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Identification and Confirmation of RAPD Markers and Andromonoecious Associated with QTL for Sucrose in Muskmelon S. O. Park, J.W. Sinclair, K.M. Crosby and K.S. Yoo G ...

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Identification and Confirmation of RAPD Markers and ...

Identification and Confirmation of RAPD Markers and Andromonoecious Associated with QTL for Sucrose in Muskmelon S. O. Park, J.W. Sinclair, K.M. Crosby and K.S. Yoo G ...

Identification and Confirmation of RAPD Markers and Andromonoecious
Associated with QTL for Sucrose in Muskmelon

S .O. Park, J.W. Sinclair, K.M. Crosby and K.S. Yoo G.E. Lester
Vegetable & Fruit Improvement Center Agricultural Research Service
Texas A&M University U.S. Department of Agriculture
Weslaco, Texas 78596
College Station, Texas 77845 USA

USA

Keywords: Cucumis me/u, glucose, fructose, su gar, total soluble solids

Abstract
Our objectives were to identify RAPD and andromonoecious (a) markers

associated with QTL for sucrose using bulked segregant analysis in an F 2 population
from the melon (Cucurnis inelo L.) cross of 'TAM Duke' (high sucrose) x TGRI55I
(low sucrose) in a greenhouse experiment, and confirm the association of RAPD and a
markers with sucrose QTL in an F 2 population from the different cross of 'Deltex'
(high sucrose) x TGRI55I in a field experiment. Continuous distributions for sucrose
were observed in the F 2 populations indicating quantitative inheritance for the trait. A
significant positive correlation was found between sucrose and total soluble solids
(TSS). Nine RAPD markers were detected to be significantly associated with QTL for
sucrose in the F2 population of the 'TAM Dulce' x TGRI55I cross in the greenhouse
based on simple linear regression. Five unlinked markers associated with QTL were
significant in a stepwise multiple regression analysis where the full model explained
39% of the total phenotypic variation for sucrose. Three to five of the nine RAPD
markers were also observed to be significantly associated with QTL for glucose,
fructose and TSS respectively, suggesting that in this cross four sugar traits are
controlled by the same QTL. Four RAPD markers were confirmed in the F 2 popula-
tion of the Deltex' x TGRI55I cross in the field to he consistentl y associated with
sucrose QTL. A significant association of a with sucrose was consistently expressed in
our populations under greenhouse and field environments.

INTRODUCTION
Sucrose, glucose and fructose are major factors determining mature melon fruit

sweetness. Due to consumer preference for sweet fruit, sugar content is a highly important
quality trait of different melon classes. The improvement of sugar content is one of the
most significant goals in breeding progranis of most melon types worldwide. Bulked
segregant analysis (BSA) is an efficient method to rapidly identify molecular markers
linked to a specific geDnNeAubfurlkosmfroFF,plants (Michelmore et al., 1991).
Molecular markers linked to genes for sweetness traits may improve the breeder's ability
to recover sugar genotypes and aid in the development of sugar cultivars. However.
markers associated with QTL for sucrose present in TAM Dulce' have not been reported.
Our objective was to identify RAPD and a markers associated with QTL for sucrose by
means of BSA in an F 2 population derived from the melon cross of -TAM Dulce' (high
sucrose) x TGR I 55 I (low sucrose) in a greenhouse experiment. Park et al. (1999)
emphasized the importance of confirming the marker-QTL associations in different
populations and environments before using molecular markers lor marker-assisted
selection in breeding programs. Thus, our additional goal was to confirm the association
of RAPD and a markers with sucrose QTL in an F2 population from the different cross of
'Deltex' (high sucrose) x TGRI551 in a field experiment.

MATERIALS AND METHODS
For identification of QTI,. 105 F 2 plants derived from the cross of' 'lAM Dulce' x

TGR 1551 were planted in a Texas A&M University-Weslaco greenhouse. 'TAM Dulce' is
a western shipper muskmelon type with high fruit quality, while TGRI55I is a wild type

