The potato: Evolution, biodiversity and genetic resources. Smithosian
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Jacobs, J.M.F., van Eck, H.J., Arens, P., Verkerk-Bakker, B, te Lintel Hekkert, B.,
Bastiaanssen, H.J.M., El-Kharbotly, A., Pereira, A., Jacobsen, E. And Stiekema,
W.J. 1995. A genetic map of potato (Solanum tuberosum) integrating molecular
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Kreike, C.M., De Koning, J.R.A., Vinke, J.H., van Ooijen, J.W. and Stiekema, W.J. 1994.
Quantitatively-inherited resistance to Globodera pallida is dominated by one major
locus in Solanum spegazzinii. Theor Appl Genet 88. 764-769
Leister, D., Ballvora, A., Salamini, F. and Gebhardt, C. 1996. A PCR-based approach for
isolating pathogen resistance genes from potato with potential for wide application in
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Lukowitz, W., Gillmor, C.S. and Scheible, W.R. 2000. Positional cloning in Arabidopsis.
Why it feels good to have a genome initiative working for you. Plant Physiol 123: 795-
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Malcolmson, J.F. and Black, W. 1966. New R genes in Solanum demissum Lindl. and their
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Marano, M.R., Malcuit, I., De Jong, W. and Baulcombe, D.C. 2002. High-resolution genetic
map of Nb, a gene that confers hypersensitive resistance to potato virus X in Solanum
tuberosum. Theor Appl Genet 105: 192-200
Meksem, K. Leister, D., Peleman, J., Zabeau, M., Salamini, F. and Gebhardt, C. 1995. A
RFLP and AFLP markers. Mol Gen Genet 249:74-81
Meyers, B.C., Chin, D.B., Shen, K.A., Sivaramakrishnam, S., Lavelle, D.O., Zhang, Z.
and Michelmore, R.W. 1998. The major resistance gene cluster in lettuce is highly
duplicated and spans several megabases. Plant Cell 10: 1817-1832
Sci USA 88: 9828-9832
Michelmore, R.W. and Meyers, B.C. 1998. Clusters of resistance genes in plants evolve by
divergent selection and a birth-and-death process. Genome Res 8: 1113-1130
Ritter, E., Debener, T., Barone, A., Salamini, F. and Gebhardt, C. 1991. RFLP mapping on
potato chromosomes of two genes controlling extreme resistance to potato virus X
(PVX). Mol Gen Genet 227: 81-85
Rouppe van der Voort, J., Lindeman, W., Folkertsma, R., Hutten, R., Overmars, H., van
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van Os, H., Buntjer, J., Stam, P. and van Eck, H.J. 2000. Evaluation of two algorithms for
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Voorrips, R.E. 2002. MapChart: Software for the graphical presentation of linkage maps and
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Wu, K.K., Burnquist, W., Sorrells, M.E., Tew, T.L., Moore, P.H. and Tanksley, S.D. 1992.
The detection and estimation of linkage in polyploids using single-dose restriction
fragments. Theor Appl Genet 83: 294-300

61
Chapter 4

62
Chapter 5

The late blight resistance locus Rpi-blb3 from Solanum
bulbocastanum belongs to a major late blight R gene
cluster on chromosome 4 of potato

Tae-Ho Park, J ack Gros, Anne Sikkema, Vivianne G.A.A. Vleeshouwers,
Marielle Muskens, Sjefke Allefs, Evert J acobsen,
Richard G.F. Visser and Edwin van der Vossen

Accepted by Molecular Plant-Microbe Interaction

Chapter 5

Abstract

Late blight, caused by Phytophthora infestans, is one of the most devastating
diseases in cultivated potato. Breeding of new potato cultivars with high levels
of resistance to P. infestans is considered the most durable strategy for future
potato cultivation. In this study we report the identification of a new late blight
resistance (R) locus from the wild potato species Solanum bulbocastanum.
Using several different approaches, a high-resolution genetic map of the new
locus was generated, delimiting Rpi-blb3 to a 0.93 cM interval on chromosome
4. One AFLP marker was identified that co-segregated in 1396 progeny plants
of an intraspecific mapping population with Rpi-blb3. For comparative
genomics purposes, markers linked to Rpi-blb3 were tested in mapping
populations used to map the three other late blight R loci Rpi-abpt, R2 and R2-
like also to chromosome 4. Marker order and allelic conservation suggest that
Rpi-blb3, Rpi-abpt, R2 and R2-like reside in the same R gene cluster on
chromosome 4 and likely belong to the same gene family. Our findings provide
novel insights in the evolution of R gene clusters conferring late blight
resistance in Solanum.

Introduction

Late blight, caused by the oomycete Phytophthora infestans, is one of the most
serious diseases in worldwide potato production. It was responsible for the Irish
potato famine of the mid-19
th
century, resulting in the death of one million people.
Although a lot of effort has been invested to control the pathogen, chemical control of
P. infestans is still the main crop management strategy, but environmental safety is
becoming more important and the pathogen is sometimes able to evolve chemical
resistance (Goodwin et al., 1996). Therefore introduction of resistance into modern
potato varieties is the most durable strategy to control the disease.
In the last century, S. demissum, which is a hexaploid Mexican species, was
extensively used in breeding for late blight resistance in potato. Initially, a series of
eleven R genes derived from S. demissum was described (Mastenbroek, 1953;
Malcolmson and Black, 1966). Of these, R1, R2, R3a/b, R6 and R7 have been
localized on the genetic maps of potato (Leonards-Schippers et al., 1992; El-
Kharbotly et al., 1994 and 1996; Li et al., 1998; Ewing et al., 2000; Huang et al.,
2004). However, these R genes confer race-specific resistance and those which were
introgressed into potato varieties, mainly R1, R2, R3, R4 and R10, were quickly
overcome by the pathogen (Wastie, 1991). Hence new sources for resistance are
required and currently several other wild Solanum species have been reported as
being potential sources of resistance (summarized by J ansky, 2000). Of these, S.
64
Rpi-blb3 / comparative genetics

microdontum (Sandbrink et al., 2000) and S. phureja (Ghislain et al., 2001) conferring
quantitative resistance and S. berthaultii (Ewing et al., 2000), S. pinnatisectum (Kuhl
et al., 2001) conferring monogenic resistance have been genetically characterized.
In addition, S. bulbocastanum, a self-incompatible diploid species from Mexico
is thought to be one of the most promising sources for late blight resistance
(Niederhausen and Mills, 1953), despite its sexual incompatibility with S. tuberosum
(Hermsen and De Boer, 1971). Introduction of S. bulbocastanum-derived resistance
has been achieved through interspecific bridge crosses between S. bulbocastanum, S.
acaule, S. phureja and S. tuberosum (Hermsen and Ramanna, 1973), resulting in so-
called ABPT material which is widely used for potato late blight breeding. Additionally,
Helgeson et al. (1998) generated somatic hybrids between S. bulbocastanum and
cultivated potato. The somatic hybrids led to fertile plants that retained the resistance
and could be used for breeding. Molecular cloning of the genes responsible for the
resistance and subsequent introduction of the genes into potato varieties is a third
method which circumvents many of the problems encountered in the previous two
strategies.
To date, two R genes from S. bulbocastanum have been cloned, the allelic
genes RB and Rpi-blb1 on chromosome 8 (Song et al., 2003; van der Vossen et al.,
2003) and Rpi-blb2 on chromosome 6 (van der Vossen et al., 2004). RB was mapped
using BC
2
populations derived from somatic hybrids (Helgeson et al., 1998; Naess et
al., 2000), whereas Rpi-blb1 was cloned using an intraspecific S. bulbocastanum F1
population (van der Vossen et al., 2003). Rpi-blb2 was mapped both in an
intraspecific F1 population and in tetraploid BC
2
and BC
3
populations derived from
interspecific ABPT hybrids (van der Vossen et al., 2004).
In this study, we have identified a third locus in S. bulbocastanum. The Rpi-
blb3 locus was mapped to an R gene hotspot on chromosome 4 in an intraspecific S.
bulbocastanum BC
1
population. Furthermore we show that a marker co-segregating
with Rpi-blb3 also co-segregates or is closely linked with late blight resistance in three
other mapping populations harboring the Rpi-abpt, R2-like and R2 loci (Chapters 3
and 4; Li et al., 1998).


Plant material
The interspecific S. bulbocastanum mapping population Blb00-20 was developed by
crossing a P. infestans resistant clone Blb99-256-3 with the susceptible clone Blb48-5.
The resistant parental clone Blb99-256-3 was obtained from a cross between a
resistant clone from accession BGRC7999 and the susceptible clone Blb48-5. The
susceptible parental clone Blb48-5 was derived from a cross between two susceptible
clones from the S. bulbocastanum accessions BGRC8005 and BGRC8006.
65
Chapter 5

Resistance assay
Detached leaf assays were used to determine the resistance phenotypes of BC
1

progeny plants. Two complex isolates IPO-655-2A (race 1.3.4.7.8.10.11) and IPO-
82001 (race 1.2.4.5.10.11), which were kindly provided by Dr. W. Flier of Plant
Research International, Wageningen, The Netherlands, were used for the resistance
assays. Both are A2 type and originate from The Netherlands and Belgium,
respectively. Inoculum preparation and inoculation were performed as described by
Vleeshouwers et al. (1999). Six days after inoculation, plant phenotypes were
determined. Leaves showing no symptoms or a localized necrosis at the point of
inoculation were scored as resistant and those with clear sporulating lesions as
susceptible.

DNA isolation and marker development
Genomic DNA was isolated from young leaf tissue according to Bendahmane et al.
(1997). PCR markers were created using primers designed on specific tomato or
potato genomic DNA sequences, BAC end sequences or cloned AFLP fragments. An
overview of the PCR-based markers used to construct the high-resolution map, is
presented in Table 1.
AFLP analysis was performed as described by Vos et al. (1995). Primary
templates were prepared using the restriction enzyme EcoRI/MseI and PstI/MseI
combinations. Bulked Segregant Analysis (BSA; Michelmore et al., 1991) was carried
out by performing AFLP analysis on resistant and susceptible bulks comprising
secondary template of eight resistant and susceptible genotypes. The bulks were
screened with 256 Eco+3/Mse+3 and 256 Pst+2/Mse+3 primer combinations.
Markers names include original marker names from public databases
(http://www.sgn.cornell.edu/cgi-bin/mapviewer/mapviewerHome.pl), the original
accession number of a tomato BAC clone followed by L or R and RGH (resistance
gene homologue). Microtitre plate, row and column numbers of the potato BAC library
8005-8 (van der Vossen et al., 2003) were used for BAC-end sequence markers.
AFLP markers were named by acronyms of enzyme combinations (EcoRI/MseI and
PstI/MseI) used to prepare the template DNA followed by three or two selective
nucleotides and the size of each marker as described in reference autoradiograms
created by Keygene NV, Wageningen, The Netherlands.

Map construction
Genetic linkage maps were constructed according to recombination frequencies
between marker loci and or the Rpi-blb3 locus. J oinmap software was used to
determine linkage groups and to select the Rpi-blb3-linked markers (Stam, 1993).
Map distances were re-calculated following marker order determination using Record
(van Os et al., 2000).
66

Table 1. PCR based-markers, primers, annealing temperature and restriction enzymes used for the
genetic mapping of Rpi-blb3 in this study. A: markers used for recombinant selection, B: markers for
high-resolution map, C: markers for marker-saturated map D: marker co-segregating with Rpi-blb3.
Aim Marker Type PCR primer (5`-3`) Tm Enzyme
CT229 SCAR F: TTGTGAGTGGTGAACTACGGGC

R: CGGCAATGGTTATGGGAACG
65 a.s. A
TG506R CAPS F: ATGCCAGCAGTCCAGTTTCC
R: TTCCTTCCTGAAGTCAACCC
54 HhaI
cLPT5B19 CAPS F: CCTTCATCAGATCTGGTCG
R: CAGCTGTCACAATTGCCAAC
55 AluI
AF411807L CAPS F: CGTTCCATGACATGTTCAAGC
R: GCTCCAGAATCAATCTGAGG
56 DdeI
RGH1 CAPS F: GGSAAGACCACTCTTGCAAG
R: GGTTTTTAAGCTGCTAATGTTG
50 HpyCH4IV
RGH2 SCAR F: GGSAAGACCACTCTTGCAAG
R: TGGTYATAATYACTCTGCTGC
50 a.s.
RGH3 CAPS F: ATGRCTGATGCMTTTRTGTC
R: CCYAAGTASAGAAAACACTGC
50 HaeIII
AF411807R CAPS F: GCTAAGTTGTCTGAGGTTAG
R: TGTTCTGGCTCTTCACAATC
48-54* AluI & HaeIII
TG370F CAPS F: TCGAAGCTCTGTTTCTGCTC
R: CCCATGTTATGCCATTCAGC
59 HpaII
T1430 CAPS F: TCCTTTGTCATGGATATGCC
R: CTGTGTAAAAGTAGAGCCAC
55 HpyCH4IV
T1261 CAPS F: CAATTCCCGTGAATCCTTCG
R: GTACCAATAGCCCAAACAGG
55 HpyCH4IV
TG339R SCAR F: ACTCTTTCGGCCTACAAGTC
R: ATCCTTGTAGGACTCCTCTC
58 a.s.
B
P1433 CAPS F: CACAGAACCATCAATGGCG
R: ACCACTGAGAAATTGAGAGC
56 DpnII
B57R CAPS F: CCGTTTATGGTTCTTACCGC
R: GTAGGGCATGAAGCATGATG
58 AluI
54I8L CAPS F: GGTGTCTTGAGTATTGTCG
R: CCACTTTTTCCTTTGCCTG
58 TaqI
54I8R CAPS F: GCTTCTTAATCTTAACAAAATA
R: CATATACTGTTTTAAATGTCAC
52 Sau96I
139K15L CAPS F: GGTAAGAAAGAAGAGGAGAT
R: CCTTTCCTACTGTCTTTCT
58 DdeI
154D15R CAPS F: CGAGGACGAGTGACATTTT
R: CCATTTTGACCCATTTTCTC
58 AciI
B10L CAPS F: TCAGAGGCATGACACCTGTG
R: GTTGACCCAGTTGATTAAGTC
58 TaqI
20D11R CAPS F: AGCATCCGGAGGCAAATC
R: TAGGCTTAACTGTCAAATGG
56 HinfI
B175L CAPS F: GAAATGTGAACTACTCCAGG
R: TCATTTGCCAATCCCTCACC
57 MseI
C
28I12L CAPS F: AGCATCCGGAGGCAAATC
R: TAGGCTTAACTGTCAAATGG
56 HpyCH4IV
D Th21 SCAR F: ATTCAAAATTCTAGTTCCGCC
R: AACGGCAAAAAAGCACCAC
56 a.s.
* Annealing temperature. In AF411807R, annealing temperature was 48
o
C for the first 7 cycles and 54
o
Restriction enzymes that reveal polymorphism between resistant and susceptible linked alleles of the marker. a.s. means allele
specific marker showing polymorphism without digestion.
67
Chapter 5

