Construction of a RAPD Marker-Based Linkage Map in Ananas Melon
Soon O. Park and Kevin M. Crosby
Texas Agricultural Research and Extension Center, Texas A&M University, Weslaco, TX 78596
and Vegetable & Fruit Improvement Center, Texas A&M University, College Station, TX 77843
Introduction: Melon (Cucumis melo L.)
fruit flesh is a significant source of
carbohydrates, ascorbic acid, beta-carotene,
folic acid, and potassium. Sucrose is a major
factor to determine mature melon fruit
sweetness. Ascorbic acid and beta-carotene
are major nutrients for human health. Due to
consumer preference, these are highly
important quality traits of different melon
classes. The improvement of these traits is
important to breeding programs of most
melon types worldwide. Diseases of melon
are primary constraints limiting melon
production. A yellowing disease, caused by
cucurbit yellow stunting disorder virus that
is transmitted by Bemisia tabaci, is common
in South Texas. Molecular tagging and
mapping information for these fruit quality
traits and disease resistance is expected to be
useful to melon breeders because of the
possibility to use molecular markers for
marker-assisted selection in their breeding
programs. Therefore, our initial objective
was to construct a RAPD marker-based
genetic linkage map in an F2 population
derived from the melon cross of >Deltex= x
TGR1551 for conducting research on the
genetics of melon fruit quality and disease
resistance.
Materials and Methods: One hundredeight F2 plants derived from the cross of
>Deltex= x TGR1551 were planted in a
greenhouse at the Texas Agricultural
Research and Extension Center-Weslaco in
2003. The >Deltex= parent is a commercial
ananas cultivar with high fruit quality, while
the TGR1551 parent is a wild type with low
fruit quality. Total genomic DNA was
extracted from the leaf tissue of the 108 F2
plants along with their parents (4). A total of
360 random 10-mer primers (Operon
22
Technologies, Alameda, Calif.) were used
for the RAPD analysis (5). PCR was
performed on 96-well plates in an MJ
Research thermalcycler (model PTC-0100;
MJ Research, Waltham, Mass.). Protocols
for PCR and the composition of the final
volume of reactants were the same as those
described previously (4). A 100-bp DNA
ladder (Life Technologies, Grand Island,
N.Y.) was used to estimate the length of
RAPD markers. The name of each RAPD
marker is derived from an AO@ prefix for
Operon primers, the letters identifying the
Operon kit, Operon primer number, and the
approximate length (bp) of the marker (3).
The 360 primers were used to screen
between the parents >Deltex= and TGR1551.
Primers
that
generated
marker
polymorphisms between the parents were
tested in the F2 population from the cross
between >Deltex= and TGR1551 to assess
genetic linkage of RAPD markers to the
traits of interest. To detect segregation
distortion of markers, F2 population marker
data were tested for goodness-of-fit to a 3:1
ratio using the chi-square test. The RAPD
marker-based linkage map was constructed
on the data for the 108 F2 plants of the
>Deltex=
x
TGR1551
cross
using
MAPMAKER version 3.0 (2). On the basis
of a LOD score of 3.0 and a linkage
threshold of 0.4, linkage groups were
displayed using the Group command. To
establish a linkage group, a subset of
markers was initially selected based on LOD
scores and pairwise linkages. The best
linkage order within the subset was
calculated using the Compare command and
then, additional markers were inserted using
the Try command. LOD scores of at least
2.0 were considered different between the
most and second most likely position for the
marker. The Ripple command was finally
Cucurbit Genetics Cooperative Report 28-29: 22-25 (2005-2006)
used to check the marker order. Map
distances (cM) between ordered loci of
markers
were
calculated
using
recombination fractions and the Kosambi
mapping function (1).
