Molecular Plant Breeding 2015, Vol.6, No.15, 1
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used for marker analysis (Kumar et al., 2008). For
example, (Hormaza et al., 1994) found only one
female-specific RAPD marker in pistachio after
testing 700 primers. On the other hand, Kumar et al.
(2012) reported five bands from 41 random decamer
primers were associated with sex expression. In
present study, the polymorphic fragments from
OPC05 appear to have the potential for development
of a molecular marker based technique for early
identification of pointed gourd sex expression. This
indicates that sex determination in
T. dioica
is under a
simple genetic control, possibly by a single gene or
few tightly-linked genes. Similarly Sakamoto et al.
(1995) screened only 15 decamer primers and found
two markers, viz., Pri-08500 and Pri-11730 that
appeared to be tightly linked to the male sex. If the
sex expression is governed by heteromorphic
chromosomes, a large number of sex-specific bands
should be appeared (Jiang and Sink., 1997). Similar to
present result, Kumar et al. (2008) also reported a
male sex associated RAPD fragment of 1000bp from
OPC-05 in pointed gourd. Cloning and sequencing of
OPC-05
1000
and subsequent BLAST analysis revealed
that this sequence shared 93% identity with
Salix
Chamissonis
.
SCAR primer SCF/R-3 proved to be highly effective
for the discrimination of male and female accessions.
This result suggests that the SCAR marker is located
in a region of the chromosome responsible for
maleness as it is present only in male. However, this
marker failed to discriminate one accession among 44
female accessions. This may be due to crossing over
of male linked gene from Y chromosome with X
chromosome or amplification of unspecific product in
female may also occur due to presence of some
sequence similarity. The same pattern of result was
obtained by Chaves-Bedoya and Nunez (2007) in their
study to discriminate male and female plants in
papaya. In case of female specific
OPK-07
300
amplicon, the marker was failed to discriminate male
and female accessions. This may be due to either
small size of the fragment obtained during initial
characterization or a single base differences or single
base mutation at the primer. The result is in agreement
with Niroshini et al. (2008) in papaya. However, the
SCAR marker SCF/R-01 was monomorphic and did
not discriminate female from male plants. This loss of
polymorphism during RAPD to SCAR conversion
was most likely due to site mutation at the original
RAPD primer binding site (Paran and Michelmore,
1993). This is the first report on the development of
SCAR marker in pointed gourd that can be used to
discriminate male plants from female plants at early
stages of development to support breeding programs.
The results presented in this study show a possible
new SCAR marker that can be used. This reduces the
time required differentiating male and female plants
which depends on the evaluation of morphological
characters. The marker can also be used to discriminate
male and female plants produced at commercial scales
in tissue culture laboratories at seedling stage.
3 Materials and methods
3.1 Plant material and DNA extraction
Plant materials used in this study included 10 male
and 44 female genotypes, currently being cultivated in
Gujarat,
were sampled from the different area of
Gujarat state. Genomic DNA from three weeks old,
field grown plants (raised from stem cuttings) was
extracted using a modified cetyltrimethyl ammonium
bromide (CTAB) protocol described by Singh et al.
(2002). The genomic DNA quality and quantity were
assessed through Nano drop V2.02 spectrophotometric
measurement and uncut lambda DNA.
3.2 Screening of RAPD
For the initial screening, only
two bulk DNA samples
(male and female) were prepared by pooling an equal
amount of DNA from 10 individual male and female
cultivars. These bulks were amplified with 104 RAPD
primers. Once polymorphism was detected with a
particular primer, then the primer was used to amplify
the DNA samples from individual plants for
sex-typing. The polymorphic markers were verified
for their consistency and reproducibility using DNAs
from 10 random male plants and 10 female plants
from field. Putative sex-linked markers which
differentiated the male and female bulks as well as the
individuals of each sex are used for developing the
SCAR marker. PCR
Amplifications were carried out
in a 25µl reaction volume containing 2µl DNA (50 ng),
12.5 µl Master Mix (Genei, Bangalore, India), and 1µl
of 10 pmol of primer. Primer concentrations
viz
., 5, 10
and 20pM were used to optimize the amplification
conditions. Based on the results, 10pM concentrations
of primers were used for RAPD analysis. Amplification
was performed in following steps: initial denaturation