Results

Linkage analysis
Segregation of late blight resistance was initially analyzed in 40 BC
1
progeny plants
derived from the S. bulbocastanum intraspecific BC
1
mapping population Blb00-20 in
detached leaf assays with the complex P. infestans isolate IPO-655-2A. Clear 1:1
segregation ratio for late blight resistance was observed suggesting the presence of a
dominant resistance allele at a single locus. To localize the R locus, we selected ten
clearly resistant and ten clearly susceptible BC
1
genotypes as a core population and
tested randomly selected markers of different chromosomes on these plants. The
chromosome 4 marker GP180 (Tanksley et al., 1992) was found to be genetically
linked to the resistance phenotype, indicating that the R locus was different from Rpi-
blb1 or Rpi-blb2 (van der Vossen et al., 2003 and 2004). This novel locus was
therefore designated Rpi-blb3. Further marker development showed the GP180
flanking CAPS marker TG506R and the SCAR marker CT229 (Table 1A) to be
proximal and distal to the Rpi-blb3 locus, respectively (Figure 1).

Figure 1. High-resolution genetic linkage map of the Rpi-blb3 locus on
chromosome 4. Genetic distances and marker names are indicated on
the left and right of the map, respectively. The number of recombinants
is shown in brackets. The magnification shows the map between
cLPT5B19 and TG370F where BAC-end sequence markers and RGH
markers from tomato were localized.
68

High-resolution mapping
To be able to construct a high-resolution genetic map of the Rpi-blb3 locus, the initial
Blb00-20 mapping population was extended to 1400 genotypes and screened with
markers CT229 and TG506R. A total of 334 CT229/TG506R recombinants were
identified and phenotyped for late blight resistance resulting in 183 resistant and 133
susceptible recombinants. The remaining 18 genotypes gave unclear phenotypes.
In an attempt to develop additional informative markers in the Rpi-blb3 interval,
several existing RFLP markers from the CT229-TG506R interval in tomato were
targeted for PCR marker development in the Blb00-20 mapping population (Table 1B).
Markers for which specific polymorphisms were identified between the parental
genotypes of Blb00-20 were tested on the set of CT229-TG506R recombinants,
delimiting the Rpi-blb3 locus to a 4.5 cM genetic interval between markers TG370F
and T1430 (Figure 1). Interestingly markers AF411807L and AF411807R, which were
based on the BAC-end sequences of the tomato BAC clone AF411807L (van der
Hoeven et al., 2002), harboring several R gene analogues (RGA), were mapped
distal to TG370F. In an attempt to identify RGA specific markers within the Rpi-blb3
interval, primers were designed on conserved regions of the RGA sequences on the
tomato BAC and used to develop RGA specific CAPS markers in Blb00-20.
Unfortunately, all the identified RGA markers also mapped distal to TG370F (Figure
1).

Figure 2. Bulked segregant analysis (BSA). A: Graphical
genotypes of individuals composing the resistant bulk (Br) or
the susceptible bulk (Bs). The presence and absence of
marker alleles is indicated by solid bars and lines,
respectively. The number of different genotypes for each bulk
is shown on the top. B: Gel images of an example of an AFLP
marker identified by the BSA method. Eight resistant
genotypes (R) and 8 susceptible genotypes (S) were checked
individually. The marker band is indicated by the arrow.
69
Chapter 5

To estimate the cM/kb ratio within the Rpi-blb3 interval, the S. bulbocastanum-
derived BAC library Blb8005-8, which was developed for the cloning of the Rpi-blb1
gene (van der Vossen et al., 2003), was screened with the two closest markers
TG370F and T1430. Both right (R) and left (L) end sequences of the selected BAC
clones were used to develop PCR markers and to start a chromosome walk to the
Rpi-blb3 locus (Table 1C). In this way the genetic interval harboring the Rpi-blb3 gene
was reduced to 2.6 cM (36 recombinants) between B10L and B175L (Figure 3).
Interestingly, several of the BAC end sequences, including B10L, were highly
homologous to the RGA sequences present on the tomato BAC sequence AF411807
(van der Hoeven et al., 2002), indicating that the RGA cluster that was mapped distal
to TG370F, extended into the Rpi-blb3 interval.

Figure 3. Graphical genotypes of recombinants
which define the Rpi-blb3 interval. The presence
and absence of the markers or resistance allele
is indicated by solid bar and line, respectively.
The numbers of recombinants belonging to
certain classes are presented on the top and
markers are shown on the left. Map distances
between markers and the number of
recombinants (brackets) are shown on the right.
As the physical map extended towards the Rpi-blb3 locus, the efficiency of the
chromosome walk was reduced. In attempt to circumvent this problem, we decided to
carry out a BSA (Michelmore et al., 1991) in order to identify AFLP markers that
closely flanked or fully co-segregated with the resistance. A resistant bulk (Br) and
susceptible bulk (Bs) were composed according to the graphical genotypes depicted
in Figure 2A. Each bulk consisted of eight DNA samples derived from genotypes that
had a recombination event within the relevant interval. In total 256 Eco+3/Mse+3 and
256 Pst+2/Mse+3 primer combinations were tested on the bulks, 80 of which
produced candidate markers putatively linked to resistance. Linkage to Rpi-blb3 was
70

confirmed by screening the individuals used to compose the bulks (Figure 2B).
Subsequently, 36 primer combinations were used to screen 63 recombinants from the
TG370F and T1430 region, resulting in the identification of 23 informative AFLP
markers. One of these markers, EATAMACG_199, fully co-segregated with
resistance, and the interval harboring Rpi-blb3 was reduced to 0.93 cM between
markers PCAMAGA_260 and EAACMAAC_99 (Figure 3). The co-segregating AFLP
marker was successfully converted into SCAR marker Th21 (Table 1D and Figure 4).

Figure 4. PCR marker (Th21) developed from the sequence of the AFLP marker (EATAMACG_199)
co-segregating with the Rpi-blb3 locus. A marker size ladder (1kb) is indicated. Pr, Ps, Br, Bs, R and S
indicate the resistant parent, susceptible parent, resistant bulk, susceptible bulk, resistant offspring and
susceptible offspring, respectively.

Comparative genomics at the Rpi-blb3 locus
To study the syntenic relationship between the Rpi-blb3 locus and the three late blight
R loci Rpi-abpt (Chapter 3), R2 (Li et al., 1998) and R2-like (Chapter 4), which were
mapped in different genetic backgrounds to the same genetic interval on
chromosome 4 (Figure 5), we screened each mapping population with the co-
segregating AFLP markers developed in the other mapping studies. Interestingly,
several markers segregated in more than one mapping population, facilitating the
alignment of the four genetic maps (Figure 5). AFLP marker EATA/MACG_199, which
co-segregates with Rpi-blb3 in 1396 BC
1
genotypes also co-segregated with
resistance in the R2 and R2-like backgrounds and was separated from Rpi-abpt by
only one recombination event. These data suggest that Rpi-blb3, Rpi-abpt, R2 and
R2-like are members of the same R gene cluster on chromosome 4 and may be
allelic.

Discussion

In this study we have identified and genetically characterized a new late blight R locus
in S. bulbocastanum. Initially, CAPS markers developed from chromosome specific
RFLP markers (Tanksley et al., 1992, Meksem et al., 1995) were used to determine
the chromosomal position of a dominant late blight R locus which segregated in an
71
Chapter 5

intraspecific S. bulbocastanum BC
1
population. Linkage to the chromosome 4 specific
markers CT229 and TG506 indicated that the R locus was different from Rpi-blb1 and
Rpi-blb2, located on chromosomes 8 and 6, respectively (van der Vossen et al., 2003
and 2004). This new locus was therefore designated Rpi-blb3. A recombinant
analysis of 1396 BC
1
progeny plants with CT229 and TG506 identified a total of 334
recombinants which were subsequently used to genetically fine map the Rpi-blb3
locus, a prerequisite for future positional cloning of the gene.

Figure 5. Integrated genetic linkage map of the different late blight resistance loci Rpi-blb3, Rpi-abpt
(Chapter 3), R2-like (Chapter 4) and R2 (Li et al., 1998) on chromosome 4.

Marker saturation of any genetic interval can be achieved by several methods
(Meksem et al., 1995), four of which were used in this study in an attempt to saturate
the Rpi-blb3 interval: CAPS marker development from published RFLP markers
(Table 1B), the RGA candidate gene approach (Table 1B), BAC chromosome walking
(Table 1C), and bulked segregant analysis (BSA). Initially, several PCR-based
markers were successfully converted from RFLP markers and genetically mapped
between CT229 and TG506 (Figure 1). The marker order was consistent with that of
previously published tomato and potato maps (Tanksley et al., 1992).
The majority of R genes characterized to date belong to the NBS-LRR class
(Dangl and J ones, 2001). This class of genes contains several conserved motifs
within the NBS which can be targeted for candidate gene marker development,
resulting in the amplification of resistance gene analogs (RGA). As R genes are often
part of complex loci (Hulbert et al., 2001), RGA specific marker strategies often lead
72

to R gene cluster landing. This approach has successfully been used in wheat (Yan et
al., 2003), barley (Madsen et al., 2003), sugar beet (Hunger et al., 2003), but also in
potato (Leister et al., 1996; Paal et al., 2004). In this study we specifically targeted
RGA sequences from the tomato BAC clone AF411807 (van der Hoeven et al., 2002)
for RGA specific marker development at the Rpi-blb3 locus. This BAC clone was
identified through BLAST analysis of a cloned AFLP fragment that was linked to the
syntenous late blight R locus Rpi-abpt (Chapter 3). Unfortunately this did not lead to
the identification of a co-segregating RGA specific marker, suggesting either that Rpi-
blb3 does not belong to the same NBS-LRR gene family as the RGAs on the tomato
BAC or that the degree of RGA polymorphism at the Rpi-blb3 locus is reduced.
Subsequently, a transgenotype BAC chromosome walk was initiated. The most
closely linked CAPS markers were used to screen an existing S. bulbocastanum
derived BAC library which was previously used to clone Rpi-blb1 (van der Vossen et
al., 2003). Although this BAC library did not contain Rpi-blb3 we expected the allelic
variation within S. bulbocastanum to be lower than that between S. bulbocastanum
and S. tuberosum, of which there are also BAC libraries available (Rouppe van der
Voort et al., 1999; Ballvora et al., 2002; Paal et al., 2004). Several positive BAC
clones were indeed identified, the BAC end sequences of which were used to develop
markers more closely linked to Rpi-blb3. However, progress was very slow, mainly
due to the fact that the level of polymorphism reduced as we approached the Rpi-blb3
gene. In addition, the different origin of the BAC library did not facilitate the mapping
of repetitive sequences. Although some BAC markers were polymorphic in the BC
1

mapping population, often we were unable to verify the BAC origin of the polymorphic
allele as the BAC specific allele was not present in the parental genotypes of the BC
1

mapping population.
BSA (Michelmore et al., 1992) in combination with the AFLP technique (Vos et
al., 1995) is a powerful tool for marker enrichment in a given region of a plant genome
(Ballvora et al., 1995; Thomas et al., 1995; Meksem et al., 1995). To make the BSA
more efficient and to focus on the Rpi-blb3 interval, we designed the resistant and
susceptible bulks based on informative recombinants from the Rpi-blb3 interval
(Figure 2). This resulted in the identification of 28 AFLP markers within the B57R-
T1430 interval (Figure 3) and the mapping of the Rpi-blb3 locus to a 0.93 cM interval
which is flanked by the AFLP markers PCAMAGA_260 and EAACMAAC_99. In
addition, AFLP marker EATAMACG_199 was identified to co-segregate with the Rpi-
blb3 locus. Although AFLP analysis is a powerful technique, the markers are costly
and technologically demanding. Conversion to simple PCR-based markers is
therefore necessary for use in genotype screening and progeny selection. In this
respect we succeeded in converting the co-segregating AFLP marker
EATAMACG_199 into SCAR marker Th21 (Table 1D and Figure 4) which can be
used for breeding applications and for BAC landing.
73
Chapter 5

