Identification Of Y Chromosome Genes And Y-SNPs In Sheep

The male specific region of the mammalian Y chromosome(MSY)does not recombine with X chromosome during meiosis,which is an important genetic resource for analyzing paternal genetic diversity due to its strict father to son inherited character.Since it is extremely difficult to sequence entire Y chromosome,many species have very few Y chromosome sequences.Most of genes on MSY are found to be exclusively or predominantly expressed in the testis,indicating they may play important roles in spermatogenesis and male reproduction.Therefore,the current study was conducted to select ovine Y chromosome specific primers and Y-SNPs based on the previous primer information used in bovidae species and newly designed primers based on the bovine Y chromosome sequences.The aim of this study is to identify the ovine Y-gene fragments and their expression profiles,compare sequence identity among sheep,cattle,goat,yak and bharal.Meanwhile,the Y-SNPs were screened to find association with ram reproductive traits,which will supply scientific basis for constructing ovine Y-haplotypes,identifying molecular markers for embryo sex and male reproductive traits selection at early stage.The main results are as follows:1.A total of 27 pairs of primers were male sepefic in sheep from 73 pairs investigated.The similarity of above 27 Y-gene fragments among sheep,bharal,cattle,goat and yak ranged from 81.51% to 99.06%,indicating they are conserved among different species.In addition,four SNPs were detected in Y chromosome genes fragment by DNA sequencing of DNA pooling and PCR-RFLP,including g.61 G>A within SRY11 fragment in Suffolk and White Suffolk sheep,g.88A>G within SRY16 in South African Mutton Merino,g.403 G>Awithin ZFY16 fragment in Suffolk sheep and South African Mutton Merino,and g.215 C>G within EIF2S3Y2 in Hu and Tan sheep.The SNPs found in SRY11,EIF2S3Y2 and ZFY16 were identified for the first time in this study.Five haplotypes were identified,and frequencies of haplotypes ?,?,?,? and ? were 0.052,0.290,0.071,0.432 and 0.155,respectively.The association analysis between the Y-SNPs and the reproductive traits revealed that g.61 G>A within SRY11 fragment was significanty correlated with the testicular size of white Suffolk sheep,and g.215G>C within EIF2S3Y2 fragment,was significantly correlated with the testis weight and epididymis weight of Hu sheep.2.The full length of DDX3 Y CDS region was cloned using RT-PCR,which is 1983 bp that encoded 660 amino acids.In addition a DEAD-box conserved domain was found to locate 199-418 amino acid.The sequence identity between DDX3 Y and DDX3 X was 86.45% and 90.33% at nucleotide and protein level,respectively.Additonally,full genomic sequence of DDX3 Y was obtained using 18 pairs of primers,revealing that full genomic sequence is 12985 bp with 17 exons.3.The mRNA of UTY,ZRSR2 Y,ZFY and EIF2S3 Y was widely expressed in heart,liver,spleen,lung,kidney,longissimus dorsi and testes tissues of Hu sheep at 6 months.Of which,DDX3 Y,ZRSR2Y and EIF2S3 Y were mainly expressed in testis.The expression of Y chromosome gene in testes of Hu sheep were different at various developmental stages.The expression level of DDX3 Y and ZFY gene increased with the testicular development of Hu sheep;the expression level of UTY in testis was the highest at 3 month;the expression level of ZRSR2 Y in testes were the highly at 3 month and 6 month;and the expression level of EIF2S3 Y in testis was the highest at 3 month.The expression level of DDX3 Y and UTY in testis were positively correlated with testicular weight(r=0.960,P=0.002;r=0.935,P=0.020),while the expression level of ZRSR2 Y in testis was negatively correlated with testicular weight(r=-0.998,P=0.002).There was no correlation between the expression level of ZFY and EIF2S3 Y in testicular development and testicular weight.