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Molecular screening for identification of blast


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Indian rice germplasm (Oryza sativa...

Article in Euphytica November 2013


DOI: 10.1007/s10681-013-1024-x

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Euphytica
DOI 10.1007/s10681-013-1024-x

Molecular screening for identification of blast resistance


genes in North East and Eastern Indian rice germplasm
(Oryza sativa L.) with PCR based makers
Jahangir Imam Shamshad Alam
Nimai P. Mandal Mukund Variar

Pratyoosh Shukla

Received: 17 May 2013 / Accepted: 31 October 2013


Springer Science+Business Media Dordrecht 2013

Abstract Molecular screening and genetic diversity specific STS marker, 195R-1, for Pi9 gene produced
of major rice blast resistance (R) genes were deter- positive bands in only two germplasm, Kalchatti and
mined in 32 accessions of rice germplasm from North Bachi thima. The Uniform Blast Nursery (UBN)
East and Eastern India with ten gene based single analysis showed that out of 32, six germplasm was
nucleotide polymorphisms and sequence tagged sites resistant, ten moderately resistant and 16 germplasm
(STS) markers, namely z56592, zt56591, k39512, were susceptible. Presence of Piz-t, Pita/Pita-2 and
k3957, candidate gene marker, Pita3, YL155/YL87, Pi9 gene ensured a resistant reaction in outdoor blast
YL183/YL87, Pb28, 195R-1 which showed close-set nursery whereas germplasm carrying Pib was suscep-
linkage to nine major rice blast resistance (R) genes, tible when present alone. Presence of multiple genes,
Piz, Piz-t, Pik, Pik-p, Pik-h, Pita/Pita-2, Pib and Pi9 however, contributed to slow blasting resistance in the
and one susceptible pita gene. Among the 32 acces- field. These results are useful in identification and
sions, 13 were positive for Piz gene and six for Piz- incorporation of resistant genes from the germplasm
t gene. Six accessions were positive for Pik gene, into elite cultivars through marker assisted selection in
seven for Pik-p and 16 for Pik-h gene. One accession, rice breeding programs.
Atte thima, was positive for three of Pik multiple
genes. Out of 32, only two germplasm, Dudhraj and Keywords Indian rice germplasm  R genes
Nepali dhan, were detected with both Pita3 and screening  Blast resistance  SNP markers 
YL155/YL87 marker for Pita/Pita-2 gene. The Pib Marker assisted selection
gene appeared to be omnipresent and was detected in
31 of 32 germplasm with marker Pb28. The gene Abbreviations
R Resistance
Avr Avirulent
SNPs Single nucleotide polymorphisms
J. Imam  S. Alam  N. P. Mandal  M. Variar STS Sequence tagged sites
Central Rainfed Upland Rice Research Station (CRRI),
UBN Uniform blast nursery
Hazaribagh 825301, Jharkhand, India
e-mail: mukund.variar@gmail.com MR Moderately resistant
S Susceptible
J. Imam  P. Shukla (&) MAS Marker assisted selection
Enzyme Technology and Protein Bioinformatics
NE North East
Laboratory, Department of Microbiology, Maharshi
Dayanand University, Rohtak 124001, Haryana, India SES Standard evaluation system
e-mail: pratyoosh.shukla@gmail.com DAS Days of sowing