R genes and RGAs have been shown to be clustered in the genomes of many
different species (Meyers et al., 1998; Michelmore and Meyers, 1998), including
Solanaceae (Grube et al., 2000). At least five R genes against diverse pathogens
have been mapped in different genetic backgrounds to the GP21-GP179 interval on
chromosome 5; Gpa and Grp1 conferring resistance to potato cyst nematodes (Kreike
et al., 1994; Rouppe van der Voort et al., 1998), Nb and Rx2 conferring resistance to
potato virus X (de J ong et al., 1997; Ritter et al., 1991) and R1 conferring resistance
to P. infestans (Leonards-Schippers et al., 1992). Gro1.3, conferring resistance to
cyst nematodes (Kreike et al., 1993) and R3, R6 and R7 conferring resistance to P.
infestans (El-Kharbotly et al., 1994 and 1996) cluster on the short arm of
chromosome 11. In the current study we have identified a major cluster on
chromosome 4, to which several R genes conferring resistance to P. infestans were
mapped. Marker order and allelic conservation was observed, facilitating the
alignment of the genetic maps of the different R genes Rpi-blb3 (this study), Rpi-abpt
(Chapter 3), R2-like (Chapter 4) and R2 (Li et al., 1998) with AFLP marker
EATA/MACG_199 (Figure 5). Clearly the short arm of chromosome 4 is also a
hotspot for resistance, harboring several distinct R gene clusters with resistance
specificities to different pathogens. The recently cloned root knot nematode R gene
Hero (Ganal et al., 1995) is part of an extensive R gene cluster which is located distal
to Rpi-blb3. The cyst nematode R locus Gpa4 (Bradshaw et al., 1998), the virus R
locus Ny
tbr
(Celebi-Toprak et al., 2002) and QTLs for late blight (Leonards-Schippers
et al., 1994; Oberhagemann et al., 1999; Sandbrink et al., 2000) and Erwinia
carotovora subsp. atroseptica (Zimnoch-Guzowska et al., 2000) are also located on
the short arm of chromosome 4.
Rpi-abpt and Rpi-blb3 confer resistance to P. infestans isolates with complex
race-specficities. Although Rpi-blb3 was tested with only two isolates, it displays a
different resistance reaction to one of the two tested isolates, compared to Rpi-abpt.
Rpi-blb3 confers a partial resistance to isolate IPO-82001 (data not shown), whereas
Rpi-abpt confers complete resistance to this isolate (Chapter 3). Also the wild
species accessions and the geographical regions from which the above two genes
originate are different from each other (http://www.cgn.wageningen-ur.nl/pgr/). Rpi-
blb3 is derived from S. bulbocastanum subsp. dolichophyllum (CGN17688) whereas
Rpi-abpt is derived from S. bulbocastanum subsp. bulbocastanum (CGN17693 or
CGN21306). These findings suggest that they are distinct genes. On the other hand,
the R2-like locus displays the same specificity as the R2 locus from S. demissum (Li
et al., 1998). The introgression fragment harboring the R2-like locus could be derived
from either S. edinense, S. tuberosum subsp. andigena or S. vernei, which are all
present in the pedigree of the mapping population (Chapter 4). Interestingly, S.
edinense is a natural hybrid between S. tuberosum and S. demissum, suggesting that
R2 and the R2-like loci may in fact have originated from the same gene pool.
74

Alternatively, syntenic loci in different wild Solanum species may harbor genes which
display the same resistance specificity without sharing a common origin.
Recently comparative genomics between potato and tomato facilitated the
isolation of the late blight R genes R3a and Rpi-blb2 from potato as these genes
mapped to regions of the potato genome that were syntenic to the I2 and Mi loci in
tomato, respectively (Huang et al., 2005; van der Vossen et al., 2004). The synteny of
markers and sequences at the late blight R gene hotspot on chromosome 4 will
facilitate the cloning of the genes Rpi-blb3, Rpi-abpt, R2 and R2-like. Recently,
Huang (2005) demonstrated that eight (R3, R5-R11) of the eleven classical late blight
resistance specificities characterized so far (Black et al., 1953) are either allelic
versions or duplicated members of the same R gene (super-) cluster on the distal
region of the short arm of chromosome 11. Furthermore, it was demonstrated that the
R3 specificity is not based on a single R gene but on the joint effect of two R genes,
currently named R3a and R3b (Huang et al., 2004). These findings provide novel
insights in the evolution of R gene clusters conferring late blight resistance in
Solanum. The prevailing hypotheses on the evolution of R gene clusters include
unequal crossover which can result in expanding numbers of paralogs with novel
specificities (reviewed in Hulbert et al., 2001). These observations are mainly based
on studies in selfing organisms (Anderson et al., 1997; Parker et al., 1997; Parniske
et al., 1997; Ellis et al., 1999). The genus Solanum harbours both outbreeding and
polyploid species. In the latter case, allelic variation is higher. Not only can additional
paralogs acquire novel specificities, but also the homeologous chromosomes of the
different genomes within the disomic pairing hexaploid S. demissum or the
heterozygosity in outbreeders can accommodate additional R gene specificities. Our
data and those of Huang (2005) demonstrate that this indeed has occurred in
Solanum in both the chromosome 4 and chromosome 11 R gene clusters.

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80
Chapter 6

Genetic positioning of centromeres using half-tetrad
analysis in a 4x-2x cross population of potato

Tae-Ho Park, J ong-Bo Kim, Herman J . van Eck,
Evert J acobsen and Richard G.F. Visser

Submitted for publication

Chapter 6

Abstract

From a genetic standpoint, centromeres play an important role in the delivery
of the chromosome complement to the daughter cells at cell division. The
position of the centromeres of potato was determined by half-tetrad analysis in
a 4x-2x population where the male parent produced 2n pollen by FDR (First
Division Restitution). The genetic linkage groups and locations of 95 male
parent-derived AFLP markers could be determined by comparing them with the
reference markers of the ultra high density (UHD) map of potato. Ten
chromosomes contained 100 % heterozygous alleles transmitted from the 2n
heterozygous gametes of the paternal parent into the tetraploid offspring. The
position of these centromeric alleles was in accordance with those predicted by
the marker density approach of the UHD map. The centromere positions of two
chromosomes could not unambiguously be determined because of the absence
of the expected 100 % simplex descendants. However, their positions could be
anticipated by the increasing or decreasing rate of heterozygosity and by the
marker density in the UHD map. Additional investigations on recombination of
three chromosomes with sufficient numbers of markers showed that only one
crossover occurred per chromosome arm, proving total interference of
recombination between centromere and telomere.

Introduction

The centromere is a functionally important region of the chromosome that interacts
with the spindle fibers and ensures proper chromosome movement during nuclear
division. When this process fails, the daughter cells will have abnormal chromosome
numbers, which can eventually lead to death or phenotypic abnormality. Centromeres
facilitate chromosome segregation during mitotic and meiotic division by nucleating
kinetochore formation, providing a target for spindle attachment and maintaining
sister chromatid cohesion (Copenhaver et al., 1999). Centromeres contain densely
methylated, highly repetitive, heterochromatic DNA that nucleates kinetochores,
promotes sister chromatid cohesion and suppresses recombination (Hall et al., 2004).
Centromere mapping can be carried out by the fact that a meiosis leads,
because of the absence or occurrence of crossover between non-sister chromatids,
to different allelic patterns in the resulting tetrad. Half-tetrad analysis, which is
possible with unreduced gametes, is a powerful tool for understanding chromosomal
behavior during meiosis (Lindner et al., 2000). When a single restitution mechanism
occurs in a diploid parent leading to unreduced gametes, the 4x-2x cross is
advantageous in centromere mapping studies (Mendiburu and Peloquin, 1979;
Wagenvoort and Zimnoch-Guzowska, 1992; Bastiaanssen et al., 1996; Bastiaanssen,
82
Centromere positioning in potato chromosome

1997). In potato, 2n gametes derived from both FDR (First Division Restitution) and
SDR (Second Division Restitution) mechanisms have been utilized for gene-
centromere mapping by using 4x-2x or 2x-4x cross derived tetraploid populations
(Mendiburu and Peloquin, 1979; Bastiaanssen, 1997). In the present research, we
used a 4x-2x cross-derived tetraploid population to identify the genetic position of the
centromeres using half-tetrad analysis in potato.
In general, diploid species can produce haploid (n) gametes. However,
unreduced or 2n gametes commonly occur in Solanum species (Carputo et al., 2000).
In diploid potato several meiotic restitution mechanisms that lead to 2n gamete
formation have been reported (reviewed by Veilleux, 1985 and Ramanna and
J acobsen, 2003). First division restitution (FDR) and second division restitution (SDR)
have been considered as the two basic types of them (Mok and Peloquin, 1975;
Ramanna, 1979). In the absence of crossover in the meiocyte, all parental
heterozygous loci will be heterozygous in FDR gametes. In those cases of FDR
where crossovers occur, the loci from the centromere to the first crossover point will
stay heterozygous. On the contrary, in the case of SDR all the loci that are situated
between the centromere and the first crossover will be homozygous in the 2n
gametes. However, a single crossover between a locus and centromere will produce
50 % heterozygous and 50 % homozygous gametes in FDR, but all these loci will be
heterozygous in SDR gametes. (Wagenvoort and Zimnoch-Guzowska, 1992; Hutten
et al., 1994; Lindner et al., 2000). Therefore, the percentage of heterozygosity or
homozygosity of the 2n gametes is indicative for determining the genetic distance
between marker and centromere (Figure 1). In FDR, if the heterozygosity of an allele
increases to 100 %, the allele is getting closer to the centromere, but if it decreases to
50 %, the allele is getting closer to the telomere. In SDR, if the heterozygosity of an
allele is 0 %, the allele is located on the centromere but if it is 100 %, the allele is on
the telomere.

Figure 1. Probability of heterozygosity.
This depends on the FDR or SDR
mechanism of unreduced gamete
formation and on the position of the
centromere on the chromosome.

The positions of centromeres were putatively proposed in the Ultra High
Density (UHD) genetic map of potato where over 10,000 AFLP markers were used to
83
Chapter 6

construct a genetic linkage map (van Os et al., submitted). They were determined by
the observation that in some species AFLP markers tend to be clustered in
centromeric regions because of recombination suppression (Alonso-Blanco et al.,
1998; Keim et al., 1997; Qi et al., 1998). The aim of this research was to identify and
localize the genetic positions of centromeres using half-tetrad analysis in the 4x-2x
cross population and to compare them with those identified by marker density in the
UHD map.


Plant materials
A tetraploid (2n=4x=48) mapping population RH4X-103 consisting of 233 genotypes
was used. This population was created from a cross between tetraploid 707TG11-1
and diploid RH89-039-16 (2n=2x=24). The male parent RH89-039-16, which
produces 2n pollen, is one of the advanced clones widely used for creating mapping
populations at the Laboratory of Plant Breeding, Wageningen University, The
Netherlands (Rouppe van der Voort et al., 1997a, 1997b, 1998, 1999; Huang et al.,
2004). This clone was the male parent to produce the population that was used to
generate the Ultra High Density (UHD) genetic linkage map in potato (Isidore et al.,
2003; van Os et al., submitted). In the UHD genetic map, the genetic position of more
than 10,000 AFLP markers has been determined (http://www.dpw.wageningen-
ur.nl/uhd/).

DNA isolation
DNA isolation was performed as described by van der Beek et al. (1992) using a
Retsch machine (Retsch Inc., Haan, Germany) that can grind the fresh leaf tissues
with two steel balls and 2 % CTAB buffer composed of Tris (pH 7.5), 5 M NaCl and
0.5 M EDTA (pH 8.0) in 96 wells Coster plates (Corning Inc., Corning, NY, U.S.A.).
After incubation of the Coster plates at 65 in a water bath for one hour, ice-cold
chloroform isoamyl alcohol (24:1) was added. After centrifugation, the supernatant
was transferred to new tubes followed by adding one volume of isopropanol. Another
centrifugation step allowed the precipitation of DNA. After the DNA pellet was dried,
the DNA was dissolved in T
0.1
E-buffer (+0.5 g RNAse).

AFLP marker analysis
To generate AFLP (Amplified Fragment Length Polymorphism) markers (Vos et al.,
1995), primary templates were prepared by using two different restriction enzyme
combinations, EcoRI/MseI and PstI/MseI. After digestion of DNA with the enzymes,
adaptors fitting to the EcoRI, PstI and MseI sites were ligated to each end. The
primary templates were diluted prior to the selective pre-amplification. The first PCR
84

amplification of the adaptor-ligated restriction fragments (Primary templates) was
accomplished with single nucleotide extended primers in order to decrease the
number of restriction fragments. The pre-amplified products (Secondary templates)
were checked on a 1 % agarose gel. After 10 times dilution, the secondary templates
were suitable for AFLP reactions with selective primers. For the selective
amplification, radioactively labeled (
33
P) E+3 and P+2 primers were used in
combination with M+3 primers. The amplified DNA fragments were separated for two
and half hours on a 6 % polyacrylamide gel in 1 x TBE buffer. The
33
P labeled PCR
products were loaded on the gel after 30 minutes of pre-run. The gels were dried on
Whatman papers for two hours in a vacuum and X-ray films were exposed for 4-6
days.
AFLP markers generated from 23 E+3/M+3 and five P+2/M+3 primer
combinations were scored according to the presence/absence of band patterns and
band intensities. The heterozygous AFLP markers of the 2x male parent were only
used when they were absent in the 4x female parent. The markers derived from the
male parent, RH89-039-16, were scored as aa, ab, bb and uu indicating absence
of the bands (nulliplex), presence of heterozygous bands (simplex), presence of
homozygous bands (duplex) and unknown, respectively. Heterozygous simplex
(Aaaa) and duplex (AAaa) bands were scored on band intensity which was
determined with the naked eye. This allowed a good but not an absolute classification.
All above indicated AFLP markers were scored in the different offspring genotypes
and the frequency of each genotype within an AFLP marker was calculated.
According to the simplex, duplex and nulliplex classification, the locus-centromere
map distance could be determined for each segregating AFLP marker using the
formula [f (duplex) +f (nulliplex)] * 100 cM, where f is the frequency (Douches and
Quiros, 1987). The genetic position of individual AFLP loci within a linkage group and
a chromosome arm was determined by comparing them with those in the reference
gels of the male parent RH in the UHD map. AFLP markers were named with an
acronym of the two enzymes used, three or two selective nucleotides and the mobility
of the fragment as described in reference autoradiograms created by Keygene NV,
Wageningen, The Netherlands. MapChart (Voorrips, 2002) drew the marker saturated
genetic linkage map. Genetic linkage maps constructed in this study were compared
with those of the UHD map.

