Molecular Plant Breeding 2015, Vol.6, No.15, 1
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at 94°C for 5 min, 40 cycles of denaturation at 94°C
for 1 min, primer annealing at 38°C for 1 min,
extension at 72°C for 2 min, and final extension at
72°C for 7 min. Electrophoresis was performed in
1.5% agarose gels. Band size was estimated by
comparison to 100 bp ladder DNA standard (Fermantas).
Cloning and sequencing of sex specific fragments
To convert the sex-diagnostic RAPD band to a SCAR
marker, the bands were excised from 2% Agarose gel.
Elution and purification of PCR fragment was
performed using Qiagen-gel extraction and purification
according to manufactures protocol. The purified PCR
product was quantified by ethidium bromide spotting
method. The eluted DNA was re-amplified with the
corresponding RAPD primers to verify whether the
fragment size was amplified consistently. The purified
DNA fragments were ligated into a PCR cloning
vector pTZ57R/T vector using the TOPO-TA cloning
kit (Invitrogen) according to the manufacturer
directions. The chimeric plasmid was transformed to
E. coli
strain DH5ά competent (Sambrook and Russell
2001). Confirmation of the successful cloning was
carried out by amplifying the chimeric plasmid DNA
with universal M13F/R primers. The total DNA and
cloning vector were used as positive and negative
controls in the PCR.
PCR reactions for cycle-sequencing were performed
using the following profile: 25 cycles of 1 min at 96˚C,
10 s at 96˚C, 5 s at 50˚C and 4 min at 60˚C. Cloned
fragments were sequenced by 3130 Genetic Analyzer
(Applied Biosystems, USA) using BigDye
®
Terminator
v3.1 Cycle Sequencing FS kit (ABI). The sequence of
male and female specificity obtained was trimmed to
define PCR end points by using sequence analysis
software 5.2.0. The sequences of forward and reverse
primers were aligned using BioEdit software. The
aligned sequence was subjected to BLAST analysis, at
of the National Center
for Biotechnology Information, to find out the
homology between the sequence obtained in the
present study and sequences present at NCBI
database.
3.3 Designing and amplifications of SCAR Primers
The nucleotide sequence of each of the cloned RAPD
fragment was used to design pairs of SCAR primers.
SCAR primer sets were designed using Primer3 clone
manager software based on sequence information
from polymorphic marker. Care was taken to avoid
possible secondary structure or primer dimer
generation and false priming, and also to match
melting temperatures and to achieve appropriate
internal stability while designing SCARs. These
primers were synthesized by custom service of
Eurofin Genomics Pvt. Ltd, Bangalore, India.
These
SCAR primers were used for the amplification of
DNA obtained from female and male and plants.
Amplification of the SCAR marker was performed in
25 µl, using 10ng total DNA, 0.2 µM each SCAR
primer, 2.5 mM dNTP, 4mM MgCl
2
, and 10x-Taq
polymerase (Finnzymes) in 1× Taq
buffer. The
reproducibility of SCAR amplicon was confirmed by
amplifying the marker under a broad range of
amplification temperature (51
℃
to 57
℃
). Eventually,
cycling condition were as follows: 90
℃
for 5 min; 30
cycles at 90
℃
for 1 min, 53
℃
for 1 min and 60
℃
for 4 min, and final extension at 72
℃
for 7 min. PCR
products were electrophoresed on a 1.5% agarose gel
and; presence and absence of the SCAR band was
visually scored and compared with samples of each
sex type and its pattern recorded on Gel documentation
system.
Authors’ contributions
KPS carried out the overall experiment and SK prepared the
manuscript. KBK supervised the experiment as Chairman of
the advisory committee for the Master degree research work.
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