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Euphytica

Introduction (4 %) genotypes have been identified and documented


(McCouch et al. 1994; Ballini et al. 2008; Huang et al.
2010; Xiao et al. 2010; Sharma et al. 2012). These R
Rice blast, one of the most destructive diseases
genes are distributed throughout the 12 rice chromo-
affecting rice production worldwide, is caused by the
somes except chromosome 3 (Yang et al. 2009a; Liu
non-obligate filamentous ascomycete Magnaporthe
et al. 2010). Out of them, 22 have been cloned (Pib,
oryzae B. Couch (syn. Magnaporthe grisea). Many
Pita, Pik-h, Pi9, Pi2, Piz-t, Pid2, Pi36, Pi37, Pik-m,
races of the blast fungus are normally present in the
Pit, Pi5, Pid3, pi21, Pb1, Pish, Pik, Pik-p, Pia,
field but some predominate the others suggesting
NLS1,Pi25 and Pi54rh (Wang et al. 1999; Bryan et al.
exclusive clonality of the population (Xia et al. 1993;
2000; Sharma et al. 2005; Qu et al. 2006; Zhou et al.
Valent and Chumley 1994; Yang et al. 2009a, b).
2006; Chen et al. 2006; Liu et al. 2007; Lin et al. 2007;
Resistance to M. oryzae is known to follow a classical
Ashikawa et al. 2008; Hayashi and Yoshida 2009; Lee
gene-for-gene system where a major resistance gene (R
et al. 2009; Shang et al. 2009; Fukuoka et al. 2009;
gene) prevents infection by a race of M. oryzae
Hayashi et al. 2010; Takahashi et al. 2010; Zhai et al.
harboring the corresponding avirulence (Avr) gene
2011; Yuan et al. 2011; Okuyama et al. 2011; Tang
(Flor 1971). Generally R genes are identified in land
et al. 2011; Chen et al. 2011; Das et al. 2012). Of the
races, cultivars or wild rice collections using differen-
several known blast resistance genes, Pi40 has been
tial physiological races of M. oryzae (Tanksley et al.
identified in an indica introgression line that has
1997). Virulence analyses for detection of specific
inherited the resistance gene from an EE genome of
blast resistance genes is time consuming and cumber-
wild species O. australiensis and advanced breeding
some requiring strict control of environmental vari-
lines derived from BC progenies were used to validate
ables. When blast resistance of a cultivar is based on a
9871.T7E and 9871.T7E2b as markers completely
single resistance gene, it can be rapidly overcome by
associated with the Pi40(t) gene (Jeung et al. 2007).
the emergence of compatible races of the pathogen
Like wise Pi9 gene identified in indica rice lines has
(Hittalmani et al. 2000). Conventional breeding pro-
been derived from the BBCC geneome of wild species
gram for introgression of a blast resistance gene into
O. minuta. Most of the resistance genes are race-
commercial cultivars is slow and less efficient because
specific (Mackill and Bonman 1992; Deng et al. 2006).
of multiplicity of races of the pathogen and the
However, it is imperative to identify broad-spectrum
masking effect of R genes when they occur together
blast resistance genes for effective protection against
(Valent et al. 1991; Kang et al. 2001; Wang et al. 2007).
dynamic blast isolates of M. oryzae. Highly adaptive
With fine mapping and cloning of many blast resis-
virulent isolates/races of the pathogen often challenge
tance genes, many PCR-based markers have been
the effectiveness of deployed R genes and thus urge
developed to screen and identify different blast resis-
the need for the positive screening and identification of
tance genes. DNA markers overcome the constraints of
different blast R genes in the germplasm collection
virulence analysis, and represent a significant advan-
(Wang et al. 2010). Identification and isolation of
tage for increasing the precision of identification and
additional host resistance genes (R) and pathogen
incorporation of blast resistance genes in a breeding
avirulence (Avr) genes is now required to deepen
program (Jia et al. 2003; Wang et al. 2007; Liu et al.
understanding of molecular mechanisms involved in
2013). DNA markers closely linked to a blast R gene
the host-pathogen interaction (Kiyosawa et al. 1986;
that confers resistance to a particular race of the
Valent 1990; Inukai et al. 1994) and strategic deploy-
pathogen can be effectively employed for MAS, which
ment of resistance genes in commercial cultivars.
is much faster than traditional pathogenicity assays.
The existence of genetic diversity has special
Accurate identification of a particular R gene in diverse
significance in India, a country characterized by
elite germplasm using DNA markers and differential
highly varied agro-climates and diverse growing
blast races is an essential step for ensuring the accuracy
conditions (Mahender et al. 2012). In the North East
of R gene utilization in using MAS for different rice
(NE) and Eastern parts of India rice blast is endemic
breeding programs (RoyChowdhury et al. 2012a, b).
causing yield loss ranging from 40 to 46 % (Ngachan
To date, over 100 blast resistance genes to M.
et al. 2011). Interestingly, only few of the genes
oryzae from japonica (45 %), indica (51 %) and other