Results and Discussion: A total of 208
RAPD markers that segregated in the F2
population of the >Deltex= x TGR1551 cross
were scored for constructing the genetic
linkage map (Table 1). All markers
displayed an amplified DNA fragment in the
>Deltex= parent that was absent in the
TGR1551 parent. An example of marker
OE08.600 obtained from the >Deltex= parent
is shown in Figure 1. Of the 208 markers,
195 (94%) fit the expected 3:1 ratio in the
F2 population on the basis of the chi-square
goodness-of-fit test (Table 1). Thirteen
markers
(6%),
however,
deviated
significantly from the expected 3:1 ratio (P
< 0.05) in the genetic population. Thus, we
excluded the 13 distorted markers in
developing the linkage map.
One hundred and ninety-five RAPD markers
were used for constructing the genetic map
(Table 1). These non-distorted markers were
divided into 12 linkage groups, three
unlinked pairs (UP), and ten unassigned
markers. We developed the molecular
marker-based linkage map with 185 RAPD
markers (Figure 2). The number of nondistorted markers per linkage group ranged
from three on linkage group 12 to 36 on
linkage group 6 (Table 1). An average of
14.9 markers were mapped per linkage
group. Our linkage map included 157
marker loci spanning a total map distance of
1148 cM. The number of loci per linkage
group varied from three on linkage group 12
with a length of 36 cM to 30 on linkage
group 6 with a length of 178 cM. An
average of 12.6 loci were located per linkage
group. Each linkage group spanned an
average length of 91 cM.
This genetic linkage map will be utilized to
identify markers linked to QTL controlling
mature melon fruit sweetness, quality, size,
and shape traits as well as disease resistance,
and to determine the genetic relationships
among QTL for these important traits in the
F2 population derived from the >Deltex= x
TGR1551 cross.
Literature Cited:
1. Kosambi, D.D. 1944. The estimation of
map distances from recombination
values. Ann. Eugenics 12:172-175.
2. Lander, E.S., P. Green, J. Abrahamson,
A. Barlow, M.J. Daly, S.E. Lincoln, and
L. Newburg. 1987. MAPMAKER: An
interactive computer package for
constructing primary genetic linkage
maps with experimental and natural
populations. Genomics 1:174-181.
3. Park, S.O., D.P. Coyne, J.R. Steadman,
K.M. Crosby, and M.A. Brick. 2004.
RAPD and SCAR markers linked to the
Ur-6 Andean gene controlling specific
rust resistance in common bean. Crop
Sci. 44:1799-1807.
4. Skroch, P.W. and J. Nienhuis. 1995.
Qualitative
and
quantitative
characterization of RAPD variation
among snap bean genotypes. Theor.
Appl. Genet. 91:1078-1085.
5. Williams, J.G.K., A.R. Kubelik, K.J.
Livak, J.A. Rafalksi, and S.V. Tingey.
1990. DNA polymorphisms amplified by
arbitrary primers are useful as genetic
markers. Nucl. Acids Res. 18:65316535.
Cucurbit Genetics Cooperative Report 28-29: 22-25 (2005-2006)
23
Table 1. Illustration of the molecular marker-based linkage map on the basis of 208 RAPD
markers segregating in 108 F2 plants derived from the >Deltex= x TGR1551 cross.
Linkage
No. of
No. of
Map
Mean
No. of distorted
group
markers
loci
distance (cM) distance (cM)
markersz
1
23
18
130
7.2
1
2
20
16
106
6.6
1
3
8
7
91
13.0
1
4
14
12
93
7.8
1
5
11
10
82
8.2
1
6
38
30
178
5.9
2
7
11
10
153
15.3
1
8
13
11
40
3.6
0
11
39
3.5
0
9
15
10
17
12
67
5.6
1
11
16
11
80
7.3
1
12
3
3
36
12.0
0
Unlinked pair 1
2
2
27
13.5
0
Unlinked pair 2
2
2
4
2.0
0
Unlinked pair 3
2
2
22
11.0
0
Unassigned markers
13
3
Total
208
157
1148
7.3
13
z
Markers deviating from the expected 3:1 ratio (P < 0.05) were not included in the map.
