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Genetic mapping of quantitative trait loci controlling fruit size and shape in
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DOI: 10.1007/s11032-011-9562-1

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Genetic mapping of quantitative trait loci
controlling fruit size and shape in papaya

Andrea L. Blas, Qingyi Yu, Olivia

J. Veatch, Robert E. Paull, Paul H. Moore
& Ray Ming

Molecular Breeding
New Strategies in Plant Improvement

ISSN 1380-3743
Volume 29
Number 2

Mol Breeding (2012) 29:457-466

DOI 10.1007/s11032-011-9562-1

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 Author's personal copy
Mol Breeding (2012) 29:457–466
DOI 10.1007/s11032-011-9562-1
Genetic mapping of quantitative trait loci controlling fruit
size and shape in papaya
Andrea L. Blas • Qingyi Yu • Olivia J. Veatch
Robert E. Paull • Paul H. Moore • Ray Ming
Received: 2 September 2010 / Accepted: 2 March 2011 / Published online: 16 March 2011
Ó Springer Science+Business Media B.V. 2011
Abstract Papaya (Carica papaya L.) is a pan-
tropical tree that bears fruit exhibiting a wide range of
size and shape. Depending on variety and environ-
ment, papaya fruit may weigh from 0.2 kg up to
10 kg. Papaya fruit shape is a sex-linked trait ranging
from spherical to ovate, cylindrical or pyriform. An
F2 mapping population, produced from a cross
between the Thai variety Khaek Dum, bearing
1.2 kg, red-fleshed fruit, and variety 2H94, a Hawaii
Solo type bearing a 0.2 kg, yellow-fleshed fruit, was
used to identify quantitative trait loci (QTLs) that
influence papaya fruit characters including weight,
diameter, length and shape. Fruit phenotype data,
Electronic supplementary material The online version of
this article (doi:10.1007/s11032-011-9562-1) contains
supplementary material, which is available to authorized users.
A. L. Blas
O. J. Veatch
Department of Molecular Biosciences and
Bioengineering, University of Hawaii, Honolulu,
HI 96822, USA
A. L. Blas
Q. Yu
O. J. Veatch
P. H. Moore
R. Ming
Hawaii Agriculture Research Center, Kunia, HI 96759,
USA
Q. Yu
Texas AgriLife Research, Texas A & M University,
Weslaco, TX 78596, USA
R. E. Paull
Department of Tropical Plant and Soil Sciences,
University of Hawaii, Honolulu, HI 96822, USA
•
collected from two subpopulations planted in succes-
sive growing seasons, showed striking differences by
year indicating significant genotype 9 environment
interactions. Fourteen QTL with phenotypic effects
ranging from 5 to 23% were identified across six
linkage groups (LGs) with clusters of two or more
QTL on LGs 02, 03, 07 and 09. These loci contain
homologs to the tomato fruit QTL ovate, sun and
fw2.2 regulating fruit size and shape. The papaya fruit
QTL provide a starting point for dissecting the
genetic pathways leading to extreme fruit size and
shape and may prove useful for papaya breeders
attempting to tailor new varieties to specific con-
sumer markets.
R. Ming (&)
Department of Plant Biology, University of Illinois at
Urbana-Champaign, 148 ERML, MC-051,
1201 W. Gregory Drive, Urbana, IL 61801, USA
e-mail: rming@life.uiuc.edu
Present Address:
A. L. Blas
Centre de Recerca Agrigenòmica, IRTA, 08348 Cabrils,
Spain
Present Address:
O. J. Veatch
Center for Human Genetics Research, Vanderbilt
University, Nashville, TN 37232, USA
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458
Keywords Carica papaya
Fruit size
Fruit shape

