This study aimed to find the SNPs in the SORBS1 gene of cattleyak, analyze the relationship between its polymorphisms and the milk fat traits, and find potential molecular markers for the milk fat traits of cattleyak. The polymorphism of the SORBS1 gene in 350 cattleyak from Hongyuan County (Sichuan, China) were detected by PCR and DNA sequencing, and the correlation between these SNPs and the milk production traits of cattleyak was analyzed. The results showed that there were nine SNPs in the CDS and their adjacent non-coding regions of the SORBS1 gene, and all SNPs have three genotypes. The correlation analysis found that the genotypes with superior milk fat traits in the other eight alleles were homozygous genotypes with a high genotype frequency except the g.96284 G > A (c.3090 G > A) (p < 0.05). However, at locus g.96284 G > A, the milk fat percentage, monounsaturated fatty acids (MUFAs), polyunsaturated fatty acids (PUFAs) and saturated fatty acids (SFAs) of the GA genotype were significantly higher than that of GG and AA genotypes (p < 0.05). Among these SNPs, three SNPs (g.6256 C > T (c.298 C > T), g.24791 A > G (c.706 A > G) and g.29121 A > G (c.979 A > G)) caused the amino acids change. The genotypes of the three SNPs consist of three haplotypes and four diplotypes. The amino acid mutation degree of diplotype H1–H1 (CCAAAA) was the highest, and its milk fat percentage, MUFAs, PUFAs and SFAs were also the highest (p < 0.05). Taken together, we found nine SNPs in the SORBS1 gene that are closely related to the milk fat traits of cattleyak. Moreover, the mutation of amino acids caused by SNPs had positive effects on the milk fat traits of cattleyak. H1-H1 is the dominant diplotype which significantly related to the milk fat traits of cattleyak. This study provides a new molecular marker and theoretical basis for screening the milk fat traits of cattleyak.

Aberrant expression of the heat shock proteins and factors was revealed to be closely associated with male reproduction. Heat shock factor 2 (HSF2) is a transcription factor that is involved in the regulation of diverse developmental pathways. However, the role and the corresponding molecular mechanism of HSF2 in male cattle-yak sterility are still poorly understood. Therefore, the aim of this study was to obtain the sequence and the biological information of the cattle-yak HSF2 gene and to investigate the spatiotemporal expression profiles of the locus during the development of cattle-yak testes. Additionally, the differential expression was analyzed between the cattle-yak and the yak, and the methylation of corresponding promoter regions was compared. Our results showed an additional 54 bp fragment and a missense mutation (lysine to glutamic acid) were presented in the cattle-yak HSF2 gene, which correlated with enriched expression in testicular tissue. In addition, the expression of the HSF2 gene showed dynamic changes during the growth of the testes, reaching a peak in adulthood. The IHC indicated that HSF2 protein was primarily located in spermatocytes (PS), spermatogonia (SP), and Sertoli cells (SC) in cattle-yak testes, compared with the corresponding cells of cattle and the yak. Furthermore, bisulfite-sequencing PCR (BSP) revealed that the methylated CpG sites in the promoter region of the cattle-yak HSF2 were more numerous than in the yak counterpart, which suggests hypermethylation of this region in the cattle-yak. Taken together, the low expression abundance and hypermethylation of HSF2 may underpin the obstruction of spermatogenesis, which leads to male cattle-yak infertility. Our study provided a basic guideline for the HSF2 gene in male reproduction and a new insight into the mechanisms of male cattle-yak sterility.

This study aimed to investigate the spatially and temporally expressed patterns and biological characteristics of TSSK1B in male yaks and explore the potential correlation between TSSK1B and male sterility of the yak hybrid offspring (termed cattle–yak). First, the coding sequence (CDS) of TSSK1B was cloned by RT-PCR, and bioinformatics analysis was conducted with relevant software. Quantitative real-time PCR (RT-qPCR) was employed to detect the expression profile of TSSK1B in various tissues of male adult yaks, the spatiotemporal expression of TSSK1B in different stages of yak testes, and the differential expression of TSSK1B between yak and cattle–yak testes. The cellular localization of TSSK1B was determined by immunohistochemistry (IHC). Furthermore, the methylation status of the TSSK1B promoter region was analyzed by bisulfite-sequencing PCR (BSP). The results showed that TSSK1B was 1235 bp long, including 1104 bp of the CDS region, which encoded 367 amino acids. It was a conserved gene sharing the highest homology with Bos mutus (99.67%). In addition, the bioinformatics analysis revealed that TSSK1B was an unstable hydrophilic protein mainly containing the alpha helix of 34.06% and a random coil of 44.41%, with a transmembrane structure of 29 amino acids long. The RT-qPCR results demonstrated that TSSK1B was specifically expressed in yak testes compared with that in other tissues and especially highly expressed in adult yak testes. On the contrary, TSSK1B was hardly expressed in the testis of adult cattle–yak. IHC confirmed that TSSK1B protein was more strongly expressed in the testes of adult yaks than in their fetal and juvenile counterparts. Interestingly, nearly no expression was observed in the testes of cattle–yak compared with the corresponding testes of yak. Bisulfite-sequencing PCR (BSP) revealed that the methylated CpG sites in the TSSK1B promoter region of cattle–yak was significantly higher than that in the yak. Taken together, this study revealed that TSSK1B was specifically expressed in yak testes and highly expressed upon sexual maturity. Moreover, the rare expression in cattle–yak may be related to the hypermethylation of the promoter region, thereby providing a basis for further studies on the regulatory mechanism of TSSK1B in male cattle–yak sterility.