| Fertility is one of the important economic traits in the meat sheep industry.Early selecting of rams could help improve the fertility of sheep populations and then improve economic benefits.Thus,the testes,as a male specific reproductive organ,received great attention because its size is closely related to the quality and quantity of sperm.Moreover,the testis size has high heritability and easy to measure,hence,testis size is a most effective index in improving male fertility.However,the testis development is a delicate and complex process.Uncovering the genetic mechanism underlying this process should be helpful for analyzing the testis development process.Therefore,this study used third-generation transcriptome sequencing and iTRAQ technology to explore the transcriptic characteristics and differentially expressed proteins in nascent(M0),3 months of age(M3),12 months of age(M12),and large(M6L)and small testis(M6S)at 6-month old,and screened proteins related to testis development.In addition,9 candidate genes were selected to scanning SNPs that related with testicular size.The results obtained in this paper are as follows:(1)A mixed sample selected from different developmental stages and different size of testes in M6,including M0,M3,M12,M6 L and M6 S,was used to perform third-generation transcriptic sequencing.A total of 209832 consensus sequences were obtained.After move redundant,86660 transcript sequences were obtained with an average length of 2337 bp.Among them,68297 were new transcripts of which 62218 were annotated in the NR,Swissprot,GO,COG / KOG,Pfam,and KEGG databases.Besides,46180 SSRs,52923 complete ORF sequences and 9465 lncRNAs were predicted by structural analysis.These results of Hu sheep testicular transcriptome are helpful for detecting the expression of transcriptome in Hu sheep,and provide reference for further research on the proteome;(2)The iTRAQ technology was used to detect differentially expressed proteins across four developmental stages(M0,M3,M6 L and M12)and between M6 L and M6 S groups.And combined third-generation transcriptome results to screening and annotation differentially expressed proteins.The results showed that 465(274 up regulated,191 down regulated),1261(689 up regulated,572 down regulated),231(94 up regulated,137 down regulated),1080(467 up regulated,613 down regulated)differentially expressed proteins were detected in M0 vs M3,M3 vs M6 L,M6L vs M12 and M6 L vs M6 S,respectively.Among these differentially expressed proteins,86 proteins were found related to reproduction and reproductive processes which were involved spermatogenesis,sperm development,and sperm flagella movement,sperm capacitation and binding of sperm and zona pellucid.(3)According to the result of iTRAQ,9 candidate genes related to reproduction were selected to screening SNPs in the exon regions and its flanking regions using the DNA pool sequencing method.The SNPs were genotyped using the iMLDR method.A total of 34 SNPs were detected,11 of them were have significant effect on testicular parameters.SNP1 was significantly affect the left testis weight,total testis weight,total epididymis weight,testicular index and left epididymis weight/testis weight;SNP5 had significant effect on left epididymis weight/testis weight;SNP10 was significantly affected right testis weight,total testis weight and left epididymis weight/testis weight;SNP11 had significant effect on right testis weight,left testis weight,total testis weight,left epididymis weight/testis weight,right epididymis weight/testis weight and total epididymis weight/testis weight;SNP12 significantly affected left testis weight,left epididymis weight and testis weight variation coefficient;SNP13 and SNP15 were significantly affected the testis weight variation coefficient;SNP16 and SNP18 had significant effect on left epididymis weight/testis weight;SNP23 had significant effect on epididymis weight variation coefficient and SNP32 had significant effect on left epididymis weight/testis weight.These SNP can be potential molecular markers for early molecular assisted selection of Hu sheep. |
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