Quantitative trait loci
Introduction
Papaya (Carica papaya L.) is a pan-tropical tree
important for production of fruit for local consump-
tion and for export to temperate climate markets.
Consumer preferences for papaya fruit traits of size,
shape and flesh color vary widely among regional
markets. Papaya fruit shape is a sex-influenced trait
with the fruit of female trees ranging from spherical
to ovoid while fruit from hermaphrodite trees are
elongate, cylindrical to pear-shaped (Chan and Paull
2008; Nakasone and Paull 1998; Paull et al. 2008).
Depending on the environment and variety, mature
papaya fruit may weigh from 0.2 to 10 kg (Chan and
Paull 2008; Nakasone and Paull 1998). The ripe color
of papaya fruit flesh ranges from yellow to orange to
red, depending on variety. Recently, a single major
gene controlling papaya fruit flesh color, CpCYC-b,
was identified and a co-dominant sequence charac-
terized amplified region (SCAR) marker, CPFC1,
was developed for use in marker-assisted selection
(MAS) in papaya breeding programs (Blas et al.
2010). However, no genetic markers are yet available
for selection of papaya fruit size or shape.
Papaya cultivars of Hawaii, including the Papaya
ringspot virus (PRSV)-resistant transgenic cultivar
SunUp whose draft genome sequence was released in
2008 (Ming et al.), are perhaps the best characterized
tropical fruit based on molecular techniques. In
Hawaii, producers prefer pear-shaped hermaphrodite
fruit of 0.3–0.5 kg in weight over fruit of any other
shape or size. Preference is based on yield per unit of
land and post-harvest handling and shipping charac-
teristics of the fruit. Since hermaphrodite trees are
self-pollinating, their exclusive culture allows for a
greater number of productive trees per unit of land
than would occur with a mix of male and female trees
(Nakasone and Paull 1998; Rieger 2006). In addition,
hermaphrodite fruit are generally full of seed and less
likely to collapse under weight than are fruit devoid
or deficient in seed. Moreover, standardization of
shape and size facilitates post-harvest handling and
shipping.
Until recently, MAS in papaya was limited to use
of SCAR markers for sex-type determination and for
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Mol Breeding (2012) 29:457–466
detection of the PRSV coat-protein gene (Chaves-
Bedoya and Nuñez 2007; Deputy et al. 2002). The
genetics of fruit size in papaya has not yet been
characterized, in part because fruit size, like many
agronomically important traits, is a quantitative trait
influenced by multiple genes. Various quantitative
trait loci (QTL) controlling fruit size or shape have
been identified in tomato (Causse et al. 2004; De
Vicente and Tanksley 1993; Grandillo and Tanksley
1996; Van der Knaap and Tanksley 2001, 2003),
pepper (Chaim et al. 2001, 2003a, b; Zygier et al.
2005), grape (Cabezas et al. 2006; Constantini
et al. 2008; Mejı́a et al. 2007) and citrus (Garcı́a
et al. 2000). QTL identified in tomato are perhaps
the best characterized for any fruit species; six QTL
responsible for 67% of the phenotypic variation in
tomato fruit size have been identified (Lippman and
Tanksley 2001), and the two most significant of
these six loci appear to control carpel and locule
number while a third locus, fw2.2, has been cloned
and is responsible for up to 30% of the fruit size
variation between wild and domesticated tomato
species. The effect of this gene on fruit size is due
to heterochronic regulatory mutation of the fw2.2
alleles (Cong and Tanksley 2006; Frary et al. 2000;
Lippman and Tanksley 2001; Liu et al. 2003).
Fruit shape in tomato is essentially determined
by flower development while fruit shape in pepper
and eggplant have been attributed to differential
and directional growth rates during fruit develop-
ment (Grandillo et al. 1999). Like tomato, papaya
fruit are fleshy and the fruit flesh is derived from
the pericarp of the ovary wall. Final fruit size in
papaya has been attributed to meristematic activity
of the central parenchyma of the pericarp tissue
(Chan and Paull 2008; Grandillo et al. 1999;
Nakasone and Paull 1998). However, some papaya
varieties have distinctly different female and her-
maphrodite flower shape even before the flowers
are fully mature, i.e. at blooming. Hermaphrodite
papaya flowers are generally cylindrical or elongate
while female flowers tend to be shorter and broader
with a bulbous base at the proximal, stem-end of
the flower. Two loci identified as controlling fruit
shape in tomato are sun and ovate. Ovate, first
reported a century ago, is a locus associated with
