Genetic Variations Of Y-STR And Y-CNV, And Transcriptome Analysis Of Testicle Tissue In Equus Animals

The mitochondrial DNA,short tandem repeat(STR)and single nucleotide polymorphism(SNP)on the Y-chromosome are considered as important roles to analyze the maternal and paternal origin.A large number of studies have been conducted to investigate genetic diversity,origins,and domestication of horse and donkey using autosomal microsatellites and mitochondrial genome.However,the Y-STR and copy number variation(CNV)on male-specific region of Y chromosome of modern equus is largely uncharacterized.CNVs,which represent a significant source of genetic diversity on mammalian Y chromosome,have been shown to be associated with development of many complex phenotypes,such as reproduction and male fertility.A number of studies were conducted on Y chromosome genes of various species and identified single-copied and multi-copied genes.However,the copy numbers of Equus Y chromosome genes are largely unknown.Testicles produce sperms,understanding the expressed genes and microRNAs in testis will improve male reproduction.Cryptorchidism is the failure of one or both testes to descend into the scrotal sacand.It is a common congenital malformation in horses.The major clinical consequences of this abnormality are impaired fertility.The identification of differentially expressed testis-specific genes of normal and undescended testes is necessary to determine the molecular mechanism of cryptorchidism and reason of impaired fertility.In this study,the paternal origin and diversity were investigated using Y-chromosome microsatellite markers in horses and donkeys.The copy number variants of Y-linked genes were measured by quantitative real-time polymerase chain reaction in horses and donkeys.The differentially expressed testis-specific genes(DEGs)and microRNAs between normal and undescended testes of horses,and DEGs and microRNAs between Chinese local donkey breeds were identified by RNA-seq and bioinformation method.The innovative results were listed as follows:1)Genetic diversity was found in Eca.YA16 markers in horses: two alleles(156 bp/152 bp)existed in modern horses,which shared among different breeds.Only 156 bp allele was found in European and American horse breeds.The results are clearly in line with multiple patrilines of Chinese horses and against the hypothesis of limited patrilines and single Y chromosome haplotype in horse.This data corroborate that the paternal origin of horses might origin from Eastern breeds.Przewalski's horses existed Y-chromosome polymorphism(160 bp/158 bp),which is valuable for the preservation of this special wild species.2)Three paternal origins were identified in donkeys: A total of 14 published equine Y chromosome-specific microsatellites were investigated in 395 male donkey samples from China,Egypt,Spain and Peru using fluorescent labeled microsatellite markers.The results showed that seven Y-STRs were male specific and polymorphic,showing 2-8 alleles in donkeys studied.A total of 21 haplotypes corresponding to three haplogroups were identified,indicating three independent patrilines in domestic donkey.3)Different distribution patterns of copy numbers of Y chromosome genes were found in horses :the CNVs of eight Y chromosome genes were investigated in Chinese horse.The EIF1 AY,ETSTY1,ETSTY4,ETSTY5,ETY4,UBE1 Y,SRY,and YIR2 had multi-copies with the median copy numbers of 1,3,8,9,26,7,1,and 12.And the variations range from 1-37,1-25,1-156,1-80,1-173,1-77,1-17,and 1-92.The CNVs of Y chromosome genes showed different distribution patterns among Chinese horse breeds.4)Copy number variants were revealed in Y-linked genes of donkeys: five genes(CUL4BY,ETSTY1,ETSTY4,ETSTY5,and SRY)were identified as multi-copied,of which the median copy numbers were 5,45,2,2,and 13 with 1-57,1-227,1-37,1-86 and 1-152 CNVs ranges,respectively.The CNVs of these five genes were shared in different breeds.Our results provided genetic variations of donkey Y chromosome genes.5)A total of 84 DEGs were identified to be associated with sperm function: transcriptome analysis of normal and undescended testes revealed 84 differentially expressed genes clustered in sperm motility,acrosomal vesicle,spermatogenesis,and sperm principal piece,11 genes(CATSPERD,NME8,SPAG6,AKAP4,CATSPER1,ENKUR,AKAP3,TXNDC8,CATSPER3)were proved to be associated with male reproduction.A total of 35 missense mutations were detected in nine out of these 11 candidate genes.6)A subset of 42 shared differentially expressed microRNAs were detected: there are 42 differentially expressed microRNAs between normal and undescended testes,of which,16 microRNAs and eight target genes were considered as candidate ones.We speculated that these microRNAs were associated with reproduction and cryptorchidism and function in male fertility.7)102 DEGs and two microRNAs were found in donkeys: transcriptome analysis of three Biyang donkeys and three Dezhou donkeys obtained the transcriptome data of donkey testicular tissue.The results revealed 102 DEGs and two microRNAs between two donkey breeds.Among 11 candidate genes in horse,nine missense mutations were detected in six genes(NME8,SPAG6,CATSPER1,CCNYL1,ENKUR,CATSPER3).A comprehensive study was conducted focus on the genetic diversity of Y chromosome and expression profile of testicular tissues in equus.Our results revealed abundant genetic diversity on Y chromosome and paternal origins,extensive CNVs,and a number of DEGs and microRNAs that associated with sperm functions in horse and donkey.These data provide genetic foundations for genetic diversity and paternal origins for Chinese horse and donkey.Our results also provide transcriptome data of DEGs and microRNAs between normal and undescended testis in horses.