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identified/cloned are effective against the different for 24 weeks for genomic DNA extraction. Rice
lineages prevalent in the region. Eastern India is leaves were collected from plants and were imme-
considered to be a rich pocket of rice genetic resources diately frozen in liquid nitrogen and stored at
in the world owing to the extremely diverse rice -80 C for DNA extraction. Genomic DNA was
growing conditions as compared to other parts of the isolated using Plant Mini Kit (Qiagen, Valencia,
country. Selection made unknowingly by various CA, USA) from 100 mg of leaf tissue according to
ethnic groups inhabiting at different altitudes and manufacturers instruction. The extracted genomic
climatic situations, practicing different forms of DNA was estimated on 1 % agarose gels for
cultivation might have contributed to some extent quality. The quantity of extracted DNA was
towards the diversity of rice crop in this region. It is measured by Nano Drop system, 2000c (Thermo,
roughly estimated that during the past more than USA) and were diluted to 100 ng/ll with TE buffer
30,000 rice cultivars were grown in North East and and stored at -20 C.
Eastern parts of India (Ngachan et al. 2011). Mahender
et al. 2012 demonstrated the presence of six to seven Markers specific for rice blast R genes
genes in rice accessions from the North Eastern state
of Manipur which was related to high level of The germplasm accessions were screened for the
resistance in the accessions. presence of nine major blast resistance (R) genes, Piz,
Molecular genetic markers are now widely used to Piz-t, Pik, Pik-p, Pik-h, Pita/Pita-2, Pib, Pi9 and one
characterize gene bank collections that contain susceptible pita gene using a set of ten SNP and gene
untapped resources of distinct alleles which will based STS markers (Table 1). PCR based allele-
remain hidden unless efforts are initiated to screen specific SNP and gene based STS markers were
them for their potential use and function. Abundance obtained for identification of blast resistance genes in
of SNP polymorphisms has made it an attractive tool the selected germplasm. All the markers were synthe-
for allele mining and marker assisted selection. SNPs sized by Operon Eurofins (USA).
can be detected using allele specific PCR primers and
typed by the presence or absence of PCR amplified DNA marker analysis
products on standard agarose gels. Presence of differ-
ent blast resistance genes in a collection of 32 rice An allele specific PCR marker assays the genotype by
germplasm from North East and Eastern India was examining the presence or absence of a PCR ampli-
examined in the present investigation using PCR fication product. The PCR analyses were conducted
based SNP markers. and templates for PCR reaction set up for 20 ll as
follows: 19 PCR buffer, 0.2 mM of dNTPs mix,
1.5 mM of MgCl2, 0.2 lM each primer, 50 ng of
Materials and methods genomic DNA template, 1 unit of taq DNA polymer-
ase (Fermentas Life Sciences, Burlington, Canada)
Plant materials and DNA isolation and MilliQ water to final volume of 20 ll. The PCR
amplification program consisted of (i) 5 min initial
Thirty-two germplasm including local land races denaturation at 94 C (ii) 35 cycles of 45 s denatur-
and primitive cultivars originating from North East ation at 94 C, 45 s primer annealing at different Tm
and Eastern India and a cultivated check Vandana (Table 1), and 2 min extension at 72 C, and a final
was selected from the genetic stock maintained at extension for 5 min at 72 C. PCR products were
Central Rainfed Upland Rice Research Station, separated in 23 % agarose gels in 19 TBE buffer,
Hazaribag, based on their partial/complete resis- stained in ethidium bromide and visualized under UV
tance to blast resistance in outdoor blast nurseries. transilluminator. All PCR reactions for each sample
The seeds were germinated in the dark on moist- were repeated at least once to confirm the results. The
ened filter papers for 2 days at 30 C in a plant amplified fragments were scored as presence (1) or
growth chamber, planted in plastic pots and shifted absence (0) of amplicon linked to each gene DNA
to greenhouse for growth at 27 C with 16 h light fragment.

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Disease evaluation in uniform blast nursery (UBN)

Jia et al. (2002, 2004a)


Jia et al. (2002, 2004a)
Hayashi et al. (2006)
Hayashi et al. (2006)
Hayashi et al. (2006)
Hayashi et al. (2006)

Hayashi et al. (2006)

Hayashi et al. (2006)


Sharma et al. (2005)

Qu et al. (2006)
The NE and Eastern India rice germplasm, selected for
this study based on earlier reports of their complete/
References

partial resistance, were re-evaluated in a uniform blast


nursery at CRURRS, Hazaribag during wet season of
2012 in two replications. Seedlings that emerged from
densely sown seeds were surrounded by rows of
Expected
size (bp)

susceptible plants CO39 and B40. Observations on

1,042

2,000
292
257
112
148
1,500

861

1,043
388
Table 1 Details of single nucleotide polymorphisms (SNP) and gene based STS markers tightly linked to the major rice blast resistant genes

disease reaction of the plants were recorded 25 days


after sowing and continued at 5 day intervals until
40 days after sowing when the susceptible checks had
Annealling
Tm. (oC)

