Identification of Polymorphism in HSP70A1A Gene in Mehraban and Romanov Sheep Breeds using PCR-SSCP Technique

The heat shock proteins (HSPs) are a family of proteins contributing to cellular resistance against a variety of thermal stresses in different climates. In this study we aimed to characterize polymorphisms of the ovine HSP70A1A gene in Mehraban indigenous sheep breed and Romanov imported sheep breed, because they are well adapted to the cold climate. Blood samples were collected from a total of 141 sheep (100 Mehraban and 41 Romanov), and then genomic DNA was extracted according to conventional instructions. Polymerase chain reaction (PCR) was performed to amplify the 247 bp and 298 bp of the exon region of the HSP70A1A gene (Oar_v4.0; Chr 20, NC_019477.2). Also, polymorphisms of the studied fragments were explored using single strand conformational polymorphism (SSCP) technique and then the amplified DNA fragments were sequenced by Sanger sequencing method. In both Mehraban and Romanov breeds, five distinct SSCP patterns were identified that two and three electrophoretic patterns were respectively related to 247 bp segment and 298 bp segments. Furthermore, frequencies of SSCP patterns showed no differences between breeds. Five single nucleotide polymorphisms (SNPs) including g.26562252C>A, g.26562261G>C, g.26562270T>C, g.26562273C>T and g.26562279C>T were identified in 247 bp segment, and also, three SNPs including g.26561745T>C, g.26561784C>T and g.26561877G>A were identified in 298 bp segment of PCR products. Results revealed that all SNPs were in Hardy–Weinberg disequilibrium (p < 0.05), except of g.26561745T>C and g.26561877G>A. Considering the role of HSPs in resistance to the environmental stresses, it can be conclude that the identification of  these SNPs can  play an important role on understanding the mechanism of adaptation  to temperature tolerance among the Iranian indigenous sheep breeds.