the tomato fruit elongation QTL fs8.1 and is
presumed to exert its effect during early ovary
development in floral organogenesis (Hedrick and
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Mol Breeding (2012) 29:457–466
Booth 1907; Ku et al. 1999, 2001). In contrast, sun
was found to affect elongation of the ovary pericarp
after fertilization (Van der Knaap and Tanksley
2001). Linkage of papaya fruit traits with fertiliza-
tion has also been recognized. In particular, female
papaya fruit with few or no seeds are produced
occasionally and these are generally smaller in size
than the female fruit with a normal compliment of
seeds (Nakasone and Paull 1998). In tomato, the
rate of successful fertilization has been shown to
determine the initial growth rate (i.e. cell prolifer-
ation) of the ovary, where a greater number of
fertilized ovules is associated with larger fruit size
(Varga and Bruinsma 1986). The parallels between
tomato and papaya fruit size development are
intriguing.
Two nearly isogenic lines (NILs) of tomato, with
either large or small fruit, were studied for fw2.2
effect. The large-fruited NIL was shown to produce
fewer fruit in relation to number of flowers per
inflorescence than the small-fruited NIL, i.e. large-
fruited lines aborted more flowers. This observation
supports the idea that intra-inflorescence competition
for photosynthates results in the production of fewer
fruits and, in terms of reproductive fitness, it is more
advantageous to the plant to produce few large seed-
filled fruit than numerous partially-filled fruit which
would decrease seed dispersal and viability (Nesbitt
and Tanksley 2001). Differential regulation and
timing of expression of fw2.2 were also shown to
be important in regulating fruit size between the
large- and small-fruited NILs. While final cell
number and rate of cell division differed significantly
between the NILs, the pattern of cell expansion did
not, supporting the concept of independent regulation
of cell proliferation and cell expansion (Cong et al.
2002).
QTL analysis of papaya fruit shape is likely to
reveal loci associated with floral development while
analysis of fruit size may reveal loci affecting cell
proliferation and expansion. The objectives of the
present study were: (1) to identify QTL for papaya
fruit size and shape attributes that could aid breeders
in specifically tailoring new varieties to consumer
preferences, and (2) to search for potential candidate
genes located in the region of the identified QTL
utilizing the growing amount of genomic and genetic
resources available for papaya.
459
Materials and methods
Mapping population
An F2 mapping population generated from a cross
between Khaek Dum (female) and 2H94 (hermaph-
rodite, pollen donor) was grown at the Hawaii
Agriculture Research Center Kunia Substation on
Oahu, Hawaii. Khaek Dum (KD) is a Thai cultivar
with large cylindrical fruit weighing an average of
1.2 kg and having red flesh color. 2H94 is a small
fruited mutant identified among a Kapoho 9 SunUp
F2 population bearing pear-shaped fruit with an
average weight of 0.2 kg and yellow flesh color.
2H94 had undergone four generations of self-polli-
nation before being used as a parent in the present
cross. Two KD 9 2H94 F2 subpopulations were
grown during the 2005 growing season: KHY
produced from a yellow-fleshed F1 hermaphrodite
and KHR from a red-fleshed F1 hermaphrodite. All
F2 individuals grown during the 2006 growing season
were collected from a yellow-fleshed F1 hermaphro-
dite. Leaf tissue and five fruit from each of 223 F2
hermaphrodite individuals were collected and data
recorded for fruit weight, fruit length, diameter and
fruit flesh color. Fruit flesh color was also recorded
for female F2 individuals that were not included in
the QTL mapping analysis. A fruit shape index was
calculated as the length: diameter ratio for each fruit.
The mean of five fruit was calculated to estimate the
central tendency of each fruit size phenotype char-
acter for each F2 hermaphrodite, for a 2H94
hermaphrodite parent, and an F1 hermaphrodite.
DNA isolation and genotyping
Freeze-dried leaf tissue was ground to a fine powder
and stored at –20°C until DNA isolation. DNA was
isolated according to the methods of Chittenden et al.
(1994) with minor modifications. Isolated DNA was
treated with DNase-free RNase and purified by
phenol: chloroform: isoamyl alcohol extraction.
DNA quality and concentration were visualized by
agarose gel electrophoresis.
KD and 2H94 were screened for DNA polymor-
phism using 706 simple sequence repeat (SSR)
markers from the AU9 9 SunUp high-density