[80 % disease. Disease was scored visually on a 09,


scale (Standard evaluation system, SES, IRRI, 2002).
55
60
60
60
60
55

59

55
60
56 Scores 03 were considered resistant (R), 45 as
moderately resistant (MR) and 69 as susceptible (S).
gcattctccaacccttttgcatgcat

Most informative score obtained when the susceptible


atctcttcatatatatgaaggccac
aggaatctattgctaagcatgac

acattggtagtagtgcaatgtca
ctgcgccaagcaataaagtc

check scored 7 (35 DAS) was used for analyses. When


atcaggccaggccagatttg
ccagaatttacaggctctgg

ctaccaacaagttcatcaaa
ctaccaacaagttcatcaaa

the score differed between the replicates, the higher


ttgctccatctcctctgtt
Reverse (5 3 )
0

score was used for analyses.


0

Results and discussion


agtcgtgcgatgcgaggacagaaac

Allelic diversity of rice blast resistance genes


atagttgaatgtatggaatggaat
gccacatcaatggctacaacgtt
ggacccgcgttttccacgtgtaa

in NE and Eastern India germplasm


catgagttccatttactattcctc

gactcggtcgaccaattcgcc
agcaggttataagctagctat
agcaggttataagctaggcc
ttgctgagccattgttaaaca

atggtcctttatctttattg
Primer sequence

All the 32 accessions of NE and Eastern India rice


Forward (5 3 )
0

germplasm possessed one or more blast resistance


0

gene as revealed by the positive bands for different


markers. One of the germplasm Atte thima showed
positive bands associated with five major rice blast
resistance genes, Piz, Pik, Pik-p, Pik-h and Pib. Five
others were positive for four markers. 11 germplasm
possessed three, nine germplasm were positive for two
Candidate gene

while six showed positive amplicon for only one of the


YL155/YL87
YL183/YL87

genic markers (Pib) (Table 2).


marker
Zt56591
K39512
Z56592

195R-1
Marker

K3957

Pita3

Pb28

Genetic diversity of Piz and Piz-t gene


Chromosome

PCR results for the Piz and Piz-t rice blast resistance
genes were determined by visualization of amplicons
number

of 292 and 257 bp, using SNP primer, z56592 for Piz
6
6

2
6
11
11
11

12

12

(Fig. 1) and zt56591 for Piz-t (Fig. 2). Piz gene was
scored on 13 accessions and Piz-t gene was detected in
Pita/Pita-

six accessions. Five accessions showed positive bands


with both z56592 and zt56591 SNP markers. 18 NE
Pik-p
Pik-h
Gene

Piz-t

Pita
Pib
Pi9
Pik
Piz

and Eastern Indian rice germplasm accessions did not

123
Table 2 SNPs and STS markers associated with ten blast resistance genes in North East and Eastern Indian rice germplasm and their reaction to blast at UBN, Hazaribag, WS
2012
Euphytica

S. No. Varieties HRC State/ Piz Piz-t Pik Pik-p Pik-h Pita/ Pita/Pita-2 Pita Pi9 Pib Highest
Location Pita-2 scorea
Z56592 Zt56591 K39512 K3957 Gene based Pita3 YL155/ YL183/ 195R-1 Pb28 (09)
marker YL87 YL87