Proc. XXVII II I('-S7 Struci. and Fund. (lenonlics off Ion. Plants 191
Ed.-in-Chief: R. Drew
Acta I Ion. 763. 1 S IS 2007

with lo fruit quality. For confirmation of QTL, 64 F 2 plants from the cross of Dcltex' x
TGRI55I were planted in a Texas A&M University-Weslaco field. 'Deltex' is a
commercial ananas cullivar. Data for sucrose were obtained from the two parental pairs as
well as the 105 and 64 F2 plants.
Total genomic DNA was extracted from the leaf tissLie of the 105 and 64 F 2 plants
using the method of Skroch and Nicnhuis (1995). A total of 500 Operon primers were
used. PCR was performed on 96-well plates in an Mi Research thermalcycler. Protocols
For PCR and the composition of the final volume of reactants were the same as those
described by Skroch and Nienhuis (1995). Low and high bulks were prepared from eight
selected greenhouse plants of the 'TAM Dulce' x TGR 1551 cross with the lowest and
highest sucrose values, respectively. The 500 primers were used to simultaneously screen
between the low and high DNA bulks, and between the parents 'TAM Dulce' and
TGRI55I. Primers that generated marker polymorphisms between the low and high hulks
were tested in the F 2 population from the cross between TAM Dulce' and TGRI55I for
identifying QTL. Eight primers were tested in the F2 population
TGR 1551 cross for confirming QTL. of the 'Deltex'

To detect segregation distortion of markers, population marker data were tested for
goodness-of-fit to a 3:1 ratio using the chi-square test. The linkage analysis was
performed using MAPMAKER version 3.0 (Lander et al., 1987). Simple linear regression
(SLR), for each pairwise combination of quantitative trait and marker locus, was used to
analyze the greenhouse and field data for detection and confirmation of QTL. Loci with
the lowest P value per QTL were chosen and then were added in a stepwise multiple
regression (SMR) to select the best set of markers for prediction of the total trait
phenotypic variation explained by the identified QTL (Paterson et al., 1991).

RESULTS AND DISCUSSION

Frequency Distribution
Clear separations for sucrose between the parents were observed. Fruits of 'TAM

Dulce' and Deltex' had high sucrose. In contrast, TGR 155 1 plants possessed low sucrose.
Frequency distributions for sucrose were skewed towards low values. Continuous
frequency distributions for sucrose were found in two F 2 populations derived from
different melon crosses in the greenhouse and field (Fig. I) indicating quantitative
inheritance for the trait.

Correlation
Sucrose was noted to be significantly and positively correlated with TSS and

sucrose percentage of total sugars in the two F populations in the greenhouse and field
experiments (Table I), indicating that selection of the three sweetness traits is feasible.
Significant negative correlations of sucrose with glucose percentage of total sugars and
fructose percentage of total sugars were found in the two genetic populations.

Identification of QTL
Nine RAPD markers were polymorphic between the two different DNA hulks. An

example of marker OAA09.350 is shown in Fig. 2. These nine marker fragments
segregated in the F 2 population of the 'TAM Dulce' x TGR 1551 cross. A goodness-of-lit
to a 3:1 ratio for band presence to band absence for each of the nine markers was
observed in 105 F 2 plants (Table 2). These markers were unlinked based on linkage
analysis. Nine RAPD markers were detected to he significantly associated with QTL for
sucrose in the F 2 population of tile TAM Dulce x TGRI55I cross in the greenhouse based
on SLR (Table 3). High sucrose alleles for these markers were contributed by the high
sucrose parent 'TAM Dulce'. Five unlinked markers associated with QTL were
significant in a SMR analysis where the full model explained 39% of the total phenotypic
variation for sucrose. Three and live of the nine RAPD markers associated with QTL for
sucrose were also observed to be significantly associated with QTL for glucose and
fructose in the population.