genetic map described by Chen et al. (2007).
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460
Eighty-four polymorphic SSRs and the two SCAR
markers, CPFC1 and CPFC2, from the recombination
hot spot flanking CpCYC-b for papaya fruit flesh
color (Blas et al. 2010) were used for genotyping in
the KD 9 2H94 mapping population. PCR condi-
tions and agarose gel electrophoresis were as previ-
ously described.
KD 9 2H94 genetic map construction
and QTL analysis
Genotype scores were proofread and only high-quality
markers were used for genetic map construction. F2
individuals with more than 10% missing genotypes
and markers with more than 10% missing datapoints
were excluded from map construction. Each segregat-
ing marker was subjected to chi-squared analysis to
test the goodness-of-fit to expected segregation ratios
of 1:2:1 for co-dominant, 2:1 for sex-linked
co-dominant, or 3:1 for dominant markers in an F2
population. JoinMapÒ 3.0 (Van Ooijen and Voorrips
2001) was used to construct a genetic linkage map with
a minimum LOD score of 4.0 and a maximum
recombination rate (h) of 0.40 using the Kosambi
mapping function. Markers of ‘‘suspect linkage’’ were
inspected again for accuracy in scoring and removed
from the map calculation if still problematic.
Phenotype data of fruit weight, length, diameter
and fruit shape index for each F2 individual and the
KD 9 2H94 genetic map were used for QTL analysis
with MapQTLÒ 5 (Van Ooijen 2004). The genome-
wide LOD significance threshold was determined for
each trait based on permutation testing (PM) with
10,000 replications. QTL were initially identified by
simple interval mapping (IM), then nearby loci with
the highest LOD score were selected as co-factors
and the data re-analyzed using the Multiple QTL
Model (MQM) mapping function. Co-factor selection
and MQM were repeated until no new QTL were
identified.
Results
Genotype data and genetic map construction
In total, 219 KD 9 2H94 F2 hermaphrodites were
genotyped with 84 SSR-specific primer sets and two
SCAR markers. Of 92 high-quality SSR markers, 68
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Mol Breeding (2012) 29:457–466
displayed co-dominant inheritance and 24 displayed
dominant inheritance in the KD 9 2H94 F2 popula-
tion. The KD parent contributed the dominant allele
for 14 of the 24 dominant SSR markers. Sixteen of
these dominant markers were co-dominant in the
AU9 9 SunUp population while eight of the 68
co-dominant markers were dominant in the AU9 9
SunUp population. Interestingly, three dominant
markers, CPM976c22, P3K2032K2 and P3K24722,
that showed the 2:1 segregation pattern typical for
sex-linked co-dominant markers did not map to
linkage group (LG) 01 which contains the sex-
determining male-specific region of the Y chromo-
some (MSY). An unexpectedly high percentage
(80.2%) of markers exhibited segregration distortion,
i.e. they showed segregation patterns outside of
expected Mendelian ratios. In total, 77 SSR markers
showed distorted segregation with 31 clearly skewed
toward the KD allele and 43 skewed toward the 2H94
allele. In three cases the distortion favored the
co-dominant allele type. By contrast, only 20.0% of
these same SSRs displayed segregation distortion in
the AU9 9 SunUp population.
Fifty-four SSR markers, the morphological flesh
color locus and the CPFC1 and CPFC2 SCAR markers
were mapped to 11 linkage groups corresponding to
LGs 01–09 and LG11 (Fig. 1, Electronic Supplemen-
tary Material Table S1). The genetic map is composed
of 11 dominant and 46 co-dominant markers and spans
a total length of 672.1 cM which corresponds to
602.0 cM (56.3%) of the AU9 9 SunUp genetic map
based on SSR locus position. Only 12 of the total 57
mapped markers followed model segregation patterns,
while the remainder displayed segregation distortion.
Inversion of locus order and expansion in genetic
distance between adjacent loci were observed in the
KD 9 2H94 genetic map. LG05 was split into two
small linkage groups designated LG05.1 and LG05.2
corresponding to the top and bottom portions, respec-
tively, of the full-length linkage group. In contrast,
the P3K7484 marker from the minor LG10 of the
high-density AU9 9 SunUp map was added to LG08
by linkage in the KD 9 2H94 mapping population.
Based on the high-density AU9 9 SunUp map, LGs
01, 03, 04, 07 and 08 of the KD 9 2H94 map represent
only portions of their respective LGs. SSRs mapped
to LG01 span the region corresponding to 73.7–109.2
cM of the full-length map (145.0 cM) while LG03 is
represented by SSRs corresponding to 20.8–76.3 cM
 Author's personal copy
Mol Breeding (2012) 29:457–466 461

