Molecular Plant Breeding 2015, Vol.6, No.15, 1
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rizing molecular markers and genes involved in plant
dioecy (Yakubov et al., 2005). Previous studies have
provided information indicating that use of molecular
markers in breeding programs can save time and
economic resources in breeding programs. Several
researchers have shown that random amplified
polymorphic DNA (RAPD) banding patterns are
linked to sex in (Esfandiyari et al., 2012). RAPD is a
rapid and inexpensive method for studies on genotypic
relationships and selection of traits of interest.
Sequence characterized amplified regions (SCARs)
are commonly derived from RAPD markers and
represent a genetically defined locus, which is
amplified from the genomic DNA by specific
oligonucleotide primers. As there is only single report
on the sex associated molecular markers of pointed
gourd (Nanda et al., 2013), this study can also be
regarded as the leading report on the use of the SCAR
technique for gender determination in pointed gourd.
1 Result
1.1 Identification of sex-specific RAPD markers
Of 104 RAPD primers evaluated, 21 produced clear
and reproducible DNA profiles with a total of 971
amplification bands for the bulked sample of female
and male plants of which 96 bands were polymorphic.
The approximate size range of the RAPD products
was 200 bp (22E1) to 2000 bp (OPA-17). Only two
markers, OPC-05 and OPK-07, were producing
distinguish banding pattern of 1.0 kb (named as
OPC-05
1000
; Fig. 1) and 300 bp (named as OPK-07
300
;
Figure 2) and were considered as associated with
maleness and femaleness, respectively. These amplicons
were extremely reproducible under a broad range of
amplification temperature without any variation in the
results. The marker OPC-05
1000
amplified a unique
band in bulk DNA of male and 10 individual male
DNA, whereas similar fragment is not observed in the
bulk and 44 individual females. Similarly, marker
OPK-07
300
amplified a unique fragment only in the
bulk and individual females. This amplification was
repeated for five times in individuals as well as bulk
genomic DNA corresponding to male and female
accessions to confirm the reproducibility and
consistency. Similar results were obtained in each
repetition. The bands were consistently presence/absent
in the bulk and male/female individuals. These unique
markers indicating that both markers was closely
linked to the sex gene and are candidate for SCAR
Figure 1 A male-specific RAPD fragment in pointed gourd
amplified using primer OPC-05. M indicated size marker. The
arrows denote the position of the 1000-bp fragment, and cause
gender differences among female and male plants
Figure 2 A female-specific RAPD fragment in pointed gourd
amplified using primer OPK-07. M indicated size marker. The
arrows denote the position of the 300-bp fragment, and cause
gender differences among female and male plants
marker development, hence will useful for determining
sex in pointed gourd.
1.2 Conversion of OPC5
1000
and OPK-07
300
to
SCAR markers
As the fidelity and the reproducibility of RAPD
marker is often questionable, the current trend is to
convert the RAPD sequence into a SCAR marker
which is more reliable and reproducible. Based on the
sequence of OPC5
1000
and OPK-07
300
, five and one
pair of specific forward and reverse SCAR markers
were designed, respectively (Table 1). The designed
SCAR primer pairs were used to amplify genomic
DNA of all 54 accessions. Among five primer pair
combinations designed from male specific sequence,
SCF/R-3 marker discriminated male and female