192

ConfirmCaotniofinrmofatQioTnLof the associations of the RAPD markers with QTL for sucrose
identified in the F 2 population of the TAM Dulce' x TGRI55I cross in the greenhouse
was needed in other populations of the same cross and or a different cross in different
environments to substantiate their merit in melon breeding. Of the nine RAPD markers
associated with sucrose detected in the greenhouse population, seven were also
polymorphic between the parents Deltex' and TGRI55I that were utilized to create the
nsrFeeo2 mvpseeoangpinrumeilngaagatritkotiwoenrnosfroamoflsmOaorAkwsePehrgOsircew3hg.e8atrh0tee0ednsaoiunntcdrttoheOssetAeeFWdtr2af0piot6or.wp6ca0uos0lnaefitnviiroamtlnhuieanotgpfeadttrhheienenmt'tsDha'eDerklfieieeelrtxl-edQxxe'TTxapnLGedaRrisTmIs5Goe5cRniItaI.t5ciD5rooInu,s.estAh.toAell
3:1 goodness-of-fit ratio for band presence to band absence for each of the seven markers,
three amplified from 'Dcltex' and four amplified from TGR155 I, was observed.
Four RAPD markers associated with sucrose in the greenhouse experiment were
Fas2spoocpiautleadtiownitohf the 'Deltex' x TGRI551 cross in the field experiment to
cboencfiormnseidstienntthley sucrose QTL on the basis of SLR (Table 4). Marker
OAA09.350 from TGRI55I associated with sucrose QTL in the greenhouse population
was also found to be significantly associated with QTL for sucrose in the field population.
A significant association of OAW06.1250 from 'Deltex' with sucrose QTL was
consistently expressed in our populations derived ftmrhoeamrgkreetrwesno(hOoduAifsfTee0rFe32n.2pt5oc0pr,ouOlsasAteiosQnuI,3nw.d7e5er0re
greenhouse and field environments. However, three
and OAS03.450) associated with sucrose identified in
not confirmed in this field F2 population.
The andromonoecious locus on linkage group 4 of the classical melon map (Pitrat,
1991) regulating stamen absence or stamen presence in female flowers was observed to
he weakly but significantly associated with sucrose in the 'TAM Dulce' x TGR155I cross
in the greenhouse (Table 3). This association was confirmed in the high sugar andro-
monoecious 'Deltex' x the low sugar monoecious TGRI55I cross in the field (Table 4).
The a locus highly associated with sucrose in the field population accounted for 15% of
the sucrose variation.

CONCLUSIONS
The sucrose trait was quantitatively inherited. A positive correlation was found
between sucrose and TSS. Nine RAPD markers were significantly associated with QTL
for sucrose in the greenhouse population based on SLR. Five unlinked markers associated
with QTL were significant in a SMR analysis where the full model explained 39 1/0 of the
total sucrose variation. Three and five of the nine markers were also associated with QTL
for glucose and fructose in the greenhouse population. Four RAPD markers were
confirmed in the field population to he consistently associated with sucrose QTL. A
significant association of a with sucrose was consistently expressed in tRwAoPdDiffaenrednta
genetic populations under greenhouse and field environments. These
markers associated with the sugar synthesis QTL identified and confirmed here could he
useful in melon breeding for improving mature fruit sweetness.

ACKNOWLEDGEMENTS
This research was funded in part by USDA Grant: 2001-34402-10543. 'Designing

Foods for Health'. We appreciate technicians, Alfredo Rodriguez and Hyun J. Kang,
Texas Agricultural Research and Extension Center-Weslaco, for their assistance.

Literature Cited P., Abrahamson, J., Barlow, A.. Daly, Mi.. Lincoln, S.E. and
Lander, ES., Green.
Newburg, L. 1987. MAPMAKER: an interactive computer package for constructing
primary genetic linkage maps with experimental and natural populations. Genomics

1:174 1 81.
Michelmore, R.W., Paran. I. and Kesseli. R.V. 1991. Identification of markers linked to

193

disease resistance genes by bulked segregani analysis: a rapid method to detect
markers in specific genoinic regions using segregating populations. Proc. NaiL Acad.
Sci. USA 88:9828-9832.
Park, SO., Coyne, D.P., Mtitlu, N., Jung, G. and Steadman, J.R. 1999. Confirmation of
molecular markers and flower color associated with QTL for resistance to common
bacterial blight in common beans. J. Amer. Soc. Hon. Sd. 124:519-526.
Paterson, Al-I., Damon, S.. Hewitt, J.D., Zamir, D., Rabinowitch, H.D.. Lincoln, SE.,
Lander, E.S. and Tanksley, S.D. 1991. Mendelian factors underlying quantitative traits
in tomato: comparison across species, generations, and environments. Genetics
127:181-197.
Pitrat, M. 1991. Linkage groups in Cucumis ,nelo L. J. Heredity 82:406-411.
Skroch, P.W. and Nienhuis, J. 1995. Qualitative and quantitative characterization of
RAPD variation among snap bean genotypes. Theor. AppI. Genet. 91:1078-1085.
Tables

Table I. Pearson correlations of sucrose with other sweetness traits including total soluble
solids (TSS) in two F 2 populations derived from different melon crosses of 'TAM
Dulce' x TGRI55I (TT) and 'Deltex' x TGRI55I (DT) in greenhouse and field
experiments.

Sweetness traits

Trait Cross Glucose Fructose TSS SPTS' GPTS FPTS

Sucrose TT 0.32** 0. 51** 0.66** 0.89** 070** _067**
DT 0.69** 0.48** 0. 57** 0.98** 0.94** 0.89**

SPTS = sucrose percentage of total sugars. GPTS glucose percentage of total sugars, FPTS - fructose
percentage of total sugars.