LG01 LG02 LG03 LG04 LG05.2

0 CPM2212CC 0 ctg-384C0 0 P3K2112K0 0 ctg-77C0 0 P3K24722
LG05.1
6 P3K5593CC
10 P3K3750CC 0 CPFC220
14 P3K71CC 14 CPM1761C0 14 P3K3968C0 3 ctg-365aK0
18 P6K1268C0 19 P3K3555C0 5 f.color20
10 ctg-365bK0
26 CPM609C0
30 P3K3187C0 29 P8K277C0
34 P3K1426C0
37 P3K4953C0 ctg-43C0 37 P6K673C0
40 39 P3K156C0
41 CPM976aK0
46 P3K6856C0 47 P3K6947C0 37 CPFC1C0
49 CPM976c22
55 ctg-375C0 56 P3K3624C0
61 P3K187C0 62 P3K2696C0
LG06 63 ctg-169C0

0 P3K411CC
71 ctg-390C0 72 P8K187C0

17 ctg-17CC 88 P3K618b23
LG09
0 P3K4311bK0

42 P3K2029C0
LG08 LG11
0 P3K7483C0 0 P3K149CC
55 CPM926C0

69 P6K175C0 LG07
22 P3K170C0 22 P6K710C0
0 CPM1550C0
48 P8K178C0
5 ctg-41C0 28 CPM1572C0
56 P3K183C0

17 ctg-03C0
46 P3K166C0

107 P3K2426aK3 56 P3K7484C0 79 P8K209CC

Fig. 1 KD 9 2H94 genetic map showing location of QTL length; bar with horizontal stripes fruit diameter; open bar fruit
affecting fruit size and shape traits. 1-LOD intervals of each shape index. Dotted lines of the bars indicate 2 LOD likelihood
significant QTL are represented on the left side of linkage groups intervals
by: solid, black bar fruit weight; bar with vertical stripes fruit

of the full-length map (132.4 cM). LG04 contains
markers corresponding to 48.3–118.0 cM, but no
markers corresponding to the top 48 cM of the full-
length map (120.6 cM); conversely, LG08 lacks
coverage of the bottom portion of the full-length
map (from 20.3 to 91.8 cM) despite linkage of the
LG10 marker at the top of the LG. LG07 corresponds
to the central portion, 16.4–67.0 cM, of the full-length
map (96.4 cM) and lacks coverage at both ends of the
linkage group for the KD 9 2H94 genetic map. No
genotyped markers from LG12 could be linked in the
KD 9 2H94 genetic map.
Phenotype data and QTL analysis
Phenotype data for QTL analysis of four papaya fruit
size and shape traits were recorded for 223 F2
hermaphrodites grown during the 2005 or 2006
growing season. Variability for each trait was
observed between growing seasons for the F2 and
the parental genotypes. Segregation for fruit length,
diameter and fruit shape index followed a normal
distribution in the full F2 population (Shapiro–Wilk
test, P \ 0.01, Figs. S1–S3). Interestingly, fruit weight
segregated according to a normal distribution during
the 2005 growing season (Fig. S4), but analysis of the
2006 and combined F2 populations showed a skewed
mass distribution toward a smaller fruit weight (coef-
ficient of skewness = 0.4889, Table 1). Transgressive
segregation, the appearance of individuals in segre-
gating populations beyond the parental phenotypes,
was evident among the F2 population for fruit length
and diameter (Figs. S1–S2). Transgressive segregation
was most evident among the F2 individuals during the

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462 Mol Breeding (2012) 29:457–466

Table 1 Descriptive statistics of the KD 9 2H94 F2 population

Weight (g) Diameter (cm) Length (cm) Fruit shape (length:
diameter)
2005 2006 Combined 2005 2006 Combined 2005 2006 Combined 2005 2006 Combined

n 132 91 223 132 91 223 132 91 223 132 91 223

Mean 847.24 653.1 768.01 8.0115 9.0951 8.4537 19.529 15.081 17.714 2.4564 1.658 2.1306
SD 256.85 242.13 268.03 0.9557 1.0376 1.1227 2.9616 3.1661 3.747 0.3714 0.2829 0.5182
Variance 65971 58626 71841 0.9134 1.0766 1.2604 8.7711 10.024 14.04 0.138 0.0801 0.2685
SE (mean) 22.356 25.382 17.949 0.0832 0.1088 0.0752 0.2578 0.3319 0.2509 0.0323 0.0297 0.0347
Minimum 175.21 304.88 175.21 4.9796 6.8981 4.9796 11.28 10.32 10.32 1.4715 1.0436 1.0436
Maximum 1833.2 1528.3 1833.2 10.403 11.475 11.475 28.749 24.198 28.749 3.5624 2.3539 3.5624
Skewness 0.3329 0.934 0.4889 -0.1731 -0.0535 0.0487 0.481 0.4108 0.0907 0.1682 -0.0364 0.1123

Table 2 Correlation (Pearson) between phenotypic fruit traits coefficients were calculated for each phenotypic trait
over 2 years pair (Table 2). As expected, fruit diameter showed
Trait Fruit weight Fruit diameter Fruit length negative correlation and fruit length showed positive
correlation with fruit shape, which is the ratio of
Fruit diameter 0.4806** length: diameter. Correlation between each pheno-
Fruit length 0.8217** 0.0255 typic trait pair was highly significant (P \ 0.01)
Fruit shape (L:D) 0.4271** -0.5179** 0.8321** except between diameter and length which appear to
** Significant at P \ 0.01 segregate independently.
Fourteen QTL affecting papaya fruit size and
2005 growing season. The calculated fruit shape index shape were mapped to LGs 02, 03, 04, 06, 07 and 09
of the parental phenotypes approximated the mean (Table 3) using the IM and MQM models. A genome-
fruit shape index of the F2 population which displayed wide LOD significance threshold for each trait of
a normal distribution (Fig. S3). Pearson correlation LOD 2.5 was determined by permutation testing with

