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Animal Molecular Breeding 2014, Vol. 4, No. 1, 1-5
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3.2 PCR amplification and sequencing
The primers for
CAPN4
(exon 4, 5 and intron 4),
CAST
(exon 12, intron 12 and partial exon 13),
DGAT1
(5’UTR, exon1 and intron1) and
FABP3
(exon
2 and intron 2) gene were taken from published
literature (Shahroudi et al. 2006; Byun et al. 2009;
Scata et al. 2009; Calvo et al. 2004). Each locus was
amplified by PCR after standardizing the annealing
temperature. The amplified products were checked on
agarose gel. Fifty microlitres of the PCR amplified
product was purified using QIAquick PCR
Purification Kit (QIAGEN). The purified PCR
amplicons were sequenced on ABI 3100 sequencer in
both forward and reverse direction.
3.3 Data Analysis
Multiple alignments of the sequences were performed
with MEGA 4.1 (Tamura et al. 2007) and DNAMAN
ver 5.2.10 (Lynnon BioSoft, Vaudreuil, Quebec,
Canada). All positions containing gaps and missing
data were eliminated from the dataset. Insertions and
deletions were excluded from all estimates. The
sequences were analysed for polymorphic sites,
nucleotide diversity (π) and haplotype diversity (h),
genetic differentiation (F
ST
) and gene flow (Nm) using
DNASPv5 (Librado et al. 2009). Allele and genotype
frequencies were estimated for the identified SNPs.
Acknowledgements
The authors acknowledge the help rendered by officials of State
Animal Husbandry Departments of different States and the
sheep farmers for their help in collection of blood samples. We
are grateful to Director, National Bureau of Animal Genetic
Resources (NBAGR, Karnal), Indian Council of Agricultural
Research (ICAR, New Delhi) for providing necessary facilities
and Mr. Rakesh Kumar (Senior Technical Assistant, NBAGR
Karnal) for technical assistance.
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