Determination of allelic frequency in parts of Leptine gene in Holstien cattle.

Most of the quantitative traits are influenced by polygene and environmental effect. Although the accurate numbers of genes, which are affecting the quantitative traits, are unknown, but according to the Finite locus models, there are some a few genes with substantial effect on genetic variations in quantitative traits. Detecting and sequencing this kind of genes was facilitated by developments in molecular genetics in the last decades. Gene expression and quality and quantity of the gene products are influenced by variation of the sequence of the coding and regulatory regions of the above genes. The Leptin gene and the Leptin receptor gene are the most important major genes that influence the milk production related traits. The objective of this study was to investigate the association of Intron 2, Exon 2, Exon 3 of the Leptin gene and Exon 20 of the Leptin receptor gene with milk production related trait's breeding value in Iranian Holstein proven bulls. Frozen semen of 141 internal proven bulls were collected and genomic DNA was extracted using Phenol-Chloroform method. Extracted DNA was diluted up to 50 ng/bl, after evaluating the quality and quantity of DNA by Agaros gel and Spectrophotometery. The PCR- RFLP method was carried out to amplify and digests the interested sequence. The AluI restriction enzyme was used to digest Intron 2 and Exon 2 and 3. The HinfI restriction enzyme was used to digest Intron 2 and Exon 3 and 20. The TaqI and Kpn2I restriction enzymes were used to digest Exon 2 and the HindIII and HphI restriction enzymes were used to digest Exon 3. All above loci were digested by Sau3AI restriction enzyme. Amplified and enzyme digested products were electrophoresis on acryl amide gel. According to the results, Polymorphism was seen only on Intron 2 when it was digested by Sau3AI and three genotypes AA, AB and BB with frequency equal to 0.823, 0.135 and 0.042 were distinguished respectively for this locus. In addition the allele frequency of B, which was digested by restriction enzyme, was equal to 0.110. The breeding value of milk yield, fat yield and fat percentage traits, with detected genotype of each animal, was used in statistical analysis based on the General Linear Models and no significant effect was observed for the least square means of different level of genotypes. A transitional mutation, that was converted C to T, was detected at position 115 bp of exon 20 of Leptin receptor gene in all DNA samples by using direct sequencing technique. The frequency of the allele T was rare and equal to 0.04 in the Iranian Holstein proven bulls. Both locus of Intron2 and exon 20 were deviated from Hardy-Weinberg equilibrium, according to their observed and expected frequency. However there wasn't any significant interaction between Intron2 and exon 20 but the average of breeding value for milk yield and fat yield were higher, about 109.5 and 1.88 kg respectively, in animal with genotype AAT than another's. Key words: Leptin Gene, Leptin Receptor Gene, Milk related traits, Sequencing, PCR-RFLP