Results

AFLP marker scoring
In order to generate AFLP markers for centromere mapping, 28 EcoRI/MseI and
PstI/MseI primer combinations were used. When the markers were scored, intensity
of the bands and presence/absence of the markers were considered. The number of
85
Chapter 6

markers scored ranged between one and 11 in each primer combination. A total of
130 markers, derived from the diploid male parent RH89-039-16, was scored as aa,
ab, bb and uu indicating absence of a band (nulliplex in the tetraploid progeny),
presence of a heterozygous band (simplex in the tetraploid progeny), presence of a
homozygous band (duplex in the tetraploid progeny) and the unknown, respectively.
The genotype of individual tetraploid progeny plants was dependent on the
unreduced gametes of the male parent RH89-039-16. The gametes of the tetraploid
female parent 707TG11-1 were always aa indicating absence of the band. When the
2n gamete from the male parent was heterozygous ab, the genotype of the offspring
was simplex aaab scored as ab. When it was homozygous aa or bb, the genotype
of the offspring was nulliplex aaaa or duplex aabb scored as aa or bb,
respectively. An image of a part of three AFLP gels is shown as an example in Figure
2. It contained four segregating markers derived from the 2x male parent (A1 to A4)
which could be scored. Comparison of the A1 and the A2 markers indicated that there
were more nulliplex and duplex genotypes in the A1 marker than in the A2 marker
meaning that the A2 marker is closer to the centromere than the A1 marker because
of the lower frequency of recombination.

Figure 2. An image of AFLP gels containing segregating markers produced by FDR. There are four
AFLP markers A1, A2, A3 and A4 shown in the picture. The first lane, Pr, is the tetraploid female
parent, 707TG11-1 and the second lane, Ps, is the diploid male parent, RH89-039-16 which
produced unreduced gametes. All other lanes are tetraploid progeny plants.

Genetic map and centromere mapping
From 28 primer combinations, 130 male parent-derived markers were scored with an
average of 4.3 markers per primer combination. The location of 95 markers was
86

determined by comparison with the reference gels of the RH parent in the UHD
genetic linkage map (http://www.dpw.wageningen-ur.nl/uhd/). The rest of them could
not be directly identified in the UHD map. According to their location in the UHD map,
they were grouped and arranged by bin numbers in each linkage group. Co-
segregating markers belonged to the same bin. Strings of bins were formed by
connecting bins that were separated by one recombination event (van Os et al.,
submitted). In Table 1, the segregation of the 95 markers is presented and the
frequencies of the different alleles were calculated. Based on these observations,
heterozygosity and homozygosity of each AFLP locus was determined. The
frequency of homozygosity was used as the criterion to localize the AFLP markers
within linkage groups. There were 17 (HxH) markers which were derived from the
male parent RH89-039-16 in this population but they were previously derived from
both parents, SH83-92-488 and RH89-039-16 in the UHD map (van Os et al.,
submitted). The chromosome number of them was assigned, but not the bin number.
According to the results of the percentage of homozygosity of each AFLP
marker in the different chromosomes that were calculated using the formula,
[f(a)+f(b)]*100, the genetic map of the 4x-2x male parent was created (Figure 3). The
positions of centromeres of the 4x-2x male parent map were compared with those
identified by the marker density approach in the UHD map. The AFLP markers whose
positions were determined were bridged with the bin where the markers belonged to
in the UHD map (van Os et al., submitted). The percentage of heterozygosity of all
AFLP markers allowed the assignment of the genetic position of the centromeres on
each chromosome. Heterozygosity of the markers increases from the distal parts of
chromosomes to the expected centromeric regions where the heterozygosity is
expected to be 100 %. These expected centromeric regions were good candidates for
positioning the centromeres on each chromosome. For ten chromosomes, they were
located in Bin number 13, 1, 35, 46, 17, 68, 22, 31, 64 and 49 on chromosome 1, 2, 4,
5, 6, 7, 8, 9, 10 and 12, respectively. Most of the AFLP markers that belonged to
those bins showed 100 % heterozygosity although exceptions were found. Like on
chromosome 1, one AFLP marker (EACTMCAA_207) that belonged to Bin 13, where
the UHD map-based centromere is expected to be located, showed 0.4 %
homozygosity, while another AFLP marker (EACTMCTC_217.1) showing 100 %
heterozygosity belonged to Bin 12 instead of Bin 13. Similarly one AFLP marker
(EACTMCAA_366.0) on chromosome 5 with 100 % heterozygosity was located in Bin
44 instead of Bin 46 where the other 100 % heterozygosity markers belonged to on
this chromosome. However, the position of centromeres of these two chromosomes
remained in Bin 13 and 46, respectively, because more markers with 100 %
heterozygosity were present in those bins. The accurate position of centromeres on
chromosomes 3 and 11, unfortunately, could not be exactly assigned by the 4x-2x
male parent approach because there were no markers showing 100 % heterozygosity.
87
Chapter 6

Table 1. Segregation of AFLP markers on the 12 chromosomes of potato and determination of the
genetic position of the centromeres
Allele segregation Map postion
AFLP loci aa(freq)* ab(freq) bb(freq)
Hetero-
zygosity
[f(a)+f(b)]
*100
Chromosome
& Bin
EACTMCAA_197 16(0.09) 158(0.90) 1(0.01) 0.903 9.7 RH01(HxH)

+
EACAMCCT_90 1(0.00) 222(0.96) 8(0.03) 0.961 3.9 RH01B10
EACTMCTC_217.1 0(0.00) 233(1.00) 0(0.00) 1.000 0.0 RH01B12
EAACMCAG_261.4 0(0.00) 233(1.00) 0(0.00) 1.000 0.0 RH01B13
EAACMCAG_196.4 0(0.00) 233(1.00) 0(0.00) 1.000 0.0 RH01B13
EAACMCAG_143.6 0(0.00) 233(1.00) 0(0.00) 1.000 0.0 RH01B13
EAACMCAG_139.9 0(0.00) 233(1.00) 0(0.00) 1.000 0.0 RH01B13
EAACMCCA_189.0 0(0.00) 233(1.00) 0(0.00) 1.000 0.0 RH01B13
EAACMCCA_136.8 0(0.00) 233(1.00) 0(0.00) 1.000 0.0 RH01B13
EAACMCCT_82.7 0(0.00) 233(1.00) 0(0.00) 1.000 0.0 RH01B13
EACAMCAA_203.2 0(0.00) 233(1.00) 0(0.00) 1.000 0.0 RH01B13
EACAMCAA_142.1 0(0.00) 233(1.00) 0(0.00) 1.000 0.0 RH01B13
EACTMCAG_127.1 0(0.00) 233(1.00) 0(0.00) 1.000 0.0 RH01B13
EACTMCAG_65.0 0(0.00) 233(1.00) 0(0.00) 1.000 0.0 RH01B13
EACTMCTC_196.8 0(0.00) 233(1.00) 0(0.00) 1.000 0.0 RH01B13
EACTMCTC_219.5 0(0.00) 233(1.00) 0(0.00) 1.000 0.0 RH01B13
EACTMCAA_207 1(0.00) 230(1.00) 0(0.00) 0.996 0.4 RH01B13
EACAMCCT_145 33(0.14) 177(0.77) 19(0.08) 0.773 22.7 RH01B36
EACTMCAA_287 6(0.03) 208(0.96) 2(0.01) 0.963 3.7 RH02(HxH)
EACTMCAA_134.5 0(0.00) 139(1.00) 0(0.00) 1.000 0.0 RH02B01
EACAMCAA_466 6(0.03) 219(0.96) 4(0.02) 0.956 4.4 RH02B02
PACMAAC_269 7(0.03) 218(0.94) 6(0.03) 0.944 5.6 RH02B02
EACTMCAG_81 31(0.13) 177(0.77) 22(0.10) 0.770 23.0 RH02B24
EAACMCCT_140.3 39(0.17) 163(0.71) 27(0.12) 0.712 28.8 RH02B29
PACMAAC_279 35(0.16) 145(0.63) 51(0.22) 0.628 37.2 RH02B35
PCGMAGA_234 45(0.19) 119(0.51) 69(0.30) 0.511 48.9 RH02B53
EACTMCAG_128 43(0.20) 151(0.70) 22(0.10) 0.699 30.1 RH03(HxH)
PCGMAGA_249 5(0.02) 209(0.90) 17(0.07) 0.905 9.5 RH03B30
EACAMCCT_588 7(0.03) 216(0.94) 7(0.03) 0.939 6.1 RH03B32
EAGAMCTA_133.1 1(0.01) 108(0.99) 0(0.00) 0.991 0.9 RH03B37
EAGAMCAT_246.6 9(0.08) 96(0.89) 3(0.03) 0.889 11.1 RH03B37
PCGMAGA_175 8(0.04) 206(0.90) 14(0.06) 0.904 9.6 RH04B15
EAACMCGA_322 9(0.04) 213(0.92) 10(0.04) 0.918 8.2 RH04B20
EAGTMCAG_130 5(0.02) 217(0.94) 9(0.04) 0.939 6.1 RH04B20
PACMAAT_146 7(0.03) 220(0.94) 6(0.03) 0.944 5.6 RH04B22
EAACMCTG_234 2(0.01) 225(0.98) 3(0.01) 0.978 2.2 RH04B25
EACAMCAC_72 3(0.01) 228(0.99) 0(0.00) 0.987 1.3 RH04B25
EACAMCAA_244.5 0(0.00) 233(1.00) 0(0.00) 1.000 0.0 RH04B35
EACAMCCT_492.8 0(0.00) 233(1.00) 0(0.00) 1.000 0.0 RH04B35
EAACMCGA_256.6 42(0.19) 175(0.77) 9(0.04) 0.774 22.6 RH04B73

88

Table 1. continued
Hetero-
zygosity
[f(a)+f(b)]
*100
Chromosome
& Bin
PACMAAT_314 2(0.01) 228(0.99) 1(0.00) 0.987 1.3 RH05(HxH)

PACMAAT_98 28(0.12) 187(0.81) 17(0.07) 0.806 19.4 RH05B02
EATGMCAC_181.8 37(0.17) 160(0.73) 22(0.10) 0.731 26.9 RH05B03
EACAMCAC_317.0 7(0.03) 220(0.97) 0(0.00) 0.969 3.1 RH05B43
EACTMCAA_366.0 0(0.00) 233(1.00) 0(0.00) 1.000 0.0 RH05B44
EAACMCAG_231.8 0(0.00) 233(1.00) 0(0.00) 1.000 0.0 RH05B46
EAACMCAG_135.2 0(0.00) 233(1.00) 0(0.00) 1.000 0.0 RH05B46
EAACMCCA_410.3 0(0.00) 233(1.00) 0(0.00) 1.000 0.0 RH05B46
EATGMCAC_137 5(0.02) 208(0.96) 4(0.02) 0.959 4.1 RH06B01
PATMAGA_335 2(0.01) 224(0.97) 4(0.02) 0.974 2.6 RH06B03
EAACMCCT_377.0 1(0.00) 226(0.99) 2(0.01) 0.987 1.3 RH06B15
EAACMCCA_231.3 3(0.01) 228(0.99) 0(0.00) 0.987 1.3 RH06B16
EAACMCGA_535.5 0(0.00) 233(1.00) 0(0.00) 1.000 0.0 RH06B17
EACAMCAC_553.6 0(0.00) 233(1.00) 0(0.00) 1.000 0.0 RH06B17
EACTMCAG_365.4 0(0.00) 233(1.00) 0(0.00) 1.000 0.0 RH06B17
EATGMCAC_185 13(0.06) 205(0.92) 4(0.02) 0.923 7.7 RH06B19
EATGMCAC_187 13(0.06) 205(0.92) 4(0.02) 0.923 7.7 RH06B19
EAACMCTG_153 25(0.11) 167(0.73) 38(0.17) 0.726 27.4 RH06B28
EACAMCAC_90 28(0.12) 159(0.69) 44(0.19) 0.688 31.2 RH06B31
EAACMCCT_144.2 45(0.20) 138(0.60) 46(0.20) 0.603 39.7 RH06B50
EAACMCCA_112 15(0.06) 194(0.84) 22(0.10) 0.840 16.0 RH07(HxH)
EACAMCCT_529 1(0.00) 227(0.98) 3(0.01) 0.983 1.7 RH07(HxH)
EACTMCAG_293 40(0.17) 136(0.59) 54(0.23) 0.591 40.9 RH07(HxH)
EACAMCAA_386 55(0.24) 155(0.67) 21(0.09) 0.671 32.9 RH07B34
EAACMCAA_188.5 0(0.00) 93(1.00) 0(0.00) 1.000 0.0 RH07B68
EAACMCGA_70.5 10(0.04) 223(0.96) 0(0.00) 0.957 4.3 RH07B77
PACMAAT_413 24(0.10) 194(0.85) 11(0.05) 0.847 15.3 RH08(HxH)
EATGMCAG_262 39(0.17) 156(0.68) 34(0.15) 0.681 31.9 RH08B09
EAACMCCA_437.7 0(0.00) 233(1.00) 0(0.00) 1.000 0.0 RH08B22
EAACMCCA_141.0 2(0.01) 228(0.98) 3(0.01) 0.790 2.1 RH08B26
EATGMCAC_278 8(0.04) 207(0.94) 6(0.03) 0.937 6.3 RH08B34
EAACMCAG_130 16(0.07) 155(0.67) 62(0.27) 0.665 33.5 RH09(HxH)
EACAMCAA_125 38(0.17) 140(0.62) 49(0.22) 0.617 38.3 RH09(HxH)
EACTMCAG_546 39(0.17) 138(060) 53(0.23) 0.600 40.0 RH09(HxH)
EACAMCAA_158 18(0.08) 194(0.85) 16(0.07) 0.851 14.9 RH09B03
EACTMCTC_228.6 0(0.00) 233(1.00) 0(0.00) 1.000 0.0 Rh09B31
PACMAAT_76 11(0.05) 216(0.93) 5(0.02) 0.931 6.9 RH09B37
EAACMCTG_89 27(0.12) 172(0.75) 31(0.13) 0.748 25.2 RH09B60
EAACMCAG_167.8 34(0.15) 160(0.69) 39(0.17) 0.687 31.3 RH09B60
EAACMCCA_222 23(0.10) 187(0.81) 21(0.09) 0.810 19.0 RH10(HxH)
PACMAAT_140 21(0.09) 196(0.84) 16(0.07) 0.841 15.9 RH10(HxH)
EACAMCAA_230 39(0.17) 157(0.68) 35(0.15) 0.680 32.0 RH10B19
EATGMCAC_199 13(0.06) 204(0.92) 4(0.02) 0.923 7.7 RH10B56
EATGMCAG_140.3 0(0.00) 233(1.00) 0(0.00) 1.000 0.0 RH10B64

