GENETIC POLYMORPHISM AND EXPRESSION PROFILE OF BOVINE TRANSMEMBRANE PROTEIN 95 GENE (TMEM95)
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Date
2019-09
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SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA
Abstract
TMEM95 gene is known to be involved in idiopathic male subfertility in cattle. The present study was aimed at identifying polymorphism of TMEM95 gene in Ongole, Jersey, HF, Jersey crossbred, HF crossbred and Murrah bulls and to study tissue specific expression profile of the gene in buffalo. A total of 270 blood samples were collected from bulls across all the genetic groups maintained at different Frozen Semen Bull Stations maintained in Andhra Pradesh and University Livestock Research Station. The available sequences of Murrah buffalo, Mediterranian buffalo, Ongole cattle, Gir cattle, Herford cattle were used for identification of putative SNP variants. Three SNP loci namely, SNP1100 (rs209226692), SNP1275 and SNP2192 (rs41904693) were identified for genotyping. RFLP was employed to detect the polymorphic patterns to genotype the bulls for each locus. TMEM95/NlaIII digestion of 600 bp amplicon in Jersey crossbred and Ongole bulls resulted in all the three genotypes, whereas in Jersey, HF, and HF crossbred resulted in two genotypes for the C/T polymorphism. In none of the exotic breed cattle bulls studied CC genotype was observed. TMEM95/AvaII polymorphism at rs41904693 in all the cattle genetic groups revealed three genotypes i.e. TT, GG and TG. The frequency of G allele was observed to be more except in HF bulls. The TMEM95/TaqI genotyping revealed that all the cattle genetic groups are monophorphic (TT). The fixation of T allele at rs209226692 and rs41904693 loci in Murrah bulls was observed, but polymorphic at SNP1275.
The effective number of alleles (Ne) across the studied genetic groups ranged between 1.069 (Jersey) and 1.471 (HF). In Ongole bulls the same value was much higher 1.957. The PIC values varied from 0.057 (Jersey) to 0.370 (Ongole) for SNP1100 and 0.269 (HF) to 0.374 (Jersey) for SNP2192 among the different cattle populations. The PIC value for SNP1275 estimated in Murrah was 0.360. The FIS values were negative ranging from – 0.0345 to – 0.2875 for SNP1100 in the cattle genetic groups except Jersey crossbred cattle. At SNP2192 the HF crossbred and Ongole were with negative FIS values. The only estimated locus in Murrah bull population was SNP1275 which showed a negative FIS value. Expression of TMEM95 gene was studied in testis and brain to understand the functional role of TMEM95 gene in bovine and bubaline male fertility. Seven primers were designed spanning various intron exon boundaries, that could differentiate the isoform that is being expressed in those tissues. Total RNA was isolated from testis and brain tissues. The successful amplification by all the primer combinations indicated the expression of all the isoforms (X1 to X7) in testis of cattle, but not in brain tissue. In buffalo, the primer combinations were amplified in testis. No amplification is observed in the brain tissue of buffalo with any of the primer combinations. However, the expression profile has not matched with any of the isoforms that are predicted in buffalo using orthologous information from cattle.
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