The Relationship Between The Polymorphisms Of Y Chromosome And Bovine Paternal Origin And Bull Fertility

China is particularly rich in genetic resource on cattle,and the origin of Chinese cattle is very complex.Clarifying the genetic diversity and evolutionary origin of cattle breeds is essential for strengthening the conservation and utilization of cattle genetic resources.The mammalian Y chromosome is restricted to males,make this chromosome a powerful tool for tracing the genetic diversity and paternal origin of Chinese cattle.A large number of studies have confirmed that the genes located in the male specific region of the Y chromosome(MSY)are usually expressed predominantly in the testis,indicating that they may play important roles in spermatogenesis and testicular development.Therefore,it is an ideal candidate locus to identify molecular markers associated with bull fertility.The current study was conducted to achieve above goals:(1)The male specific primers for ZFY(Zinc finger protein,Y-linked)and USP9Y(Ubiquitin specific peptidase 9,Y-linked)on the bovine MSY were verified to identify SNP(single nucleotide polymorphism)using PCR amplification,DNA-pooling sequencing and PCR-RFLP(restriction fragment length polymorphism)methods,thus,combination analysis of Y-SNPs identified in this study and Y-SNPs in previous study was applied to investigate the paternal genetic diversity and origin of 421 bulls from 22 cattle breeds;(2)The qPCR method was applied to estimate the CNV(copy number variation)of ZNF280 AY in 715 bulls across 21 cattle breeds and to further investigate the association of the CNV of ZNF280 AY with bull reproductive traits of Holstein and Simmental bulls;(3)The whole genome resequence method was used to investigate the Y-SNPs on the MSY signle copy gene region of 30 Simmental bulls,including 16 low and 14 high fertility individuals,and thus construct Y-haplotype with bull fertility.The main results were as follow:(1)A total of 42 male specific primers were identified for ZFY and USP9 Y from 60 pairs of primers designed,and 24 novel SNPs in ZFY and USP9 Y were identified by DNA-pool sequencing.Y-haplotype analysis using 3 SNPs identified in this study and 2 Y-markers in previous study revealed that foreign cattle breeds ususlly contained limited Y haplotype,while Chinese cattle breeds had relatively abundant Y haplotype.In addition,Y2 haplotype was predominant in cattle breeds from Northern China,Y3 was predominant in Southern China,and Central China is mixture of Y2 and Y3.Significantly,Y1 haplotype was firstly identified in cattle breeds from Sothern China in current study.(2)The qPCR method was used to measure the CNV of ZNF280 AY in 715 bulls across 21 cattle breeds.The results revealed that the median copy number(MCN)of ZNF280 AY was 47,and the copy number(CN)varied from 11 to 154,showing significant CNV between and within the cattle breeds investigated.Furthermore,bulls belonging to Y3 lineage had a significantly higher MCN(57)than bulls belonging to the Y1(40)and Y2 lineages(52).Association analysis revealed that the CNV of ZNF280 AY was correlated negatively with the percentage of normal sperm(PNS)(r =-0.206,P = 0.008)and sperm concentration(SPC)(r =-0.148,P = 0.042)in Holstein bulls,while no significant correlation was observed with ejaculation volume(EV),total sperm count(TSC),sperm motility(SM),postthaw motility(PTM)and scrotal circumference(SC)in Holstein and Simmental bulls.(3)Whole genome resequencing of 30 Simmental bulls showed that 504 Y-SNPs were detected on the single copy genes of bovine Y chromosome,and the most SNPs were located in UTY.Seven Y chromosome haplotypes were constructed based on 504 Y-SNPs,and haplotype H4 was unique to low fertility bulls,indicating Y haplotype may be used to select elite bulls at early stage.According to the comprehensive analysis of the polymorphism of bovine Y chromosome,this study found that Chinese cattle breeds have diverse Y chromosome haplotypes,which provide a theoretical basis for the development,utilization and protection of bovine genetic resources.The CNV of ZNF280 AY was correlated negatively with PNS and SPC in Holstein bulls,and Y-haplotype was found to be associated with Simmental bull fertility,which may serve as a valuable marker for early bull fertility selection.