89
Chapter 6

Table 1. continued
Hetero-
zygosity
[f(a)+f(b)]
*100
Chromosome
& Bin
EACAMCGT_163 20(0.09) 189(0.83) 20(0.09) 0.825 17.5 RH11(HxH)

EACAMCGT_161 20(0.09) 189(0.83) 20(0.09) 0.825 17.5 RH11(HxH)
EACTMCAG_190 2(0.01) 227(0.98) 2(0.01) 0.983 1.7 RH11(HxH)
EACTMCAA_194 39(0.22) 129(0.72) 10(0.06) 0.725 27.5 RH11B01
EACTMCAA_190 31(0.17) 131(0.72) 19(0.10) 0.724 27.6 RH11B01
EACAMCAC_162 13(0.06) 199(0.86) 19(0.08) 0.861 13.9 RH11B17
EACTMCAG_203 5(0.02) 207(0.90) 18(0.08) 0.900 10.0 RH11B21
EAGAMCCT_132.3 4(0.04) 103(0.94) 2(0.02) 0.945 5.5 RH11B61
EACAMCAC_131 19(0.08) 180(0.78) 32(0.14) 0.779 22.1 RH12(HxH)
PACMAAT_306 32(0.14) 190(0.82) 9(0.04) 0.823 17.7 RH12B06
EAACMCCT_231.1 0(0.00) 233(1.00) 0(0.00) 1.000 0.0 RH12B49
* freq indicates allele frequency
Map distance is calculated using the frequency of a homozygous allele times 100 cM genetic distance of each chromosome
indicating genetic distance from the centromere
Chromosome and Bin are indicated. For instance, RH01B10 means the marker belongs to Bin 10 in chromosome 1 of RH.
+
HxH means that the marker is derived from both parents SH and RH in the UHD map.

However, they could be assigned on the most probable location according to the
increasing or decreasing rate of heterozygosity in the 4x-2x map and based on
marker density in the UHD map (Figures 3 and 4). On chromosome 3, the centromere
appeared most probably to be positioned in Bin 35 and on chromosome 11 in Bin 60.
The chromosomes 2 and 12 showed predominantly a terminal location of the
centromere which is the best indicated to be telocentric. All the other positions of the
centromeres were in different degrees metacentric.

Dissection of three chromosomes
In order to investigate the assumption that only one crossover occurs per
chromosome arm, genetic marker data of 233 genotypes of three chromosomes
(chromosome 2, 4 and 6) were arranged by marker order in a spreadsheet (Figure 5).
Those three chromosomes had enough markers to analyze one arm of the telocentric
chromosome 2 and the metacentric chromosome 4 and both arms of the metacentric
chromosome 6. One, two and three markers on chromosomes 2, 4 and 6,
respectively, which were located in the centromeric regions, were all heterozygous
indicating that 2n pollen originated through FDR, but not through SDR mechanism or
a mixture of both. On the telocentric chromosome 2, there was as expected no
marker localized on the north arm. No crossover and only one crossover on the south
arm were observed in 120 genotypes (group a in Figure 5A) and 113 genotypes
(group b in Figure 5A), respectively. On the metacentric chromosomes 4 and 6,
crossover did not occur in 167 and 135 genotypes (group a in Figure 5B and 5C), a
single crossover was observed on one of the chromosome arms in 58 and 93
90

genotypes (group b in Figure 5B and 5C), and eight and five genotypes were found
with two crossovers per chromosome, but with only one crossover per chromosome
arm (group c in Figure 5B and 5C), respectively.

Figure 3. Comparison of the genetic maps of the male parent RH obtained by the 4x-2x cross
population and the UHD mapping population, respectively. The two maps of each chromosome have
been connected to each other by joint markers. The position of the centromeres of each chromosome
is indicated by small gray squares between the two maps.
91
Chapter 6

Figure 4. The distribution of AFLP markers on the RH map of the UHD map (adopted from van Os et
al., submitted). The amount of non-recombining markers in each bin is represented by shades of gray
according to the figure legend. According to the marker density, the potential position of centromeres
was determined. Based on the results in the present study, the accurate position of individual
centromeres is indicated by a bold capital letter I on each chromosome and a regular capital letter I
with an asterisk indicates the expected position of the centromere on chromosome 3 and 11.
92

Figure 5. Marker distribution and graphical genotyping on three chromosomes: Chromosome 2 (A), 4
(B) and 6 (C). Heterozygous (he) and homozygous (ho) markers were formatted by different intense
shades. Bold typed regions are centromeric regions where all markers are heterozygous. The location
indicated is adopted from the UHD map and m.d. is the map distance from the markers to the
centromeric region indicating the frequency of homozygosity. Progenies are classified by the graphical
genotypes and a number of crossovers. In group a, b and c, no crossover, one and two crossovers
occur, respectively.

The frequency of non-crossover for the telocentric chromosome 2 was 120 out
of 233 and for the metacentric chromosomes 4 and 6, 167 and 135, respectively. This
could be an indication that a telocentric chromosome could show more crossover
than a metacentric chromosome. However, testing of the hypothesis of no difference
in crossover frequency using the
2
-test did not show a significant difference for non-
93
Chapter 6

crossover in the three chromosomes 2, 4 and 6 (data not shown) indicating that the
crossover frequency for these chromosomes was essentially the same.
The observation on the single arms of all three chromosomes showed total
interference indicating that a second crossover is prevented. The frequency of two
crossovers per chromosome could be tested in chromosomes 4 and 6 where 8 and 5
occasions were found, respectively. The number in both chromosomes was
accordingly to the expectation on the basis of two independent events (data not
shown).

Discussion

In the UHD map, the centromere positions on the map of the RH parent could
tentatively be determined by using marker density as indicator (Figure 4). van Os et al.
(submitted) observed in the UHD map that AFLP markers were not evenly distributed
over the genetic map. Several centromeric bins contained high numbers of co-
segregating markers, while other regions of the map contained much higher numbers
of recombination events with much less co-segregating markers per bin. It has been
reported that suppression of recombination at a centromere could be 10 to 40-fold
higher than that at the rest of a chromosome (Tanksley et al., 1992; Centola and
Carbon, 1994). The marker density observations of each chromosome of the RH map
have been adopted from van Os et al. (submitted). The bins, which were most dense
in each chromosome and, therefore, candidates for centromeric positions were
indicated by arrows (Figure 4). They were the same as obtained from the present
study where the centromere position on the chromosomes of RH was identified by
using half-tetrad analysis in a 4x-2x cross population (Figure 3). In the present study,
it was also proven that normally only one crossover occurs per chromosome arm as
proposed by van Veen and Hawley (2003) and Hillers and Villeneuve (2003). This
confirms the earlier results in which RFLP analysis was used for localizing
centromeres by 2x-4x populations in which 2n eggs originated exclusively through
SDR (Bastiaanssen, 1997). van Veen and Hawley (2003) and Hillers and Villeneuve
(2003) suggested that crossover interference could act over large distances along the
length of meiotic chromosomes to limit the number of exchanges but the crossover
interference signal could not be transmitted through the centromere or telomere. Our
observations support both suggestions, the occurrence of a second crossover per
chromosome arm was prevented and the number of simultaneous crossovers in both
chromosome arms was occurring at the expected frequency (Figure 5).
The first half-tetrad analysis was performed in attached-X chromosomes in
Drosophila (Beadle and Emerson, 1935). In the last decades, gene or genetic marker
related centromere positions, called gene-centromere mapping, have been identified
using half-tetrad analysis in some plants (Mendiburu and Peloquin, 1979; Douches
94

and Quieos, 1987; Wagenvoort and Zimnoch-Guzowska, 1992; Lindner et al., 2000),
fishes (Liu et al., 1992; J ohnson et al., 1996) and animals (J arrell et al., 1995; Baudry
et al., 2004). However, access to several products of the same meiosis is
indispensable to map centromeres. The number of species where centromeres can
be genetically mapped, therefore, is relatively limited (Baudry et al., 2004). In half-
tetrad analysis using progenies created from a 4x-2x cross, the male parent produces
2n pollen resulting in tetraploid and not in triploid progenies because of the existence
of a so-called triploid block (Marks, 1966; Peloquin et al., 1989). In potato it has been
suggested that because of the type of meiosis SDR 2n egg cells should be
predominant under normal synaptic conditions and transfer a high degree of
homozygosity to the progeny, whereas the occurrence of FDR 2n eggs is an
exception (J ongedijk, 1985; Douches and Quiros, 1988; J ongedijk et al., 1991;
Werner and Peloquin, 1991). In contrast, FDR 2n pollen in synaptic diploids should
prevail and transfer a high degree of heterozygosity to the progeny, whereas the
occurrence of SDR 2n pollen should be excluded (Ramanna, 1983; Peloquin et al.,
1989; Watanabe and Peloquin, 1993). Therefore, FDR was considered as a
mechanism to produce unreduced 2n gametes via 2n pollen and tetraploid progeny in
a 4x-2x cross of potato. Depending on the percentage of heterozygosity of the
gametes at certain AFLP loci, the centromere position of each chromosome could be
localized. The position of 100 % heterozygous AFLP loci, where heterozygous
gametes were transmitted from the male parent to all of its progeny of the population,
was determined as the position of the centromere. In this case, all tetraploid
progenies had a simplex genotype. This was also an indication for the occurrence of
only FDR gametes.
The centromere is one of the most important functional elements of eukaryotic
chromosomes. It ensures proper cell division and stable transmission of the genetic
material (Wang et al., 2000). Elucidating the composition and structure of
centromeres can be of importance to understand its functional roles including
chromosome segregation, karyotypic stability and artificial chromosome-based
cloning (Wu et al., 2004). Centromeres of higher eukaryotes are composed of densely
methylated, recombination suppressed and cytologically constricted DNA. Its region
consists of tandemly repeated DNA surrounding transposons, retroelements and
pseudogenes (Hall et al., 2004; Houben and Schubert, 2003). Recently, centromeres
were sequenced and studied in several plant species including Arabidopsis
(Copenhaver et al., 1999; Kumekawa et al., 2000; Kumekawa et al., 2001; Hosouchi
et al., 2002) and main crops such as Maize (Nagaki et al., 2003; J in et al., 2004), Rice
(Wu et al., 2004) and Wheat (Kishii et al., 2001). However, a similar study has not
been reported in potato. Although centromere functions are highly conserved, the
sequence among the centromeres of related species is not homologues (Hall et al.,
2004).
95
Chapter 6

Identification of the genetic position of centromeres, which is important for
distinguishing chromosome arms, identifying proximal and distal markers or genes
and providing fixed positions in genetic maps (Bastiaanssen et al., 1996), is the first
step to understand the composition and structure of the centromeric region. In the
present research, we localized centromeres of most chromosomes of potato by half-
tetrad analysis and we could confirm them with those indicated in the UHD map (van
Os et al., submitted). This proves that 1. the marker density approach in ultra high
density maps can be used for positioning of centromeres and 2. half-tetrad analysis in
potato is powerful for the same purpose. The identification of the accurate genetic
position of centromeres described in this paper is a good starting point for future
research on the construction of physical contigs of centromeric regions as well as for
further research in sequencing and analyzing centromeres.

Acknowledgement

We thank Dr. Munikote Ramanna for critical reviewing the manuscript.

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99
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100
Chapter 7

General discussion

Chapter 7

Identification of the resistance genes

In potato breeding, resistance to late blight caused by Phytophthora infestans is one
of the most important traits. To introduce resistance into the cultivated potato,
identification of resistance is the first essential step. In this research, we identified
three different foliage resistance (R) genes derived from different sources. All these R
loci were localized on chromosome 4. High-resolution maps of the R loci were
required to be constructed to find markers closely linked to or co-segregating with the
R loci for marker assisted selection and for map based gene cloning. In Chapter 3, a
broad-spectrum R gene termed Rpi-abpt, which is most probably from the wild
species Solanum bulbocastanum, was identified. The Rpi-abpt locus co-segregated
with one of the AFLP markers that was converted into a PCR based SCAR marker
and flanked two AFLP markers in a 0.5 cM interval. In Chapter 4, a race-specific R
gene termed R2-like that is phenotypically indistinguishable from R2 but most
probably not derived from S. demissum was identified. The R2-like locus co-
segregated with four AFLP markers and flanked with other AFLP markers in a 0.4 cM
interval. In Chapter 5, another S. bulbocastanum-derived R gene named Rpi-blb3
was identified. The Rpi-blb3 locus co-segregated with one AFLP marker and flanked
two AFLP markers in a 0.9 cM interval.
The three R genes are conferring resistance to late blight, are located on the
same region of chromosome 4 and could belong to the same R gene cluster.
However, they differ from each other. We investigated the origin of the R genes using
pedigree information, race-specificity and AFLP marker assessment. According to the
pedigree information, we could identify the origin of only Rpi-blb3, because the
mapping population for this gene is an intraspecific hybrid and all genotypes still exist
(Chapter 5). The origin of the two other genes, Rpi-abpt and R2-like, could not
unambiguously be confirmed using the pedigree information, because the pedigrees
of the two populations are complex and not all genotypes involved in the pedigrees
are available (Chapters 3 and 4). However, the experiments of race-specificity and
AFLP marker assay supported the conclusion as to the origin of the R genes. Both
Rpi-blb3 and Rpi-apbt originate from the wild species S. bulbocastanum, but they
differ from each other. The wild species accessions involved in pedigrees of the
mapping populations for the two R genes are different. Another evidence is the race-
specificity. Rpi-blb3 conferred resistance to isolate IPO-655-2A, but confer partial
resistance to isolate IPO-82001. Rpi-abpt showed complete resistance to the isolate
IPO-82001 (Chapters 3 and 5). The origin of R2 is S. demissum (Li et al., 1998) and
the introgression fragment of the R2-like gene that is phenotypically indistinguishable
from R2 could be of wild species origin. S. edinense, S. tuberosum subsp. andigena
and S. vernei, which were involved in the pedigree of the mapping population, could
102
General discussion

be the source for this R gene (Chapter 4). S. edinense, a natural hybrid between S.
tuberosum and S. demissum, and the other wild species are known to contain late
blight resistance (Hawkes, 1990). The true origin and identity of R2-like will only be
proven if R2-like and R2 are cloned and compared.
To date, most breeding efforts for late blight are made in foliage. However,
tuber late blight is also of importance. Potato tubers can be diseased from sporangia
that are washed down from the infected potato foliage into the soil. The pathogen can
be transmitted from one season to the next, and the pathogen can lead to rotting of
the potato tubers during storage. Therefore, we also investigated foliage as well as
tuber blight resistance in four mapping populations and studied whether foliage or
tuber specific resistance occurred (Chapter 2). R1 and a QTL derived from S.
microdontum were observed to be active in both tuber and foliage. Interestingly Rpi-
abpt and R3a appeared to be foliage specific R genes. The R3 resistance in the
SHRH population was active in both foliage and tuber, but this could be explained by
R3b or by allelic variation as proposed by Hulbert et al. (2001), Huang et al. (2004)
and Huang (2005).

