Amplification, Copy Number Variation, Expression and Association of Non-synonymous SNP of Bovine Beta-defensin 129 Gene With Distinct Fertility of Cattle Bull

Abstract

\n BackgroundThe male reproductive specific class-A β-defensins are adsorbed on sperm surface and enrich sperm functioning thus considered vital for maintaining male fertility. The primate DEFB129 play role in sperm maturation, motility, and fertilization but its contribution to bovine fertility is still unexplored. MethodRLM-RACE and RT-qPCR approaches were used to characterize and expression analysis of Indian cattle BBD129 gene. The polymorphism analysis of the BBD129 gene was done by PCR, sequencing, and absolute RT-qPCR on sperm gDNA from distinct fertility cattle bulls. Bioinformatic analysis was performed to understand the structural and functional implications of SNP on BBD129 protein. ResultsThe complete coding sequence of the BBD129 gene consists of 582 bp mRNA including UTRs and conserves all beta-defensin-like characteristics. Sequencing results revealed two conserved non-synonymous T169G (rs378737321, S57A) and A329G (rs383285978, N110S) SNPs in the functional protein-coding exon. Based on SNP position and linkage, BBD129 gene haplotypes were categorized into four groups: TA haplotype (169T & 329A), GA haplotype (T169G polymorphism), TG haplotype (A329G polymorphism), and GG haplotype (when T169G & A329G polymorphisms present together). The frequencies distributions of BBD129 haplotypes in the high fertile group (n=105 clones) were: TA (71.42%), GA (1.90%), TG (2.8%), and GG (24.76%), while in the low fertile group of bulls, the frequencies distributions of observed BBD129 haplotypes (n=149 clones) were: TA (36.24%), GA (0%), TG (2.68%), and GG (61.07%). The distributions of TA haplotype were majorly distributed in bulls with a high conception rate (P=0.5256) while double mutated GG haplotype was significantly more abundant in bulls with a lower conception rate (P=0.0001). BBD129 exist as a single-copy gene in the bovine genome and found higher expression in the corpus-epididymis region. Bioinformatic analyses found nsSNPs as neutral and non-deleterious but their structural-distorter could result in altered mRNA secondary structure, protein conformations decreased protein stability, and compromised biological functionalities. The polymorphisms resulted in altered O-glycosylations (deletion S57A and insertion N110S) and an increase in phosphorylations (52T-Threonine and 110S-Serine) post-translational-modifications. ConclusionBBD129 gene polymorphism could be associated with the fertility performance of cattle bulls.