1 Dudhkanti 1,294 Sikkim 1 1 0 0 1 0 0 1 0 1 4


2 Atte thima 1,300 Sikkim 1 0 1 1 0 0 0 1 0 1 5
3 Chirakey-C 1,295 Sikkim 1 1 0 0 1 0 0 1 0 1 3
4 Dudhraj 1,343 Sikkim 1 0 0 0 1 1 1 0 0 1 3
5 Jhapaka 1,322 Sikkim 0 0 1 1 0 0 0 1 0 1 4
6 Brown gora 14 Jharkhand 0 0 0 1 0 0 0 1 0 1 5
7 Krishnabhog 1,333 Sikkim 1 0 0 0 1 0 0 1 0 1 4
8 Tonak-1 1,344 Sikkim 0 0 0 0 0 0 0 1 0 1 5
9 Kalchati 1,285 Sikkim 0 0 0 1 1 0 0 0 1 1 3
10 Basmati 1,319 Sikkim 1 0 0 0 1 0 0 1 0 1 5
11 Adde-1 1,361 Sikkim 0 0 0 0 1 0 0 1 0 1 5
12 Atte Basmati 1,340 Sikkim 0 1 0 0 1 0 0 1 0 1 4
13 Phaudel 1,335 Sikkim 1 1 0 0 1 0 0 1 0 1 1
14 Bachi thima 1,293 Sikkim 1 0 0 0 1 0 0 1 1 1 1
15 Nepali dhan 1,309 Sikkim 1 1 0 0 1 1 1 0 0 1 1
16 Darmali 1,316 Sikkim 0 0 1 1 0 0 0 1 0 0 4
17 Tulsi 1,281 Sikkim 1 0 0 0 0 0 0 1 0 1 4
18 Thule Atte 1,279 Sikkim 0 0 0 1 1 0 0 1 0 1 5
19 Chain Gora (Black) 811 Jharkhand 0 0 0 0 0 0 0 1 0 1 4
20 Gora 1,494 Chattisgarh 0 0 0 0 0 0 0 1 0 1 5
21 Batasi-1 1,431 Chattisgarh 0 0 1 0 1 0 0 1 0 1 5
22 Sathia- 2 1,439 Chattisgarh 0 0 0 0 0 0 0 1 0 1 4
23 Dani gora-3 1,489 Chattisgarh 1 0 0 1 1 0 0 1 0 1 5
24 Vandana Indica Jharkhand 0 0 0 0 1 0 0 1 0 1 4
25 Krishnabhog 1,506 Tripura 1 1 0 0 0 0 0 1 0 1 4
26 Atte 1,507 Sikkim 1 0 1 1 1 0 0 1 0 1 4
27 Phulbadam 1,508 Tripura 0 0 0 1 1 0 0 1 0 1 5
28 Garo malati 1,509 Tripura 0 0 0 0 0 0 0 1 0 1 6
29 Kataktara 1,510 Tripura 0 0 0 1 1 0 0 1 0 1 4

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Highest
scorea
(09)

5
5
4
Pb28
Pib

1
1
1
195R-1
Pi9

0
0
0
0 means the absence of amplicons linked to the rice blast resistant genes 1 means the presence of amplicons linked to the rice blast resistant genes
YL183/
YL87
Pita

1
1
1
Pita/Pita-2

YL155/

Fig. 1 Agarose gel photograph of 32 North East and Eastern


YL87

India rice germplasms to estimate the presence or absence of


292 bp of Piz rice blast resistance gene amplified with SNP
0
0
0

marker z56592. M, molecular marker (Fermentas 100 bp


Pita-2
Pita3

ladder); lane 1 IRBLZ-Fu (positive control); lane 2, CO39


Pita/

(negative control); lanes 334, 32 accessions of rice germplasms


0
0
0
Gene based
marker
Pik-h

0
0
0
K3957
Pik-p

0
0
0
K39512

09 SES scale: score 03 resistant, 45 as moderately resistant, 69 as susceptible


Pik

0
1
0
Zt56591
Piz-t

0
0
0

Fig. 2 Agarose gel photograph of 32 North East and Eastern


Z56592

India rice germplasms, to estimate the presence or absence of


Piz

257 bp of Piz-t rice blast resistance gene amplified with SNP


0
0
0

marker zt56591. M, molecular marker (Fermentas 100 bp


ladder); lane 1 IRBLZT-t (positive control); lane 2 CO39
Jharkhand
Jharkhand

(negative control); lanes 334 32 accessions of rice germplasms


Location

Tripura
State/

1,511
1,002

amplify either of the two SNP markers and were hence


HRC

808

negative for the two genes.

Genetic diversity of Pik multi gene


Black Gora
Chain gora
Chandmori

Among the three genes of the multi-gene family located


Table 2 continued
Varieties

on chromosome 11, Pik-h was the most predominant


one, having been detected in 18 of the 32 germplasm
using a candidate gene marker. Pik was detected in six
S. No.