Comparative genetics

Comparative genetics can be used for the analysis of linkage groups. In this research,
we localized R loci using several reference maps. For instance, the Rpi-abpt locus
(Chapter 3) was mapped on chromosome 4 according to the position of a PCR
marker developed from the co-segregating AFLP marker in the ultra high density
(UHD) map (van Os et al., submitted) and AFLP bridge markers with those in the R2
map (Li et al., 1998). The position of the R2-like locus (Chapter 4) was also found
using the AFLP bridge markers, which are present in the R2 map (Li et al., 1998). In
addition to these, since the R loci were expected to be clustered at the same region
on chromosome 4, screening each mapping population with the co-segregating AFLP
markers developed in the other mapping studies allowed to study the syntenic
relationship among four late blight resistance loci, Rpi-abpt (Chapter 3), R2-like
(Chapter 4), Rpi-blb3 (Chapter 5) and R2 (Li et al., 1998). See also Figure 5 in
Chapter 5.
Another potential of comparative genetics is the rapid identification of R genes
similar to those already mapped in related genera, based on the conserved position
of R loci and similar function and specificity of these R loci for the same or related
pathogens (Grube et al., 2000). In total 12 R gene clusters were identified in two or
more genera. However, only two of them had specificity for Phytophthora at the
corresponding positions in different genera, Solanum and Capcisum. The two
103
Chapter 7

positions are chromosome 4 and 11 where two major R gene clusters for P. infestans
in potato and QTLs for P. capsici in pepper (Lefebvre and Palloix, 1996) are located.

R gene cluster and allelic diversity

We identified three different R genes in foliage against P. infestans which we named
Rpi-abpt (Chapter 3), R2-like (Chapter 4) and Rpi-blb3 (Chapter 5) originating from
different sources. According to the comparative genetic study, these genes are
located on the same location on chromosome 4 as the S. demissum-derived R gene
R2 is (Li et al., 1998). This location on chromosome 4 seems to be a hot spot of R
genes or to be an R gene cluster.
In plant species, most characterized R genes are clustered (reviewed by
Gebhart and Valkonen, 2001 and Hulbert et al., 2001). It is often reported that the R
genes in the same clusters confer resistance to the same pathogen, but different
pathotypes. For instance, three rust R genes were mapped to the complex N locus of
flax (Dodds et al., 2001) and at least 10 Dm genes conferring resistance to downy
mildew were mapped to the major R cluster in lettuce (Meyer et al., 2001). However,
R genes for resistance to different pathogens are also clustered within an individual
plant species (de J ong et al., 1997; Ashfield et al., 1998; Speulman et al., 1998).
When literature was reviewed for R gene clusters in potato, 12 disease R gene
clusters on 10 potato chromosomes are apparent (Table 1). Two major R gene
clusters for late blight resistance are present on chromosome 4 and 11. In view of the
high homology among species in the Solanaceae family, the R gene clusters can be
extended to other solanaceous genera. Grube et al. (2000) made a comparative
analysis of genomic organization of R genes in Solanaceae and R gene homologues.
In total, 18 Solanaceae R gene clusters were defined by the presence of two or more
R genes within 15 cM from one or more solanaceous genera.
When the R gene clustering region on chromosome 4 that we identified is
investigated in more detail, the region seems to contain a wide range of R gene
clusters. At least four R genes Rpi-abpt, R2-like and Rpi-blb3 (this thesis) and R2 (Li
et al., 1998) against P. infestans, Ny
tbr
for potato virus Y (Celebi-Toprak et al., 2002)
and QTLs against P. infestans, E. carotovora subsp. atroseptica and G. pallida
(Leonards-Schippers et al., 1994; Oberhagemann et al., 1999; Zimnoch-Guzowska et
al., 2000; Bradshaw et al., 1998) are located in this region (Table 1). If we include
tomato R genes found in this region like Hero conferring resistance against G.
rostochiensis (Ganal et al., 1995; Ernst et al., 2002), then there are nine members in
this R gene cluster.
R genes that cluster in the same region could be the result of allelic variation
created by mutation and recombination between alleles (reviewed by Hulbert et al.,
104
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General discussion

2001). As we observed that the R3 locus in two different populations conferred
different specific resistance in foliage and tuber (Chapter 2), it is tempting to
speculate that this is due to allelic diversity. The R3 locus consists of two functionally
distinct R genes, R3a and R3b for foliage blight resistance (Huang et al., 2004). Also
the differentials R5-R11 all contain allelic versions of the R3 locus, and these
functionally diverse R3 allelic versions interact with P. infestans in a gene-for-gene
manner (Huang, 2005). The allelic diversity could be the result of the allelic variation
of R genes occurring to generate new R genes as the pathogen acquires new
virulence alleles (reviewed by Hulbert et al., 2001). Therefore the four R genes we
compared in this study are expected to be allelic and the isolation of one of the R
genes and characterization of this R gene could facilitate the isolation of the other
resistance alleles.

Resistance breeding to P. infestans in potato

The resistance to P. infestans causing the severe disease late blight is one of most
important targets in breeding potato. Due to the pathogen that destroys the potato
crop, crop production is reduced and a lot of money is needed to control the pathogen.
Even though fungicides are still needed for the control of potato late blight,
introduction of R genes into potato is the most promising method to combat the
disease and enable a more durable resistant potato culture.
The cultivated potato (Solanum tuberosum) originates from the Andean region
of South America where many other species of the genus Solanum occur. The
utilization of those Solanum species could broaden the genetic base of the cultivated
potatoes and facilitate the introgression of new R genes into the cultivated potato
(Hermunstad and Peloquin 1985). Late blight resistance has been reported to be
found in S. acaule, S. chacoense, S. berthaultii, S. brevidens, S. bulbocastanum, S.
demissum, S. microdontum, S. sparsipilum, S. spegazzinii, S., stoloniferum, S.
sucrense, S. toralapanum, S. vernei and S. verrucosum (Hawkes, 1990). However,
approximately 75 % of the species have a diploid constitution (2n =2x =24) (Hawkes,
1990), while the majority of the cultivated potatoes is tetraploid (2n =4x =48). To
overcome the difficulties to use those wild species in potato breeding that are caused
by ploidy difference, chromosome doubling can be accomplished by making use of 2n
gametes (meiotic doubling) and colchicine treatment or in vitro explant culture (mitotic
doubling). In addition to these techniques, somatic hybridization and gene
transformation are also important methods to introgress desirable genes into the
cultivated potato in breeding (Hermsen, 1994).
In this research, we used three different populations containing R genes which
have different origins. To create those populations, different breeding methods such
107
Chapter 7

as meiotic doubling and mitotic doubling were applied with the final aim to lead to
gene identification and gene cloning. After gene isolation, gene transformation
techniques should be used to improve resistance to P. infestans in the cultivated
potato.

Chromosome doubling in potato breeding
The cultivated potato, Solanum tuberosum, is tetraploid (2n =4x =48) and shows
tetrasomic inheritance. However, diploid (2n =2x =24) potatoes are often used in
potato breeding programs, because many wild species, which include useful
characteristics such as disease resistance, are diploid and the genetic study of the
tetraploid potato is much more complicated than that of the diploid potato. Additionally,
breeding at the 2x level may shorten the time required to produce a new variety, more
rapidly eliminate deleterious recessive alleles and enable a more efficient introduction
of desired characters from 2x species (Ortiz and Peloquin, 1994; Hutten, 1994). The
tetraploid level can be restored by using different methods such as the use of 2n
gametes and colchicine treatment. For instance, the RH4X-103 population we used
(Chapter 2, 3 and 6) is tetraploid, but diploid wild species and diploid potato clones
were involved to create this population. In the process of developing ABPT clones,
the AB clone (3x), which was derived from a cross between tetraploid wild species S.
acaule and diploid wild species S. bulbocastanum, was treated with colchicine (Figure
1a in Chapter 3). 2n gametes were also used in the RH4X-103 population which is a
4x-2x cross-derived population. The female parent 707TG11-1 is tetraploid and the
male parent RH89-039-16, which produces 2n gametes, is diploid (Figure 1b in
Chapter 3).
In the Solanaceae family, unreduced 2n gametes commonly occur (Carputo et
al., 2000). The discovery that 2x potato can produce 2n gametes has allowed various
breeding approaches to synthesize highly heterozygous 4x clones and these
breeding methods have led to introgress valuable traits from related species to the
tetraploid potato. Therefore, those breeding methods have been used extensively to
introduce resistance against several pathogens such as bacterial wilt (Pseudomonas
solanacearum), late blight (Phytophthora infestans) and potato cyst nematode
(Globodera pallida) from diploid Solanum species to the cultivated tetraploid potato
through 4x-2x and 2x-2x crosses (Werner and Peloquin, 1991; Ortiz et al., 1997;
Watanabe et al., 1999). Mating between 4x and 2x parents gives rise to almost
complete 4x progeny due to the triploid block mechanism (Marks, 1966). Mating
between 2x parents produces both 2x and 4x progeny. In potatoes, first division
restitution (FDR) and second division restitution (SDR) are the two important modes
of 2n gametes formation (Tai, 1994). Watanabe et al. (1991) investigated the genetic
consequences for different populations of tetraploid progenies that were produced by
heterozygous diploid populations through breeding methods with the use of 2n
108
General discussion

gametes. About 80 % (FDR) and 40 % (SDR) of the parental heterozygosity was
transmitted to their progenies by 2n gametes. The breeding value of both types of 2n
gametes have also been investigated in different interploidy crosses and it was
concluded that the breeding value of FDR 2n gametes is superior to that of SDR 2n
gametes (reviewed by Douches and Maas, 1998).
In addition to the breeding aspect, these 2n gametes can be used to identify
the position of the centromere on chromosomes. It is of importance to know the
chromosome arms, to identify proximal and distal markers or genes and to provide
fixed positions in genetic maps (Bastiaanssen et al., 1996). The centromeric regions
can be determined according to the rate of heterozygosity and homozygosity. In FDR,
if the heterozygosity of an allele increases to 100 %, the allele is getting closer to the
centromere, but if it decreases to 50 %, the allele is getting closer to the telomere. In
SDR, if the heterozygosity of an allele is 0 %, the allele is located on the centromere
but if it is 100 %, the allele is on the telomere (Chapter 6). By using this analysis
method, we localized the centromeres on all 12 potato chromosomes. Another
possibility for positioning of the centromere is to make use of the density of AFLP
markers in genetic linkage maps. AFLP markers tend to be clustered in centromeric
regions. 51 % of the AFLP markers were clustered in the centromeric region in barley
(Qi et al., 1998). Similarly Keim et al. (1997) and Alonso-Blanco et al. (1998) reported
a clustering of AFLP markers in soybean and in Arabidopsis, respectively. This could
be caused by the fact that crossover is preferentially located at the tips of the
chromosomes indicating fewer loci become homozygous per recombination event
(Baudry et al., 2004). In potato, centromeres were tentatively determined in the ultra
high density map (UHD map; van Os et al., submitted). These results are identical
with the results obtained by half-tetrad analysis (Chapter 6).

