SNP exploring in the middle and terminal regions of the IGF-1 gene and association with production and reproduction traits in Holstein cattle

• This study did not report SNP in exons 2 to 6 in the IGF-1 gene of cattle.
• A SNP in the place of 48th nucleotide after end point of exon 4 was observed.
• HRM had higher accuracy to genotype SNP compared to PCR-RFLP.
• The CT genotypes showed more milk fat yield and content than the CC genotype.
• More studies are needed to consider variants of region in larger population.

Five primer sets were designed in order to identify single nucleotide polymorphisms (SNPs) in middle and terminal exons (2 to 6) and in some flanking intronic regions of the bovine insulin-like growth factor 1 (IGF-1) gene. Sequencing results of PCR products for 10% of animals showed no variant in exons but a SNP at intron 4 was occurred. Both polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) and high resolution melting (HRM) methods were developed to genotype samples. The PCR–RFLP results showed the presence of three fragments on agarose gel for the C allele due to two cleavage sites while two fragments for the T allele were observed. Melting curves of 123 bp fragments in HRM analysis showed a difference between temperature melting (Tm) of two homozygous genotypes as the CC genotypes had higher Tm than the TT genotypes. Melting curve of the CT genotype was different and crossed two parallel patterns of homozygous genotypes. The frequencies of the CC, CT and TT genotypes were 0.6, 0.37 and 0.03, respectively. Also, the estimated allele frequencies were 0.785 and 0.215 for the C and T alleles, respectively. Results showed higher accuracy of the HRM analysis compared to the PCR–RFLP method. Least square means (LSMs) comparison of the different genotypes in the SNP showed significant association with milk fat yield trait in the first lactation and open days after the second calving. The polymorphism did not have a significant effect on other milk production or reproduction traits. It seems that other variants or QTLs known in this region underlie genetic variation in the production and reproduction of dairy cattle.