(SNP marker k3957) and Pik-p (SNP marker k39512) in


30
31
32

ten germplasm. All together, 25 accessions had one or


a

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Euphytica

Fig. 3 Agarose gel photograph of 32 North East and Eastern


India rice germplasms, to estimate the presence or absence of Fig. 5 Agarose gel photograph of 32 North East and Eastern
112 bp of Pik rice blast resistance gene amplified with SNP India rice germplasms, to estimate the presence or absence of
marker k39512. M, molecular marker (Fermentas 50 bp ladder); 1,500 bp of Pik-h rice blast resistance gene amplified with gene
lanes 132 32 accessions of rice germplasms; lane 33 IRBLZK- based marker marker. M, molecular marker (Fermentas 1 kb
Ka (positive control); lane 34 CO39 (negative control) ladder); lane 1 IRBLKH-k3 (positive control); lane 2 CO39
(negative control); lanes 334 32 accessions of rice germplasms

Fig. 4 Agarose gel photograph of 32 North East and Eastern


India rice germplasms, to estimate the presence or absence of Fig. 6 Agarose gel photograph of 32 North East and Eastern
148 bp of Pik-p rice blast resistance gene amplified with SNP India rice germplasms, to estimate the presence or absence of
marker k3957. M, molecular marker (Fermentas 100 bp ladder); 861 bp of Pita/Pita-2 rice blast resistance gene amplified with
lane 1 IRBLZKP-k60 (positive control); lane 2 CO39 (negative SNP marker Pita3. M, molecular marker (Fermentas 100 bp
control); lanes 334 32 accessions of rice germplasms ladder); lane 1 IRBLTA-KI (positive control); lane 2 CO39
(negative control); lanes 334 32 accessions of rice germplasms

the other of the three genes, singly or in combination of


two and three. Atte (HRC 1507) was the only accession produced positive bands of 861 bp with Pita3
which showed positive bands for all the three Pik multi (Fig. 6) and 1,042 bp with YL155/87 (Fig. 7) primer
genes. Seven germplasm did not amplify any of the Pik sets which were tightly linked to the resistant Pita/
multi genes (Figs. 3, 4 and 5). Pita-2 allele. 30 NE and Eastern Indian rice germ-
plasm did not have Pita/Pita-2 genes with allele
PCR profiling of Pita/Pita-2 gene specific dominant marker YL155/87, but 29 of them
had the 1,043 bp amplicon corresponding to the
PCR based screening of Pita/Pita-2 genes on chro- dominant marker YL183/87 (Fig. 8) marker for sus-
mosome 12, showed that only two (Dudhraj and ceptible pita allele. Only one accession Kalchati was
Nepali dhan) of the 32 accessions under study negative for both the markers.

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Fig. 7 Agarose gel photograph of 32 North East and Eastern


India rice germplasms, to estimate the presence or absence of Fig. 9 Agarose gel photograph of 32 Eastern India rice
1,042 bp of Pita/Pita-2 rice blast resistance gene amplified with germplasms, to estimate the presence or absence of 2,000 bp
dominant marker YL155/YL87. M, molecular marker (Fermen- of Pi9 rice blast resistance gene amplified with gene based STS
tas 1 kb ladder); lanes 132 32 accessions of rice germplasms marker 195R-1. M, molecular marker (Fermentas 1 kb ladder);
lane 1 IRBL9-w (positive control); lane 2 CO39 (negative
control); lanes 334 32 accessions of rice germplasms

Fig. 8 Agarose gel photograph of 32 Eastern India rice


germplasms, to estimate the presence or absence of 1,043 bp
of pita rice blast susceptible gene amplified with dominant
marker YL183/YL87. M, molecular marker (Fermentas 100 bp Fig. 10 Agarose gel photograph of 32 Eastern India rice
ladder); lanes 132 32 accessions of rice germplasms germplasms, to estimate the presence or absence of 388 bp of
Pib rice blast resistance gene amplified with SNP marker Pb28.
M, molecular marker (Fermentas 50 bp ladder); lane 1 IRBLB-b
Genetic diversity of Pi9 gene (positive control); lane 2 CO39 (negative control); lanes 334
32 accessions of rice germplasms
Presence of major rice blast resistance gene Pi9 on
chromosome 6 was determined by visualization of sequence on chromosome 2. Screening of Pib blast
2.0 kb positive fragments using 195R-1 primer. The resistance gene was determined by visualization of
gene-specific marker, 195R-1 amplified positive 388 bp positive fragments from 31 accessions of NE
bands in only two accessions namely Kalchati and and Eastern India rice germplasm using this marker
Bachi thima (Fig. 9). In other 30 accessions it did not (Fig. 10). This gene fragment was the most prevalent
produce bands indicating the absence of the gene in among the germplasm. Only one accession, Darmali is
these germplasm. negative for Pib gene.