Somatic hybridization and transformation for rapid introgression
In addition to classical breeding methods, genetic engineering methods will allow
plant breeders to introduce desirable genes into cultivars that are outside
conventional and sexual hybridization (crossing) techniques and will complement
traditional breeding efforts.
Somatic hybridization provides possibilities to introduce valuable genes from
sexually incompatible wild species into cultivated plants (Umaerus and Umaerus,
1994). Helgeson (1992) and Mattheij et al. (1992) reported the potential of somatic
hybridization between some recalcitrant species (S. brevidens, S. bulbocastanum, S.
circaeifolium and S. polyadenium) and S. tuberosum cultivars. Some of the somatic
hybrids have the ability to sexually backcross to cultivars that will allow further
breeding. Introgression of resistance to PLRV and Erwinia soft rot could be
demonstrated in the backcross population derived from somatic hybrids (Helgeson
and Williams, 1991). For late blight resistance, somatic fusion approaches were also
109
Chapter 7

reported. Mattheij et al. (1992) combined S. circaeifolium with S. tuberosum to
introduce late blight resistance and found that some fertile resistant progenies were
produced. Other achievements were made by Helgeson et al. (1998) and Horsman
(2001). Helgeson et al. (1998) produced somatic hybrids between tetraploid potato
and diploid S. bulbocastanum, which is extremely difficult to cross with potato, with
the aim to transfer late blight resistance. Sexual backcrosses of those somatic hybrids
were easily obtained and late blight resistance was successfully transferred to their
progenies. Horsman (2001) also generated somatic hybrids between S. tuberosum
and S. nigrum that contains resistance to P. infestans (Colon et al., 1993;
Vleeshouwers et al., 2000).
Genetic transformation is another possibility for introgressing R genes from
wild species to cultivated potato. This method is potentially the shortest procedure for
transferring genes into susceptible potato without sexual hybridization problems and
the transformation efficiency in potato is relatively high using Agrobacterium-mediated
transformation techniques (Hermsen, 1994). However, it has several barriers, which
have to be overcome previous to gene transformation. First of all, targeted genes for
resistance should be isolated from the plants that contain these R genes.
Furthermore the ability of transformation and regeneration is of importance and a
sufficient number of transformants is needed for selection of transformants, which are
identical to the untransformed clones combined with R gene (Hermsen, 1994). To
date, four R genes against P. infestans have been isolated. R1 (Ballvora et al., 2002)
and R3a (Huang, 2005) that are of S. demissum origin, and Rpi-blb1 (van der Vossen
et al., 2003) / RB (Song et al., 2003) and Rpi-blb2 (van der Vossen et al., 2004) that
are of S. bulbocastanum origin were cloned. These R genes were reported to be
successfully transformed to susceptible cultivars making them resistant to the
respective P. infestans strain. With the R genes we identified in this thesis, similar
approaches will be followed after isolation of the R genes.

Future research

As mentioned in the general introduction, the main goal of this research was the
identification, mapping and isolation of R genes against P. infestans in potato. We
have accurately localized three new R genes Rpi-abpt (Chapter 3), R2-like (Chapter
4) and Rpi-blb3 (Chapter 5) and constructed high-resolution maps. These maps
containing AFLP and PCR-based markers, which are flanking or co-segregating with
the R loci, can be used for map-based gene cloning. Additionally, the R2 gene, which
is a member of the R gene cluster on chromosome 4, will be included in further gene
cloning studies. The process of map-based gene cloning is now in progress to isolate
those R genes. For Rpi-abpt, we have already selected a single BAC clone containing
flanking markers and a co-segregating marker indicating that the BAC clone spans
110
General discussion

the R gene. This BAC clone is now being sequenced. For Rpi-blb3, the construction
of a BAC library has been completed and we are now developing PCR based flanking
markers and selecting a single BAC clone, which could include the R gene. For R2-
like and R2, the BAC libraries are in development. The four R genes are members of
the same R gene cluster and their positions are comparable. These R loci have
different genetic haplotypes, but the structure of the R genes could be similar.
Therefore, the selection of a single BAC clone that contains candidate R genes from
any of these BAC libraries could facilitate the isolation of the other R genes. The
BAC-end sequences of the selected BAC clone can be used for marker development.
Subsequently those markers can be localized on the other maps and used for
selection of other BAC clones that contains other candidate R genes. When the
selected BAC clones include several R gene homologues, these can be used for
gene isolation by the candidate gene approach. After cloning the R genes, the
sequence analysis of the R genes in the same R gene cluster might allow to
understand the evolution of the R genes.

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115
Chapter 7

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st
Solanaceae Genome
116
General discussion

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117
Chapter 7

118
Summary

Summary

Potato (Solanum tuberosum L.) is one of the most important crops in the world. The
oomycete Phytophthora infestans (Mont. de Bary) is the causal agent of late blight
which is the most devastating disease of the cultivated potato. It causes economic
losses of several billion US dollars in crop production and protection annually
worldwide. To control this disease, the application of enormous amounts of chemicals
is required, which is expensive and environmentally unsafe. Also, late blight is
increasingly more difficult to control because the pathogen is developing tolerance to
the chemicals and new strains of the pathogen are evolving. Therefore, introduction
of resistance genes from wild Solanum species into potato cultivars is considered as
the most promising and environmental-safe approach to achieve durable late blight
resistance.
Late blight resistance exists in a wide range of Solanum wild species of which
75 % have a diploid constitution (2n=2x=24) although the majority of the cultivated
potato is tetraploid (2n=4x=48). Therefore, several different approaches such as the
use of 2n gametes, colchicine treatment, intraspecific hybridization, somatic
hybridization, embryo rescue and gene transformation are used to overcome the
difficulties caused by different ploidy levels. Of these approaches, gene
transformation is the most controlled and time-efficient method to introduce resistance
into the cultivated potato. However, gene identification and gene isolation are the first
prerequisites for this approach.
During the last century, most effort for late blight resistance was focused on
the wild species S. demissum which contains at least 11 race-specific resistance
genes (R1-R11). However, these resistances were rapidly overcome by new virulent
pathogens which evolved. Therefore, new sources of late blight resistance are
required and several resistances from different wild species such as S. microdontum,
S. bulbocastanum, S. berthaultii and S. pinnatisectum were characterized. In the
present research, we investigated both foliage and tuber blight resistances in different
mapping populations derived from different sources of Solanum species.
The pathogen P. infestans can infect both foliage and tuber of potato. To
study tuber blight resistance, a wounded tuber assay was developed and the
correlation between tuber and foliage blight resistance in four different mapping
populations CE, SHRH, RH94-076 and RH4X-103, in which foliage blight resistances
were identified, was examined (Chapter 2). Tuber blight resistance was found to be
correlated with foliage blight resistance in three populations CE, SHRH and RH94-
076, while it was independent in the RH4X-103 population. Further investigation to
explore late blight resistances using molecular markers and resistance assays on
both foliage and tuber with four different isolates was focused on the RH4X-103
population. Three specific resistance genes in foliage, tuber or both were segregating.
The tuber blight resistance locus was closely linked to marker GP179 and co-
segregating with the R1-derived marker 76_2s on chromosome 5. The R1 gene
120
Summary

functioned both as a foliage and tuber blight R gene, whereas the Rpi-abpt and R3a
genes acted only on foliage.
S. bulbocastanum is one of the most promising wild Solanum species
possessing late blight resistance. ABPT material, which is a quadruple hybrid created
by bridge crosses using the four Solanum species; S. acaule, S. bulbocastanum, S.
phureja and S. tuberosum, to overcome incompatibility between S. bulbocastanum
and S. tuberosum were used to introduce late blight resistance. A tetraploid mapping
population (RH4X-103) containing the late blight resistance gene Rpi-abpt was
created by a 4x-2x cross and was originally derived from ABPT material (Chapter 3).
Resistance assay and AFLP marker analysis allowed localization of the Rpi-abpt
locus on chromosome 4. The origin of the resistance was confirmed by the analysis of
Rpi-abpt locus-linked AFLP markers and the resistance assay of ABPT-related wild
species accessions. An extended population of 1383 offspring was screened to
construct a high-resolution map where the Rpi-abpt locus was consequently localized
in a 0.5 cM interval between flanking AFLP markers and was co-segregating with one
AFLP marker. The translated protein sequence of the co-segregating AFLP marker
appeared to be highly homologues to several disease resistance proteins.
The investigation of the race-specific R2-like gene was performed in a diploid
mapping population (Chapter 4) primarily used for mapping nematode resistance. R2-
like was phenotypically indistinguishable from the S. demissum-derived R2 gene
although S. demissum is not directly involved in the pedigree of the population and
R2-like is expected to originate from another species. The R2-like locus was placed in
a 0.4 cM interval, flanked by two AFLP markers on chromosome 4 using the extended
population of 1586 offspring. Four AFLP markers were also identified to be co-
segregating with the R2-like locus.
A further late blight R gene (Rpi-blb3) on chromosome 4 was identified in an
intraspecific hybrid population derived from S. bulbocastanum (Chapter 5). The Rpi-
blb3 locus was localized in a 0.9 cM interval between flanking AFLP markers and co-
segregated with one AFLP marker in a population of 1396 offspring. According to the
results of AFLP marker allele similarity tested by all co-segregating markers with four
different R genes Rpi-abpt, R2-like, Rpi-blb3 and R2, the four R genes appeared to
be genetically very close and could be members of the same R gene cluster although
the four R genes showed different race-specificities and origins.
Additional interesting scientific results came from the 4x-2x population (RH4X-
103) used for late blight resistance. In this population, the male parent produced
unreduced 2n pollen by FDR (First Division Restitution), allowing the localization of
centromeres by using half-tetrad analysis (Chapter 6). 130 male parent-derived AFLP
markers were analyzed and the genetic linkage group and location of 95 AFLP
markers was determined by comparing them with the reference markers of the ultra
high density (UHD) map of potato. Depending on the heterozygosity (simplex) or
121
Summary

homozygosity (nulliplex or duplex) of the AFLP markers of the offspring, the position
of centromeres and the genetic distance of the AFLP markers from the centromere
were determined. In FDR 2n pollen, all the parental heterozygous loci from
centromere to first crossover are expected to be heterozygous (Aa) resulting in
simplex genotype (Aaaa) of their offspring, when they are combined with homozygous
recessive gametes (aa) from the female parent. In contrast, a single crossover
between the locus and centromere releases 50 % homozygous and 50 %
heterozygous gametes in FDR. The centromeres of 10 chromosomes were accurately
localized. The centromere positions were compared with those, which were putatively
identified in the UHD map by the fact that markers are dense due to suppression of
recombination, and proved to be identical. The centromere position of two
chromosomes could not precisely be determined by the half-tetrad analysis approach
due to the absence of the 100 % heterozygous markers. However, their positions
could be inferred based on the combination of the results between the increasing or
decreasing rate of heterozygosity in the half-tetrad analysis and marker density in the
UHD map. Additional investigation of three chromosomes with sufficient numbers of
markers proved that only one crossover occurs per chromosome arm, as a result of
interference of recombination via centromere and telomere.
In conclusion, three different R genes against P. infestans in foliage were
identified and the loci were accurately determined. These R loci are genetically very
close and could be allelic. Our research for tuber blight resistance showed that R
genes are not always active in both foliage and tuber. The results of the research
described in this thesis demonstrate a great potential for further research. This will be
directed towards the isolation of the R genes which can be used to introduce
resistance into cultivated potato and to explore tuber blight resistance using a genetic
transformation approach.

122
Samenvatting

Samenvatting

Aardappel (Solanum tuberosum L.) is n van de belangrijkste gewassen op de
wereld. De omyceet Phytophthora infestans is de veroorzaker van de meest
ingrijpende ziekte die bekend is in de aardappelteelt, namelijk de aardappelziekte.
Deze ziekte veroorzaakt wereldwijd grote economische schade tot in de orde van
enkele miljarden Euro's per jaar. Deze kosten hebben betrekking op het toepassen
van gewasbeschermingsmiddelen en op productieverliezen. Om deze ziekte
voldoende onder controle te houden moeten boeren tot wel 14 keer per seizoen
gewasbeschermingsmiddelen spuiten, wat duur is en milieubelastend. Vanwege
toenemende tolerantie van deze ziekteverwekker tegen de gebruikte middelen door
het ontstaan van nieuwe pathotypen wordt het steeds moeilijker de ziekte effectief te
bestrijden. Het toepassen van resistentiegenen (R genen) vanuit wilde Solanum
soorten lijkt daarom de meest belovende en meest milieuvriendelijke benadering om
via een resistentiestrategie te komen tot duurzame vormen van bescherming tegen
deze ziekte.
Resistentie tegen Phytophthora komt voor in veel wilde Solanum soorten,
waarvan er 75 % diploid zijn, terwijl de meerderheid van de gecultiveerde aardappel
tetraploid is. Verschillende methodieken, zoals het gebruik van 2n gameten,
colchicine behandeling, intraspecifieke hybridisatie, somatische hybridisatie, embryo
rescue en genetische modificatie zijn manieren om de problemen die veroorzaakt
worden bij het kruisen te overkomen. Van deze genoemde benaderingen is
genetische modificatie in principe de meest gecontroleerde en efficinte methode om
resistentie in de cultuuraardappel te introduceren. Hiervoor zijn echter wel eerst
gedentificeerde en moleculair gesoleerde genen nodig.
In de vorige eeuw was de meeste aandacht voor P-infestans resistentie gericht
op 11 genen afkomstig van de wilde soort Solanum demissum. Deze resistenties
werden echter snel door het pathogeen doorbroken. Daarom, waren nieuwe
resistenties uit andere wilde soorten nodig zoals gekarakteriseerd in S. pinnatisectum,
S. microdontum, S. bulbocastanum, S. berthaulthii.
Phytophthora kan zowel bladeren als knollen infecteren. Een knol assay werd
ontwikkeld, teneinde knolresistentie te bestuderen en de correlatie tussen knol- en
bladresistentie in vier verschillende populaties (CE, SHRH, RH94-076 en RH4X-103)
en hun ouders vast te stellen (Hoofdstuk 2). In drie populaties was knolresistentie
gecorreleerd aan bladresistentie, terwijl het onafhankelijk was in n populatie
(RH4X-103). Deze populatie is verder gekarakteriseerd, zowel fenotypisch als
moleculair. Drie R genen, actief in blad, knol of beide splitsten hierin uit. De
knolresistentie was nauw gekoppeld met merker GP179 en co-segregeerde met de
R1-specifieke merker 76_2s op chromosoom 5. Het R1 gen gaf zowel resistentie in
blad als in knol, terwijl de twee andere aanwezige genen (Rpi-abpt en R3a) alleen in
blad actief waren.
124
Samenvatting