Significant at P < 0.01

Table 2. Chi-square anal y ses for segregation of nine RAPD markers and andro-
monoecious (a) associated with sucrose in an F2 population derived from the melon
cross of 'TAM Dulce' x TGR 1551 in the greenhouse experiment.

F2 plants (no.)

Marker Source Presence Absence Ratio

0OOAAWIJ0I36..11325500 .TTAAMM DDu5ullccee 8705 32350 0.x022 00..P8476
887125 223340 0.53 0.46
OATO3 .250 TAM Dulce 777709 322586 0.38 0.54
OAAO9.350 TGRI55I 0.15 0.69
OAPO3. 800 TGR 1551 0.00 0.96
3.45 0.06
OAO 13.750 TGR 1551 80 25 0.08 0.78
OASO3 .450 TGRIS5I 0.02 0.87
OAUO5.600 TGRI55I 0.91 0.34

OAWO6.600 TGRI55I 74 (mono) 31(u)

a TGR155I

194

Table 3. Simple linear regression (SLR) and stcp ise multiple regression (SMR) analyses
of marker and data for detection of QTL for sucrose in an F 2 population derived from
the melon cross of TAM Dulce' (high sucrose) x TGR 1551 (low sucrose) in the
greenhouse.

SLR Average value SMR

Marker Source P R2(%) Presence Absence P R(%)

OAUI3.1350 TAM Dulce 0.000 10 9.11 4.7> 0.000 10

OAW06.1250 TAM Dulce 0.008 7 8.7 5.0

OAT03.250 TAM Dulce 0.020 5 8.7 5.6
Cumulative R = 10

OOOOOOa'AAAA0AAAWPUSQOOOOO1933356.....48.7636505500000000 TTTTTTTGGGGGGG.RRRRRRRI11IIII55SSSS5SS5555S1IIIIII00000000.......000000000010143005856 1I68674S2 0 6666767.......4718907 0111119921000..76.....69586 0000....000000041294 146838
Cumulative R2 = 39

'An average sucrose value of F2plants with hand presence for the marker.
An average sucrose value off, plants with band absence for the marker.
'andromonoecious.

Table 4. Confirmation of four RAPD markers and andromonoecious (a) associated with
QTL for sucrose in an F2 population derived from the melon cross of 'Deltex' (high
sucrose) x TGRI55I (low sucrose) in the field.

Simple linear regression Stepwise multiple
regression

Marker Source P R2(%) P

O0AW06.1250 T.AM D0ulce & Deltex00.002 92 9

0AU13.1350 TAM Dulce& Deltex 0.040 4 0.040 4

aO AAO9.350 TTGGRRII5S5SII 00..000006 175 Cumulative R= 13
OA U05 .600 TGRI55I 0.0)8 7 0.000 15
0.006 7
0.008 7

Cumulative R2 = 29

195

Figures

GreenhouseTAM Dulce 45.2 I Fie0ld.
TGRI55I 3.7 -
i 3)- Fl Mean 7.7 I)eltex 27.3
C F2 Mean 10.8 TGRI55I 1.1
Fl Mean 15.0
Li IF2 SD 6.2 " F2 Mean 13.3
F2 SD 9.2

I--.-
aii-o000 SI-ISO Nil-ISO ISI-3IO
3)1-3)0 311-3)0 3)1-11(I 00 51.100 Nil-Lift LSl-3)0 3)1-3)0 3)1-110 311-3)1) 3)14)0

Sucrose (mg-g- 1 ) Sucrose (mg-g-1)

Fig. 1. Frequency distributions for sucrose of F2 plants derived from two melon crosses of
'TAM Dulce' (high sucrose) x TGRIS5I (low sucrose) and 'Deltex' (high sucrose)
TGR155I in greenhouse and field experiments.

1 500bp

6 SOb p

4 300bp

Fig. 2. RAPD marker OAA09.350 expressing polymorphism between two DNA bulks
from high and low sucrose F 2 plants, between the high sucrose parent 'TAM
Dulce' and the low sucrose parent TGR1551, and between the high sucrose parent
'Deltex' and the low sucrose parent TGRI55I. 1='TAM Dulce', 2=TGRI55I,
3=DNA 2bpullaknftrso, m5='eDigehlttehxi',g6h=sTuGcrRos1e5F512,palnadnt7s=, 4a=10D0N-bpADbNulAk
sucrose F from eight low
marker ladder.

196


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