Genetic detection of Pib gene Uniform Blast Nursery

The Pib-specific PCR primer set, Pb28 marker was Reaction of the set of 32 germplasm to blast under
developed to produce a 388 bp amplicon based on its UBN was evaluated at CRURRS, Hazaribag. Disease

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Fig. 11 Frequency distribution of blast disease score of 32 North East and Eastern India rice germplasm

reaction after 35 days of sowing ranged from 0 to 6 in resistance in the uniform blast nursery. When the
the SES scale when the susceptible spreader CO39 resistance pattern of the germplasm is examined vis a
was completely killed in the nursery (score 9). Six vis the presence of amplicon products of the major
germplasm were resistant (score 03), ten moderately genes it was noted that though resistance is generally
resistant (score 4) and sixteen susceptible ([5) proportional to the frequency of the gene(s), certain
(Fig. 11). All the six resistant germplasm originated genes (Pi9, Pita-2, Piz-t) were more effective than
from Sikkim and were positive for the markers for Piz- others in thwarting infection. They were also less
t, Pita-2 or Pi9. Presence of Pita-2 or Pi9, invariably frequently detected by marker amplification. Con-
contributed to resistance in the nursery. None of the versely, the less effective genes were more predomi-
other 26 moderately resistant/susceptible germplasm nant but mostly ineffective against the different
harboured markers for these genes. Piz-t appeared to pathotypes/lineages that are normally present in the
be associated with moderate resistance in two germ- nursery, albeit with different frequencies. Multi-loca-
plasm in addition to the two germplasm which tion evaluation of a set of isogenic lines carrying 24
recorded resistant reaction. Though the presence of major blast resistance genes had earlier indicated that
multiple resistance genes generally appeared to have Pi9, Piz-t and Pita-2 were resistant at most locations,
higher level of resistance, there were also instances indicating their broader spectrum of resistance to
where the presence of several genes did not ensure pathotypes prevalent in Eastern India (Variar et al.
resistance. Atte thima, for example, harboured five 2009). Among the multi-genes near the waxy gene
resistance genes, but was susceptible. Pib, Pita, Pik- locus on chromosome 6 (Ballini et al. 2008) Pi9 was
p and Pik-h were the most abundant genes detected in more effective than Pi2 (Piz-t) in the present investi-
the germplasm but they were also the least effective gation. Piz, Piz-t and Pi40(t) were commonly detected
against the population of the blast fungus present in the in germplasm originating from North East India earlier
nursery at Hazaribag. It is however, noted that all the but Pi9 was not detected among them (Mahender et al.
tested germplasm possessed low levels of resistance as 2012). Since the present study was taken up with a
evidenced by their relative scores vis a vis the select set of rice germplasm covering a wider geo-
susceptible spreader, CO39 which possessed none of graphical region (Chattisgarh, Jharkhand, Sikkim and
the genes evaluated in this study (data not shown). Assam), the chances improved for detection of rare
Genetic frequency of the nine major rice blast alleles like Pi9 which was identified in two germplasm
resistance genes, Piz, Piz-t, Pik, Pik-p, Pik-h, Pita/ Kalchati and Bachi thima. While Pi9 gene conferred
Pita-2, pita, Pi9 and Pib, ranged from 6 to 97 % in the complete resistance in Kalchati and Bachi thima, Piz-t,
select set of germplasm that exhibited different level of along with other genes, contributed to complete