S. bulbocastanum is n van de meest veelbelovende wilde soorten voor wat
betreft resistentie tegen Phytophthora. ABPT materiaal, een hybride verkregen via
brugkruisingen tussen vier Solanumsoorten (S. acaule, S. bulbocastanum, S. phureja
en S. tuberosum), werd in het verleden ontwikkeld om de resistentie vanuit S.
bulbocastanum in S. tuberosum over te brengen. De tetraploide populatie (RH4X-
103), verkregen uit een 4x-2x kruising , met RH4X als resistente ABPT nakomeling,
werd geanalyseerd via resistentietoetsen en AFLP merkers. Het resistentiegen Rpi-
abpt werd op chromosoom 4 gelocaliseerd en bleek inderdaad afkomstig te zijn van S.
bulbocastanum. Een hoge resolutiekaart werd gemaakt waarbij het Rpi-abpt locus in
een 0.5 cM interval geplaatst kan worden tussen flankerende, splitsende AFLP
merkers en een co-segregerende AFLP merker. De sequentie van deze co-
segregerende merker werd bepaald en deze bleek overeen te komen met eiwitten die
normaal coderen voor ziekteresistentie.
In hoofdstuk 4 wordt fysiospecifieke R2 resistentie in een diploide mapping
populatie beschreven die oorspronkelijk voor het localiseren van andere resistenties
bedoeld was. Echter in de afstamming komt S. demissum niet direct voor en daarom
wordt er hier over een R2-overeenkomstig gen gesproken. Het R2-overeenkomstige
gen was fenotypisch niet onderscheidbaar van het S. demissum R2 gen. Deze locus
werd geplaatst in een 0.4 cM interval op chromosoom 4 waar ook het R2 gen van S.
demissum te vinden is. Vier AFLP merkers co-segregeerden met het R2-
overeenkomstige locus.
Tenslotte werd er op chromosoom 4 een ander resistentiegen gelokaliseerd
dat afkomstig is van een intraspecifieke hybride populatie in S. bulbocatanum
(Hoofdstuk 5). Dit Rpi-blb3 locus werd gelokaliseerd op chromosoom 4 in een 0.9 cM
interval dat geflankeerd is door splitsende AFLP merkers en een co-segregerende
AFLP merker in een populatie met 1396 planten. Uit resultaten verkregen met de
verschillende co-segregerende merkers in de 4 onderzochte populaties bleek, dat 4
verschillende resistentiegenen tegen P. infestans (Rpi-abpt, R2-overeenkomstig, Rpi-
blb3 & R2) genetisch zeer dicht bij elkaar liggen en hoogstwaarschijnlijk leden zijn
van n en hetzelfde resistentie cluster op chromosoom 4, alhoewel hun
fysiospecifiteit en herkomst duidelijk verschillend zijn.
In hoofdstuk 6 worden aanvullend op het resistentieonderzoek resultaten in de
4x-2x populatie (RH4X-103) beschreven die tot mapping van de centromeren van alle
chromosomen heeft geleid. In deze populatie produceerde de mannelijke ouder
ongereduceerd, zogenaamd, 2n pollen via het "First Division Restitution" (FDR)
mechanisme. Dit leidt in 4x-2x kruisingen tot 4x nakomelingen. Hierbij werden 130
AFLP merkers afkomstig van de diploide mannelijke ouder geanalyseerd en 95 ervan
konden met behulp van de eerder genoemde UHD-kaart in bekende
koppelingsgroepen worden geplaatst. Afhankelijk van de hetero (simplex, duplex) of
homozygotie (nulliplex) van de AFLP merkers in de nakomelingen kon de positie van
125
Samenvatting

de centromeren en de genetische afstanden van de AFLP merkers ten opzichte van
de centromeren op de verschillende chromosomen worden bepaald. In FDR 2n pollen
blijven al de heterozygote loci van de diploide ouder, die zich tussen centromeren en
eerste overkruising bevinden, heterozygoot (simplex) in de nakomelingen indien deze
gecombineerd zijn met homozygote (nulliplex) gameten van de moeder. En enkele
overkruising tussen de locus en het centromeer van de vader resulteert in 50%
homozygote en 50 % heterozygote FDR gameten. De centromeren van 10
chromosomen konden op basis van de recombinatiegegevens nauwkeurig worden
bepaald. Van 2 chromosomen konden door het ontbreken van de klasse met 100%
heterozygote merkers, via deze half-tetradenanalyse de centromeren minder
nauwkeurig worden bepaald. Echter de posities van deze 2 centromeren konden zeer
goed benaderd worden door de toe- en afname van de mate van heterozygotie over
het chromosoom in ogenschouw te nemen. De gevonden posities van alle 12
centromeren bleken feilloos overeen te stemmen met de merker-dichtheden die
eerder gevonden waren op de UHD kaart. De positie op het chromosoom met de
meeste niet-splitsende merkers bleek de positie van het centromeer te zijn. Een
bijkomend resultaat van dit onderzoek was dat op chromosomen met voldoende
splitsende merkers per arm slechts n enkele overkruising gevonden werd.
Concluderend kan vastgesteld worden dat er in dit proefschrift 3 nieuwe,
resistentiegenen tegen P. infestans gedentificeerd en genetisch gekarakteriseerd zijn.
Deze R loci zijn genetisch erg dicht bij elkaar gelegen en zijn samen met R2 uit S.
demissum hoogstwaarschijnlijk allelische vormen van elkaar. Het onderzoek aan R
genen in knollen heeft laten zien dat de R genen die in blad actief zijn niet altijd in
knollen werken. Het verdere onderzoek zal zich richten op moleculaire isolatie van de
verschillende R genen teneinde nduidig hun functie en specificiteit te kunnen
vaststellen.

126
()

Korean summary

(Solanum tuberosum L.) .
Phytophthora infestans
.
.
.
, race
.
.
4
75% 2 .
2n , , ,
, .
.
.
11
S. demissum .
.
S. microdontum, S.
bulbocastanum, S. bertaultii, S. pinnatisectum
. Solanum
.
P. infestans .

CE, SHRH, RH94-076,
RH4X-103
(Chapter 2). - CE, SHRH, RH94-076
- RH4X-103
. RH4X-103
isolate
. ,

. 5
GP179 R1 76_2s
. R1
Rpi-abpt R3a
.
S. bulbocastanum
. ABPT S. bulbocastanum S. tuberosum S.
bulbocastanum S. tuberosum
S. acaule, S. bulbocastanum, S. phureja, S. tuberosum
128
Korean summary

. Rpi-abpt 4 RH4X-
103 4 2 ,
ABPT (Chapter 3). Rpi-abpt
AFLP 4
. ABPT
Rpi-abpt AFLP
. 1383
Rpi-abpt AFLP Rpi-abpt
AFLP 0.5 cM
. Rpi-abpt AFLP

.
race R2-like
2 . R2-like
S. demissum
S. demissum R2
, R2-like S. demissum
. 1586
R2-like AFLP
R2-like 0.4
cM.
4 Rpi-blb3
S. bulbocastanum (Chapter 5). Rpi-blb3
1396 AFLP ,
AFLP 0.9 cM.
, Rpi-abpt, R2-like, Rpi-blb3
R2 AFLP ,
,
.
, 4 2
RH4X-103 .
FDR(First Division Restitution) (n) 2n
, half-tetrad
(centromere) . 130 AFLP
, 95 AFLP
(UHD, Ultra High Density)
. AFLP
AFLP
. FDR 2n
,
129
Korean summary

simplex(Aaaa) ,
50% 50%
.
50% nulliplex(aaaa) duplex(AAaa) 50% simplex(Aaaa)
. 10
. AFLP
UHD
.
100%
half-tetrad ,
UHD AFLP
. AFLP

. telomere
.

,
.
.

.

130
Acknowledgements

Acknowledgement

This dissertation presents the results of a PhD research performed in the Laboratory
of Plant Breeding, Department of Plant Sciences, Wageningen University, The
Netherlands for last four years. It would be impossible to complete this work without
the support, encouragement, guidance and help of many people in The Netherlands
and in my home country, Republic of Korea. The past five and half years experience
including the period for my MSc research was one of the most important pages in my
life and several important events occurred during the period.
First of all, I would like to acknowledge my promoters Prof. Richard Visser and
Prof. Evert J acobsen for providing me with the opportunity to do this research project.
They had already given the opportunity before I completed my MSc thesis. It was very
good for me to make future plans for my life at the moment. I also would like to thank
them for all their efforts including financial support, discussion and critical comments
to complete this thesis. Additionally, I am willing to acknowledge Prof. Han-Young
Park, Prof. J ong-Chun Kim, Prof. Ki-Chul Son, Prof. Doo-Hwan Kim and Prof. Sung-
Kwon Hong who is my former professor in Konkuk University, Seoul, Korea for his
encouragement and advice on my life and my research.
I am deeply grateful to other supervisors, Herman van Eck, Vivianne
Vleeshouwers and Edwin van der Vossen. I would like to include J an Draaistra who
was a supervisor for my MSc research. Herman and J an introduced me to potato
genetics and molecular biology. They taught me a lot and they were helpful to me to
get a PhD position. Vivianne and Edwin were my scientific guides. I would specially
like to acknowledge them for their critical comments and suggestions for experiments
and revision for manuscripts. I also would like to thank Sjefke Allefs, J ack Gros, Anne
Sikkema and Marielle Muskens who are working at potato breeding company Agrico
for their co-authorship of a part of this thesis. I thank Dr. Munikote Ramanna for
critical reviewing the manuscript of one of the chapters.
I would like to thank Ronald Hutten, Dirkjan Huigen and Robert Graveland who
provided me with all plant materials for my research. Ronald generated all
populations and did some field work for me. Dirkjan managed all greenhouse works to
generate and maintain plant materials. Robert who is working at potato breeding
company HZPC provided me with tuber materials of one of populations.
I am grateful to all technicians in the Laboratory of Plant Breeding for their
helps to keep my research going well, especially to Fien Meijer and Petra van den
Berg for ordering primers and all materials I needed, to Marian Oortwijn for managing
sequencing order and to Luc suurs for his help related to isotope lab work and
mechanical matter. I also want to thank Elly J anssen and Irma Straatman. I often
bothered them with questions about lab works and chemicals.
I am very thankful to Annie Marchal and Letty Dijker for their help with
administrative matters.
132
Acknowledgements

I want to thank my (ex)roommates Luisa Trindade and Sanwen Huang.
Especially Sanwen was my extra supervisor. I am thankful to Ningwen J iang and
J eroen Werij. I often met them in the office because they were Sanwens students.
They were always smiling and very kind to me. I also would like to thank all of other
lab members in the Laboratory of Plant Breeding.
I would like to thank several persons working at National Institute of Highland
Agriculture, Kwangwon, Korea. It is a major institute for potato research in Korea.
Hyo-Won Seo stayed in Wageningen for four months and helped me. He was partly
involved in my research. J ong-Nam Lee and Kwang-Soo Cho reviewed and revised
Korean summary for this thesis.
All of my family members in Korea have had great concern about the life of
my family in the Netherlands. Especially I am deeply thankful to my parents. I couldnt
complete my study without moral and financial supports of them. I always feel that I
am in debt to them. They are still doing hard works to support me. I also would like to
thank my parents-in-law for encouragement and moral support. I shouldnt forget to
thank my sister Eun-Hee and brother-in law J ong-Bo who also lived in Wageningen.
Both of them helped me a lots for my daily life and my scientific career. I also thank
Sayuri Miyatake and Tetsuro Miyatake. I am feeling that they are almost our family.
That is the reason to include their names in this paragraph. Especially they are the
best friend of J iwon, but sometimes like her uncle or aunt and grandfather or
grandmother (sorry, Sayuri and Tetsuro!! You are still young).
Finally, I should mention my wife and my daughter. During stay in
Wageningen, two of the most important events in my life occurred. I have got married
and had my daughter. I would like to express my special acknowledgement to my
dear wife Dong-Min. Dong-Min!! I thank you for all of your supports and
understanding. You never (maybe only once or twice since our marriage) complained
of little contribution for our family life. The birth of our lovely daughter J iwon brings us
much more pleasure in our life.
Once again, I thank you all who have contributed to the completion of this
thesis and wish you all the best, good healthy and happy life in the future.

Tae-Ho Park

Wageningen, April 2005

133
Curriculum Vitae

Curriculum Vitae

Tae-Ho Park was born on March 16, 1974 in Ulsan, Republic of Korea. After finishing
high school in 1989, he studied at Kon-Kuk University and received a BSc degree in
Horticultural Science in 1999. In the same year, he enrolled as an MSc student of
Wageningen University. He obtained his MSc degree in Crop Sciences in 2001. After
graduation, he could immediately start his PhD research on the project of
Identification and mapping of resistance genes against Phytophthora infestans in
both foliage and tuber of potato. The thesis is the results of four years of research
carried out in the Laboratory of Plant Breeding at Wageningen University.

134

Training and supervision plan of the Graduate School Experimental
Plant Sciences

1. Participation in postgraduate courses and workshops

a. EPS Autumn School Disease Resistance in Plants
b. Written English Course
c. Scientific Writing Course
d. Safe handling with radioactive materials
e. Scientific Artwork Word/Adobe

2. Participation in international meetings

a. 15th Conference of European Association Potato Research & Global
Initiative of Late Blight, Hamburg, Germany (2002), Poster presentation
b. 7
th
Congress of Plant Molecular Biology, Barcelona, Spain (2003), Poster
presentation
c. Crop Functional Genomics 2004, J aeju, Korea (2004), Poster presentation
d. 1
st
Solanaceae Genome Workshop, Wageningen, The Netherlands (2004)

3. Participation in national meetings

a. EPS seminars and other seminars (2001-2005)
b. Annual EPS theme symposia (2002-2004)
c. ALW (Earth and Life Sciences) meetings (2001, 2003 and 2004)

135

136

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Identification, characterization and high-resolution

mapping of resistance genes to Phytophthora

Annealing temperature. For 76_2S, the annealing temperature was 50

CT229 SCAR F: TTGTGAGTGGTGAACTACGGGC

EACTMCAA_197 16(0.09) 158(0.90) 1(0.01) 0.903 9.7 RH01(HxH)

PACMAAT_314 2(0.01) 228(0.99) 1(0.00) 0.987 1.3 RH05(HxH)

EACAMCGT_163 20(0.09) 189(0.83) 20(0.09) 0.825 17.5 RH11(HxH)

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