P1 P2 M
-1500bp
-600bp
Figure 1. Segregation of RAPD marker OE08.600 amplified from >Deltex= in an F2 population
derived from the melon cross of >Deltex= x TGR1551. Lines1-18=F2 plants of the cross,
P1=TGR1551, P2=>Deltex=, and M=a 100-bp DNA marker ladder.
24
Cucurbit Genetics Cooperative Report 28-29: 22-25 (2005-2006)
0
1
OJ04.1000
0
2
OM11.750
0
3
OC14.1400
0
4
OK10.1500
0
5
OM18.600
0
6
OF15.300
OF16.300
0
7
OB06.900
OK10.550
OG18.500
OJ09.350
OH03.500
OJ20.500
OR15.800
OH19.1300
OA07.800
OA10.1250,OA04.2400
OC18.900,OA01.450
OJ16.300,OJ09.800
OM01.500
OM09.500
OG06.750
OJ04.550
OA19.900
OM06.500
OI04.600,OR02.400
OG11.1000,OAS14.350
OL15.300
OJ19.450
OAP03.600
OA09.300
OG17.800
OC19.450
OM20.700
OH16.500
OI20.650
OG17.1050
OI06.800
OR04.900,OAS14.450
OC09.800
OL04.1500,OM14.1500
OI19.1200,OK01.600
OK03.450
OM11.1250
OG16.650
OE08.600,OB20.1500
OF10.600
OB16.1150
OB16.1000
OH16.600
OD08.750
OD08.400,OM10.1200
OA09.850
OK01.400
OC15.1400
OJ15.550
OJ15.1300
OC15.850
0
0
9
OL20.850
8
10
OM09.2200
40
39
0
0
11
OK20.700
UP1
OI03.600
0
12
OD13.700,OC02.600
OK12.700
OE01.1350
OG12.400
OF12.400
OI06.500
OI12.500
OI19.300
OK15.1000,OK10.2000
OI19.650
OH11.250
OB06.1250
OL12.800,OAT03.250
OB14.1400,OM13.300
OM01.950
OE04.300,OI16.750
OF05.500
OH07.600
0
4
OA15.850
OE14.700
OM11.500
OB12.2000
67
27
OG09.300
OK06.650
OK06.550
OL18.500
OL07.1100
OI18.350,OJ07.400
OC07.350,OK04.1050
OC02.550
OC01.800,OR06.650
ON08.350
OA15.400
OP17.900
OC09.1600
OK20.1050
OK13.600
OB12.550
OA16.450
OJ14.300
OC16.200,OM11.1150
OM11.950,OJ19.400
OK01.1250
OM11.2000
OK16.900
OA12.600
93
OO05.2100
OJ04.450
OM07.400
82
OG17.600
106
0
OK16.800
OC09.600
OI12.300
OD07.2100
OA05.1600
OJ07.900
91
130
OQ15.1500
OH07.300
OQ15.1000
OAU05.700
OK17.600
OB17.1050
OM18.300
ON08.600
OA18.650
OA16.1000
OK20.800
OA02.1500
OL12.2100
OR02.850
36
OE09.450
OF03.1600
OAA09.650
OC11.450
OM06.700
OA05.450,OA18.1300
OC20.700
153
OC11.700
OK14.1350
OB13.1750,OP17.600,OAU05.350
OA02.850,OM17.1000,OE01.250
OB04.900
OG17.250
OI10.550
OF04.600
OH07.700
OJ11.850
OG09.650
UP2
OM14.500
OD13.600
178
80
OH06.1600
0
OE17.400
UP3
OI11.1900
OC02.1250
22
OK04.600
OK17.700
Figure 2. The RAPD marker-based linkage map constructed using an F2 population of the >Deltex= x
TGR1551 cross. The marker names are given on the right and the length in cM is indicated on the
left of each linkage group.
Cucurbit Genetics Cooperative Report 28-29: 22-25 (2005-2006)
25