158. EFFECT OF A59V LOCUS IN THE LEPTIN GENE ON LENGTH OF PREGNANCY IN IRANIAN HOLSTEIN COWS

Abstract

We investigated effect of A59V polymorphism in the leptin gene on length of pregnancy. Blood was collected from 255 Holstein cattles belonging to four different herd managements in Isfahan province. Genomic DNA extracted from whole blood. Genotypes of A59V locus were identified PCR-RFLP technique. Amplified region is located in exon three of leptin gene. The genomic bovine leptin sequences, which consist of three exons, were obtained from Gene Bank (Accession number U50365). The polymerase chain reaction was used to amplify the 331 bp DNA fragments from genomic DNA. The PCR reaction contained 100 ng of genomic DNA, 0.3 µM of each primer, 1.5 mM MgCl₂, 200 µM dNTP, 10mM Tris HCl, 50 mM KCl and 1 U Taq-polymerase in total volume of 20 µL. Sequences of primers that were used in PCR were reported previously by Haegeman et al. (2000). Conditions for PCR were 94°C for 2 min, followed by 35 cycles of 94°C for 30 s, 55°C for 1 min, and 72°C for 30 s. Followed by final extension for at for 15 min 72°C. Digestion of PCR product of 331 bp with 5 U of HphI (Fermentas) in 20 µL of reaction volume at 37°c for 8 h and analyzed on 8% no denature polyacrylamyde gel. Allele A in the A59V locus was the allele not digested by restriction enzyme, allele B was the restriction enzyme-digested PCR product. Digestion revealed 3 genotypes, AA (331 bp), AB (331, 311, and 20 bp), and BB (311 and 20 bp). Significances of the genotype effects were estimated by GLM procedure of SAS. This study showed that genotype effect on length of pregnancy were significant (P<0.01). Animals homozygous for allele A had higher length of pregnancy ((P<0.01, AA=279.17±0.47, AB=276.96±0.57, BB=274.8±2.2).