Table 3 Significance, effect and location on the KD 9 2H94 genetic map of QTL detected for fruit size and shape index
Trait QTL position LOD threshold % var. Significance
a = 0.05
LG Peak (cM) Nearest marker cM 1 LOD interval LOD score

FW2.1K2 02 38.6 ctg-43C0 39.9 36.6–39.9 7.76 2.5 23.1 **

FW3.1K2 03 3.0 P3K2112K0 0.0 0.0–9.0 3.19 2.5 15.1
FW7.1K2 07 0.0 CPM1550C0 0.0 0.0–2.0 3.78 2.5 7.0 *
FW9.1K2 09 12.0 P3K4311bK0 0.0 0.0–16.0 5.33 2.5 20.5
FL2.1K2 02 30.4 P2K1426C0 33.6 28.4–32.4 15.04 2.5 17.3 **
FL4.1K2 04 51.1 P3K6947C0 47.2 42.1–54.2 6.10 2.5 6.8 **
FL7.1K2 07 1.0 CPM1550C0 0.0 0.0–2.0 6.00 2.5 5.4 **
FL9.1K2 09 15.0 P3K4311bK0 0.0 0.0–23.0 7.37 2.5 12.0
FD2.1K2 02 44.8 CPM976aK0 40.8 40.8–46.8 3.27 2.5 17.0
FD3.1K2 03 1.0 P3K2112K0 0.0 0.0–3.0 5.20 2.5 18.2
FS2.1K2 02 43.8 CPM976aK0 40.8 42.8–45.8 6.17 2.5 15.1
FS3.1K2 03 71.3 P3K187C0 61.3 53.5–76.3 7.71 2.5 9.9 **
FS6.1K2 06 93.1 P3K2426aK3 107.4 79.1–107.1 5.28 2.5 6.8
FS9.1K2 09 22.0 P3K4311bK0 0.0 0.0–32.0 3.33 2.5 7.5
* P = 0.005; ** P = 0.0001 in the non-parametric Kruskal–Wallis model

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Mol Breeding (2012) 29:457–466 463

Table 4 Papaya genomic regions with predicted homology to tomato fruit size and shape loci
Fruit trait WGS contig, GeneScan Conserved Homology % ID EST
locus accession no. prediction domains (E-score) hits
CDS Peptide

Ovate LG1 contig_13552, 573 bp 190 aa DUF623 Ovate protein, Solanum lycopersicum, 67 (8e-21) –
AAN17752.1 ABIM01013535.1 superfamily AAN17752.1
LG4 contig_17626, 1098 bp 365 aa DUF623 ATOFP13/OFP13, ovate family protein 46 (2e-37) –
ABIM01017601.1 superfamily 13, Arabidopsis thaliana,
NP_196102.1
LG5 contig_6659, 651 bp 216 aa DUF623 ATOFP14/OFP14, ovate family protein 56 (4e-30) –
ABIM01006651.1 superfamily 14, A. thaliana, NP_178114.1
LG7 contig_6797, 951 bp 316 aa DUF623 ATOFP8/OFP8, ovate family protein 8, 41 (3e-24) 1
ABIM01006789.1 superfamily A. thaliana, NP_197466.1
UN contig_30864, 1131 bp 376 aa DUF623 Ovate-like protein, Nicotiana tabacum, 44 (5e-60) –
ABIM01030815.1 superfamily ABW05088.1
Sun LG4 contig_18801, 1473 bp 491 aa – SUN, putative calmodulin binding 47 (7e-20) 4
ABO36639.1 ABIM01018776.1 protein, S. lycopersicum, ABO36630.1
UN contig_22697, 996 bp 331 aa – ELD1, elongation defective 1, 77 (1e-138) 2
ABIM01022664.1 A. thaliana, AAN33186.1
fw2.2 LG9 contig_1322, 675 bp 224 aa PLAC8 fw2.2, ORFX, Lycopersicon pennellii, 50 (1e-30) –
AAO12188.1 ABIM01001322.1 superfamily AAF74287.1
Putative homologous genes expressed when supported by EST of minimum 500 bp and 99% sequence identity

