Identification of Single Nucleotide Polymorphism(s) in Part of LEP Gene and Investigation of their Effects on Carcass Traits in Afshari and Afshari×Booroola Merino Sheep

Introduction Leptin is a 16 kDa protein produced by adipocytes. This protein controls appetite, energy balance, efficiency of production and distribution of fat storage in the body and therefore plays an important role in regulating body weight and growth in mammals. LEP gene in sheep (NC_019461) located on 4th chromosomes and has 16275 bp in length and contains 3 exons and 2 introns. The resulting protein contains 193 amino acids (the length of coding region is 579 bp). So far in this gene, 17 single nucleotide polymorphisms in the coding region were reported that seven of them are synonyms and the rest are missense. The entire exon 3 is containing 357 bp in length that starts from nucleotide 13544 till nucleotide 13903. This exon comprise 119 (or 118) codons, seven missense and a synonymous SNPs from this area of the gene is reported till now. Different alleles of this gene may lead to different phenotypic effects.
Material and Methods The aim of this study was to identify alleles of the gene in exon 3. For this purpose, 133 lambs from a flock at almost the same age in three groups of Afshari pure breed and B1 and B4 of Afshari × Booroola Merino male lambs were used. Afshari sheep is one of the heavy weight sheep in Iran, which is important breed in terms of meat production. It has notable twinning rate and birth weight, growth rate and weaning weight is remarkable compared with other breeds of sheep in this country. Given the potential of this breed suitable for the production of meat, it can play an important role in production of red meat in the area. To increase productivity of this breed, FecB gene from Booroola Merino sheep was introgressed to this breed of sheep in University of Zanjan in 2007. Following the introgression of the gene (FecB) to Afshari breed Afshari- Booroola Merino crosses as a new genetic combinations was developed. This study aimed to identify single nucleotide polymorphisms (SNP) in exon 3 of Leptin gene and its association with carcass treats in male Afshari and cross lambs. First, blood samples of all the animals were taken and phenotypic measurements on live animals were done and then 85 lambs were slaughtered. After slaughter, carcass weight was measured and after 24 hours maintaining in cold, weight of carcass, thigh, shoulder, lion muscle, back fat and waste (tail, back and visceral fat) were measured. Estimation of the carcass traits in non-slaughtered lambs, were accomplished using regression coefficients achieved before. DNA was extracted using phenol-chloroform procedure from all the samples. Then, using designed primers, the target DNA was amplified and PCR products of a number of samples were directly sequenced to identify potential SNP(s). The sequencing data were analyzed and two SNPs were detected in samples. Thereafter, all the samples were genotyped by RFLP using two restriction enzymes Hpa II and AIwNI. The association of genotypes with phenotypic and carcass traits were studied.
Result and Discussion According to this study results, one of the polymorphism was identified in nucleotide position 13713. All three genotypes of this; GG, GA and AA were observed in studied samples. This missense polymorphism led to amino acid arginine/glutamine change at codon 142 and had a significant effect on the back fat thickness and withers height. The second polymorphism observed in the nucleotide (G> A13875) and two GG and GA genotypes were found in the samples. This polymorphism also had the same amino acid changes at codon 196 and associated with body length (with probability close to significant level). The frequency of GG genotype in both SNP was higher than other genotypes. This genotype is the genotype of the reference sequence in biological data bases. The alleles locating in 13713 bp in the studied population were not in Hardy-Weinberg equilibrium. But alleles of the 13875 location were in Hardy-Weinberg equilibrium. The allele frequency of each SNP was significantly different in genetic groups. In location of 13713, the most frequent mutation allele (A) and the lowest frequency of this allele was observed in genetic group B4 and Afshari group respectively. In location 13875, Afshari group had the highest frequency of the mutant allele and B1 had the lowest frequency.
Conclusion In general, in this study polymorphisms were found out in exon 3 of the leptin gene which has been found in previous studies and sheep SNP projects and have been reported previously in biological databases. Based on the results obtained and the variations observed in some previous studies, if the changes associated with these traits be confirmed in next studies, these polymorphisms could be used in marker-assisted selection in breeding programs.