Molecular Plant Breeding 2015, Vol.6, No.15, 1
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6
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Table 1 Sequence of SCAR primer used for male and female
identification
Name of Primer
Sequences
SC F-1
GTCCGAAGCCCATATTTCCTC
SC R-1
GGACTCACTGCCAATTCTACC
SC F-2
CGAAGCCCATATTTCCTC
SC R-2
CTCACTGCCAATTCTACC
SC F-3
AGTGCCCAAACATAGTCG
SC R-3
GGACTCACTGCCAATTCTAC
SC F-4
GGAAGACCCAAAGGACTTAC
SC R-4
CCTCAGAAATGGGAGCAAAC
BOT F
TTGTGGGGAAAGGTTGAGAG
BOT R
GCCAATTCTACCAGCCCATA
SCFF-1
#
CGCTTTCTTTCCCTTCCTTT
SCFR-1
#
TGTGGAATTGTGAGCGGATA
#
Female specific forward and reverse primers designed from
OPK-07
300
accessions. This SCAR amplification produced a
single specific band of predicted size of 628 bp in all
the males without any amplification in the females.
Reduction of the annealing temperatures did not
generate any spurious fragment other than the specific
SCAR,
confirming the specificity of the SCAR primer
for maleness. However, this marker failed to discriminate
one accession among 44 female accessions. With this
exception, remaining 43 female accessions were
clearly discriminated by this marker. In case of female
specific
OPK-07
300
amplicon, with small size of
sequence, Primer3 could design only one pair of
primer. But this marker was failed to discriminate
male and female accessions.
1.3 BLAST analysis of male sex specific RAPD
amplicons
The OPC-05
1000
with 38% G+C content (A:371;
C:211; G:212; T:323) did not possess any open
reading frames in either orientations. BLAST results
revealed that the sequence has partial homology
(ranging from 54.8% to 59.8% identity over a length
of 169-232 bp) with known plant nucleotide sequences at
different sequence-similarity levels. BLAST searches
were made to determine if the sequenced fragment
might identify a known gene or mapped sequence.
The Phylogenetic tree obtained based on sequence
similarity revealed that
Solanum lycopersicum
,
Populus trichocarpa, Lycopersicon esculuntum
and
L.
pimpinellifolium
were similar and grouped together. In
the same cluster,
Salix chamissonis
and
Phoenix
dactylifera
were also present, but
Trichosanthes dioica
Roxb and
Coccidiodes posadassis
were not clustered.
This reveals that, even though sequences obtained in
present studies are similar to the sequences of above
mentioned plant species, but they are not identical to
them.
2 Discussion
Dioecy prevents intra-individual self-pollination and
is one of the most extreme inbreeding avoidance
mechanisms (Ainsworth, 2000). Efforts to identify sex
type in dioecious plant at seedling stage is important
for selecting female or hermaphrodite plants for
transfer to the field, to gain time and reduce costs
(Chaves-Bedoya and Nunez, 2007). To date, several
molecular markers including RAPD have been
exploited for sex type discrimination in dioecious
plants. RAPD, without prior DNA sequence information,
can reveal high degree of polymorphism; easy to
perform and provides wider coverage of the genome.
However, RAPD might lead to irreproducibility;
therefore RAPD marker was converted into a SCAR
to distinguish sex type in pointed gourd (Paran and
Michelmore, 1993). This would increase the reliability
and reproducibility of PCR assays. SCARs are
advantageous over RAPD markers because they are
co-dominant, detect only single genetically defined
loci, identified as distinct bands in agarose gels, are
easier to score, less sensitive to reaction conditions
and are more reproducible. The SCAR has been
successfully developed in the case of
Mercurialis
annua
(Khadka et al., 2002),
Carica papaya
(Urasaki
et al., 2002) and
Cannabis sativa
(Mondolino et al., 1999).
Pointed gourd has also been studied and found to have
a RAPD marker associated with females (Singh et al.,
2002; Kumar et al., 2012). Since pointed gourd is an
importantdiocious vegetable crop, the SCARs
developed in present study will be useful for its
improvement. To identify the sex associated genome
segment, sufficient number of RAPD primers were
tested before generating the SCAR marker. This is in
agreement with Jiang and Sink (1997) where 760
primers were used in
Asparagus officinalis
in order to
find a SCAR marker associated with the M
(male-determining) locus.
The chances of any RAPD
markers being linked to a gene or a genomic region of
interest is mainly dependent on genome size, type of
gene or genomic region and on the type of population