10,000 iterations. Six of these QTL were also
significant under the Kruskal–Wallis non-parametric
mapping model. Several QTL for fruit diameter and
length on LGs 04, 07 and 09 had significant LOD
scores but were responsible for B5% of the allelic
variance and thus were not included in the final
analysis. Four QTL clusters with effects on two to
four fruit traits are located on LGs 02, 03, 07 and 09.
Candidate genes for fruit trait QTL
The papaya genome sequence was searched for
homology to previously identified fruit size and
shape loci in tomato. Five putative orthologous
regions to tomato ovate protein sequence (GenBank
No. AAN17752.1) were identified by tblastn: one
each on LGs 01, 04, 05 and 07, and one located on an
unanchored WGS contig (Table 4). Two putative
orthologs to the tomato sun protein sequence (Gen-
Bank No. ABO36639.1) were identified on LG04 and
on an unanchored WGS contig. Only one putative
orthologous region on LG09 was identified with
tomato fw2.2 protein sequence (GenBank No.
AAO12188.1). Gene expression of the ovate ortholog
on LG07 and the sun ortholog on LG04 is supported
by expressed sequence tag (EST) evidence. The
linkage group position (cM) of each candidate gene
was estimated by locating mapped SSRs and
anchored WGS contigs to a single 2007-04-27
annotated papaya supercontig available at the Hawaii
Papaya Genome Project website (http://asgpb.
mhpcc.hawaii.edu/papaya) (Table 5). Contigs con-
taining two QTL, FW2.1K2 and FW9.1K2, responsi-
ble for greater than 20% of the allelic variance for
phenotype were searched for additional candidate
genes. Ctg-43, the SSR marker closest to FW2.1K2,
was localized to papaya supercontig_6 while the
P3K4311 locus associated with FW9.1K2 was local-
ized to papaya supercontig_19.
Discussion
A total of 14 significant QTL were detected for the
papaya fruit traits weight, length, diameter and shape.
Significant correlations were observed for each
phenotypic fruit trait pair except for between diam-
eter and length. One cluster of overlapping QTL
positioned at approximately 12 cM on LG09 affects
fruit weight, length and shape to explain 7.5–20.5%
of the allelic variance. A putative homolog to fw2.2
which has been proposed to link carbohydrate supply