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resistance in three germplasm (Nepali dhan, Chirakey- virulent on the isogenic lines carrying Pib and Pik
C and Phaudel) and moderate resistance in others. (Unpublished data).
Thirteen accessions (41 %) showed positive bands for Genetic variability among the rice accessions and
the Piz gene with z56592 SNP marker but its presence within the pathogen often leads to inconsistent marker
independently did not contribute to complete resis- and phenotype analysis. MAS have the advantage in
tance in any of the germplasm evaluated in this study. identifying R genes, but its power lies in the robustness
Both Piz and Piz-t genes have been used for conferring of the markers used. The identification of rice blast
blast resistance to Japanese cultivars, because their resistance gene Pib and analysis of rice blast resistance
importance was emphasized by Hayashi et al. (2004) in gene Piz suggests that DNA markers derived from the
rice breeding in Japan. The Piz-t and Piz genes, co- gene is a valuable tool for blast gene identification and
segregated with z56592 and zt56591 markers, were screening among the rice germplasm (RoyChowdhury
flanked on one side by z4794 and on the other side by et al. 2012a, b). In this study, the PCR based markers
z60510 and z5765 markers (Hayashi et al. 2006). employed for screening of different blast resistance
Two blast resistance genes, Pita and Pita-2, have genes are well established and effective. The consis-
been located at the Pita locus near the centromere of tent results showed with the selected SNPs and STS
chromosome 12 and are tightly linked to each other markers for respective genes was highly reliable and
(Wang et al. 2002). These two genes were interacting make them the marker of choice for molecular
in terms of their resistance specificity. Pita-2 has a screening of rice blast resistant genes among the rice
broader resistance spectrum than Pita (Rybka et al. germplasm. For the molecular identification of blast
1997; Bryan et al. 2000). In the present study, only resistance genes, isogenic lines for the selected genes
two accessions Dudhraj and Nepli dhan were positive were taken as positive control and CO39 as negative
of Pita/Pita-2 genes using the SNP marker Pita3 and control.
dominant marker YL155/YL87. These two acces- Many rice varieties have been developed as com-
sions showed resistance pattern in UBN. The pletely resistant to M. oryzae strains, but soon
isogenic lines of Pita and Pita-2 gene, evaluated breakdown of rice blast resistant genes occurred
earlier, were resistant to blast in multi environment because of the emergence of stronger virulent isolates
testing and this confirmed that the germplasm having of rice blast fungus (Bonman et al. 1986; Mackill and
Pita/Pita-2 gene is likely be effective in rice Bonman 1992). Genotyping of the germplasm with
breeding program. The 32 accessions were also allele specific markers helped to identify nine major
screened for pita susceptible gene with dominant blast resistance genes Piz, Piz-t, Pik, Pik-p, Pik-h,
marker YL183/YL87 and found that 29 accessions Pita/Pita-2, Pib and Pi9 and one recessive pita gene in
gave amplicons of 1,043 bp. The Pita/Pita-2 markers 32 North East and Eastern Indian rice germplasm. The
used in this study were based on DNA polymorphism UBN analysis revealed the blast disease pattern of
of nucleotides between the resistant indica Pita allele these germplasm and showed that germplasm con-
and the susceptible japonica pita allele (Jia et al. taining Pita/Pita-2, Pi9 are also effective against the
2002, 2003, 2004a, b). Identification of Pita/Pita-2 prevalent pathotypes of M oryzae. Interestingly,
genes and its validation with differential isolates of several germplasm had multiple disease resistance
M. oryzae reveals that the Indian rice germplasm are genes. Virulence analyses using specific isolates
diverse and potential source of blast resistant lines would help unravel the response of the resistance
which can be exploited in rice blast breeding genes against specific lineages. Results of this work
programs (Shikari et al. 2013). showed that all of the 32 accessions of North East and
Members of the Pik multi-gene family and Pib were Eastern India rice germplasm possessed more than one
the most frequently detected genes in the present study blast resistance gene. Being adapted to the locations
but neither the germplasm possessing them nor the where these germplasm originated, and having co-
isogenic lines in the previous evaluations (Variar et al. evolved with the local population of the blast fungus,
2009) exhibited resistance. Virulence analyses using use of these germplasm could have a competitive edge
50 M. oryzae isolates collected from North East and over other exotic resistance donors currently being
Eastern India also indicated that most isolates are used in the breeding programs.

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Euphytica

Acknowledgments The authors acknowledge the National Hayashi N, Inoue H, Kato T, Funao T, Shirota M, Shimizu T,
Agriculture Innovative Project- Component 4, Indian Council of Kanamori H, Yamane H, Hayano-Saito Y, Matsumoto T,
Agricultural Research (ICAR), on Allele mining blast resistance Yano M, Takatsuji H (2010) Durable panicle blast-resis-
genes for financial support for this research. This work was tance gene Pb1 encodes an atypical CC-NBS-LRR protein
performed at the Central Rainfed Upland Rice Research Station, and was generated by acquiring a promoter through local
Hazaribag, Jharkhand, India. genome duplication. Plant J 64:498510
Hittalmani S, Parco A, Mew TW, Zeigler RS, Huang N (2000)
Fine mapping and DNA marker-assisted pyramiding of the
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