Page 9 - Molecular Plant Breeding

Molecular Plant Breeding 2014, Vol.5, No.3, 10-17
http://mpb.biopublisher.ca
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Table 3 Sequence information of the RAPD and EST-SSR primers
Rapd primers
S. No. Primer Sequence (5’- 3’) Source
1 OPA- 02 TGC CGA GCT G Public domain
2 OPA- 03 AGT CAG CCA C
3 OPA- 05 AGG GGT CTT G
4 OPA- 07 GAAACG GGT G
5 OPA- 09 GGG TAA CGC C
6 OPA- 10 GTG ATC GCA G
7 OPA- 11 CAA TCG CCG T
8 OPB-10 CTG CTG GGA C
9 OPC-02 GTG AGG CGT C
10 OPC-08 TGG ACC GGT G
11 OPD- 07 TTG GCA CGG G
12 OPD- 18 GAG AGC CAA C
13 OPE- 01 CCC AAG GTC C
14 OPE- 02 GGT GCG GGAA
15 OPE- 03 CCA GAT GCA C
SSR primers
1 PBCESSRJU2 F:TTCACATCTTCTTCATCTTCC
R: TTG CTA TTC GTT CTC AGT CTC
Hopkins et al. (2007)
2 PBCESSRJU7 F: TAC CAC TCC CTAACC GCA
R: ATC ACC TTG AGA GCG AAG
3 PBCESSRJU9 F: CCC TAC CGC TGG CTA GAC TT
R: GCA TCA TGA CCAACT ATC AAC C
the primer concentration was 15pmol. PCR tubes
containing master mix and DNA template were
thoroughly mixed and subjected to the thermal profile
under following conditions: 4 min at 94°C, 35 cycles
(1 min at 94°C, 1 min at 36°C, 2 min at 72°C) and 10
min at 72°C for RAPD markers. In case of SSR markers,
annealing temperature was calculated based on primer
sequence.The same reaction mixture without genomic
DNA was run for each reaction to serve as a negative
control. PCR amplification was carried out in a 96 well
Universal Gradient Thermal Cycler (PEQLAB,
Deutschland and Osterrtich, United kingdom).
PCR Banding Profile
The amplification products were then subjected to
electrophoretic separation using horizontal agarose gel
electrophoresis. 1.5% and 3.0% agarose gels were
prepared for resolving RAPD and SSR amplified PCR
products respectively. The gel was visually examined
under UV and documented using Biometra Gel
documentation system.
Data Analysis
PCR bands were detected in the gel and their sizes
were estimated using 100bp standard marker.The
banding patterns of all genotypes against each primer
were compared. Variable bands were used to score for
polymorphism and binomial data matrix was
generated which was further used for calculating total
number of bands, number of polymorphic bands, and
monomorphic bands for each primer. In order to check
the informativeness and discriminatory power of
RAPD primers used in this study, different parameters
like polymorphism percentage, polymorphic information
content, resolving power and marker index were
calculated. Polymorphism percentage was calculated
by dividing the number of polymorphic bands by the
total number of scored bands and multiplied with 100.
Average PIC indicates the ability of utilized markers
to differentiate the genotypes. Maximum Polymorphic
information content in case of dominant markers such
as RAPD is 0.5 (De Rick et al., 2001). It is calculated
as proposed by Roldan-Ruiz et al. (2000), as PIC = 2fi
(1-fi), where fi is the frequency of bands present and
(1-fi) = frequency of bands absent. Resolving power
was calculated as proposed by Prevost and Wilkinson
(1999). Rp= ΣIb, where Rp is the resolving power and