123
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464
Table 5 Linkage group position of candidate fruit trait genes on AU9 9 SU high-density genetic map
Candidate gene WGS contig Genome supercontig
Ovate contig_13552 Supercontig_64
contig_17626 Supercontig_104
contig_6659 Supercontig_22
contig_6797 Supercontig_23
Sun contig_18801 Supercontig_120
fw2.2 contig_1322 Supercontig_2
with ovary cell proliferation in tomato flowers
(Nesbitt and Tanksley 2001) is also located on
LG09. Our current KD 9 2H94 genetic map is
incomplete, so enrichment with additional markers
may resolve the location of the QTL to the same
region as the fw2.2 homologous gene. A second
cluster of overlapping QTL having minor effects on
fruit length and shape is located on LG07 centered
around 1.0 cM and bounded by the CPM1550C0 and
ctg-41C0 SSR loci. These loci are located at 16.4
and 67.0 cM, respectively, on the AU9 9 SunUp
genetic map. A putative ovate orthologous gene is
predicted on LG07 near 85.4 cM based on EST
evidence for its position and expression. In tomato,
ovate contributes to fruit elongation through its
effect on ovary elongation during flower organogen-
esis (Grandillo et al. 1999; Ku et al. 1999, 2001).
EST evidence supporting a papaya ovate gene on
LG07 comes from a papaya flower cDNA library,
indicating the possibility of a similar role during
papaya flower development as in tomato. The
position of ovate on LG07 is approximate, as only
one locus from the genetic map could be anchored to
supercontig_23. Improving the linkage between the
papaya genetic and physical maps may eventually
show an overlapping position between ovate and the
FL7.1K2 QTL cluster.
Fruit shape in papaya has long been recognized as
a sex-related trait with female fruit exhibiting a
rounder, spherical shape and hermaphrodites exhib-
iting a more elongated, pear to cylindrical shape.
However, differences in flower shape have not been
considered as an indicator of papaya fruit shape or
size. Also, as this study examined only hermaphrodite
fruit, significant QTL on LG01 which carries the sex-
determining MSY region were not identified.
Several of the QTL detected for papaya fruit size
and shape show promise for use in MAS. The clusters
123
Mol Breeding (2012) 29:457–466
Nearest SSR locus LG cM
P6K1117CC 1 97.9
P3K1972CC 4 74.1
P6K108CC 5 56.7
ctg-456CC 7 85.4
CPM1024LCC 4 47.6
P3K6568CC 9 42.6
of QTL on LGs 02, 03 and 09 each influence two or
more fruit phenotypic traits. The QTL cluster on
LG02 is responsible for C15% of the allelic variance
for each of four traits and two of the individual QTL
(FW2.1K2 and FL2.1K2) are highly significant in the
non-parametric Kruskal–Wallis model. Exploration
of this region may reveal candidate genes for
pleiotropic effects while examination of the two
QTL responsible for 15.1 and 18.2% of the allelic
variance at FW3.1K2 and FD3.1K2, respectively, also
appears promising for MAS in papaya breeding
programs. In contrast, the QTL cluster at 1.0 cM on
LG07 contributes slightly more than 5% of the allelic
variance for fruit weight and length, probably through
minor effects on fruit length.
Identification of putative ovate, sun and fw2.2
genes on LGs containing QTL for papaya fruit size
and shape attributes is suggestive of similar functions
for these genes in papaya. A putative sun ortholog on
LG04 is supported by EST evidence; however it was
not associated with any QTL for fruit length or shape.
In tomato, sun contributes to fruit size through post-
fertilization regulation of ovary pericarp elongation
(Van der Knaap and Tanksley 2001). Population-
specific QTL are well documented (Gonzalo and Van
der Knaap 2008; MacKay 2001) and it may be that
sun does not play a major role in fruit elongation in
the papaya KD 9 2H94 mapping population. Simi-
larly, a papaya region orthologous to tomato fw2.2
was identified on LG09, but it is located outside of
the 1-LOD interval of the nearby FW9.1K2, FL9.1K2
and FS9.1K2 QTL. A putative ovate ortholog on
LG07 with EST support was not associated with any
QTL, but since the LG07 map is incomplete, an
enrichment of the KD 9 2H94 map may resolve this
position. Fine mapping of this region may provide
linkage between ovate and one of the flanking QTL
clusters.
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Mol Breeding (2012) 29:457–466
PCR tests are available to determine individual
sex-type, papaya fruit flesh color and presence or
absence of the coat protein transgene for resistance to
PRSV, though the last is more generally used in
regulatory programs to detect gene flow. Improve-
ment for phenotypic traits such as fruit size, shape
and flesh color and quality traits such as fruit
softening and sugar content is still contingent upon
physical screening of multiple generations of field-
grown populations. Cost, labor and land area further
constrain breeders’ selection for complex, quantita-
tive traits which require large breeding populations.
Application of MAS has been proven in breeding
programs for crops such as maize, wheat, rice, tomato
and grape (Dalbó et al. 2001; Harjes et al. 2008;
Huang et al. 1997; Tanksley et al. 1996; William
et al. 2007). Papaya breeding programs would greatly
benefit from MAS due to the generation time of the
species (9–15 months from planting to fruit set) and
relatively large space requirements (*5 m2 per
individual) compared to row crops such as maize
and tomato. Molecular screening of seedlings prior to
field planting could greatly reduce the land area
requirements for breeding populations and allow
breeders to selectively target specific components of
complex traits.
Worldwide fresh market consumption of papaya
fruit is on the rise, especially in developed countries
where papaya occupies a specialized exotic tropical
fruit niche market. In much of the developing world,
where nearly all of the commercial papaya supply is
produced—99.7% according to the Food and Agri-
culture Organization (FAOStat 2007)—consumption
of fresh papaya fruit is part of the World Health
Organization supported strategy to combat vitamin A
deficiency (Rodriguez-Amaya 2003; WHO 2007).
Papaya breeders are now working to develop varieties
with enhanced vitamin A nutrition that must be
tailored to local preferences for size, shape, color,
aroma, etc. Molecular characterization of the genetic
bases for these complex traits and development of
tools for MAS will enable breeders to develop and
release new papaya varieties more quickly than by
using conventional screening methods alone. The
study presented here provides a platform for further
investigation of QTL controlling fruit size and shape
characteristics. In future studies, inclusion of both
female and hermaphrodite F2 individuals may aid in
revealing major QTL affecting fruit shape.
465
Acknowledgments We thank the HARC Kunia Substation
Field Crew for their aid in set-up and maintenance of the
mapping populations. Special thanks are also given for the
assistance and technical support provided by Anne-Florence
Lava and Gail Uru. Funding for this project was provided by
the Hawaii Agriculture Research Center and a grant from the
USDA Tropical Subtropical Agricultural Research program via
the University of Hawaii.
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