DNA POLYMORPHISM OF MYOSTATIN GENE IN DIFFERENT MEAT AND NON-MEAT GOAT BREEDS
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Date
2012
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Nanaji Deshmukh Veterinary Science University, Jabalpur
Abstract
The present study was conducted on three breeds of goat viz. Black Bengal (20 samples), Barbari (20 samples) and Sirohi (20 samples). In this investigation, an attempt has been made to identify different allelic patterns of MSTN gene in these breeds of goat by PCR-RFLP technique. Two regions- i) 701 bp fragment of partial 5’UTR and partial exon-1 and ii) 272 bp fragment consisting of exon-1 of MSTN gene were amplified in each breed. Both the fragments were subjected to PCR-RFLP. The first fragment (701 bp) was digested by using DraI restriction enzyme and second fragment (272bp) was digested by using NlaIII restriction enzyme.
Various genotypes were identified and these gene and genotype frequencies were calculated. The different genotypes of MSTN gene were cloned and sequenced and the sequence data was compared with the available NCBI GenBank sequences. These sequences were submitted to NCBI GenBank data.
Genomic DNA isolated from fresh blood. All the samples yielded sufficient amount of DNA. Quality of isolated genomic DNA samples showed compact bright flouroscent band of genomic DNA under UV transilluminator indicating its good quality. Purity and concentration of genomic DNA was determined by ND 2000 spectrophotometer (Nanodrop Inc USA). DNA samples with O.D. 260 /O.D. 280 ratio ranging between 1.7 to 1.9 indicating high purity and absence of contamination of proteins and other impurities.
PCR programme is optimized and a suitable annealing temperature with consistent results were obtained at 57 0 C and 510C for MSTN-I and MSTN-II respectively. MSTN gene was amplified using thermocycler (Gene AmpTM PCR System 9700 ABI). The optimized PCR reaction mixtures contained 2x mastermix (Fermentas), 1µl forward and reverse primer of 10 pico mole concentration. PCR amplification of both the fragment MSTN-I and MSTN-II was carried out by using the sets of primers reported by Zhang et al. (2011). The PCR amplified products of MSTN-I and MSTN-II were run on, agarose gel electrophoresis (2%) at 5 V/cm for 45-60 min and found a single clear band of 701 bp and 272 bp respectively.
Restriction enzyme digestion of MSTN-I (701 bp) PCR amplified product with DraI restriction enzyme revealed two genotypes (patterns), “AA” and “AB”. The genotype AA only found in Black Bengal and AA and AB both found in Barbari and Sirohi. The homozygous genotype AA can not be digested by Dra-I (696 bp) and the heterozygote genotype AB was digested and yields three bands of 696, 504 and 197 bp. PCR-RFLP analysis of MSTN-II with Nla-III restriction enzyme revealed only one types of genotype i.e. DD (219 and 53 bp) in all three breeds.
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