Transcriptome Analysis And Genomic Characteristics Of Testicular Tissue Of Kulan Hybrids

Mongolian Kulan are an important germplasm resource in China,a country class I protected animal,mainly distributed in Xinjiang,Gansu,and Qinghai and other regions of China.Mongolian Kulan ♂× domestic donkeys ♀ can produce offspring(Kulan hybrids),but their progeny did not show any signs of fertility after long-term observation.In this study,we focused on Mongolian Kulan and Kulan hybrids to explore the causes of the sterility of semi wild blood donkeys by transcriptome sequencing,and to identify the ancestry composition of Kulan hybrids from a genomic perspective,comprehensively elaborate their genomic characteristics of autosomes,mitochondria and Y chromosomes,and explore possible signs of selection in the Kulan hybrids.The following results were obtained:(1)A total of 560 known mi RNAs and 49 new mi RNAs were identified in testicular tissues of Xinjiang donkey and Kulan hybrids after sexual maturity.Among them,there were 530 known mi RNAs and 40 new mi RNAs in Kulan hybrids.A total of 24 mi RNAs were significantly differentially expressed between the Xinjiang Donkey and the Kulan hybrids testicular tissues,among which,6 mi RNAs were significantly upregulated and 18 mi RNAs were significantly downregulated in the testicular tissues.A total of 520 target genes were predicted for the 24 significantly differentially expressed mi RNAs between Xinjiang donkey and Kulan hybrids,mainly enriched in signaling pathways related to tissue development,cell proliferation,cell cycle regulation and reproductive development.Significant down-regulation of four mi RNAs(mi R-449 a,mi R-34 c,mi R-9024,and mi R-449b),especially of mi R-34 c and mi R-449,in the testicular tissue of Kulan hybrids resulted in the failure of the spermatogonia to differentiate from mitosis to meiosis,which arrested spermatogenesis in Kulan hybrids。(2)The expression profiles of donkey in Xinjiang and Kulan hybrids sexual mature testis tissues were quite different,and a total of 3326 lnc RNAs and 19175 m RNAs were identified,resulting in a total of 342 differentially expressed lnc RNAs and 8398 differentially expressed m RNAs by the differential gene screening criteria.Among them,105 lnc RNAs and 3812 m RNAs were upregulated and 237 lnc RNAs and 4586 m RNAs were downregulated in the Kulan hybrids compared with Xinjiang Donkey.Identified 342 differentially expressed lnc RNA and 8398 differentially expressed m RNAs between sexually mature testicular tissues of Xinjiang Donkey and Kulan hybrids,and found that downregulated expression of some important differentially expressed target genes(PPP2R5C,RPA2,TEX14)were associated with abnormal DNA replication and retarded mitotic progression in the testicular tissues of half wild blood donkey,Found that the differential expression of a large number of m RNAs related to spermatogenesis(KIF2B,TEKT1,and TEKT2,etc.)most likely contributed to the seminiferous arrest.(3)Whole genome resequencing of 30 Kulan hybrids by next-generation sequencing identified their ancestry composition,as demonstrated at the autosomal genome,mt DNA genome,and Y chromosome genome levels,with 1 of 30 Mongolian Kulan or Kulan hybrids samples being a pure breed of Mongolian Kulan,and the remaining 29 Kulan hybrids samples being all Mongolian wild donkey(♂)× Xinjiang(♀)of hybrid generation;The Mongolian Kulan and its Kulan hybrids had very low genetic diversity of Y chromosome as evidenced from Y chromosome genomic SNPs and were likely to be descendents from 1 Mongolian wild donkey.(4)Using Guanzhong donkeys as the reference population and four selection methods to find the subject genome of Kulan hybrids,we found that the Kulan hybrids group had a large number of immune related genes and gene pathways,indicating that the Kulan hybrids may inherite the excellent stress resistance properties of Mongolian Kulan,and also found that kitlg genes had completely different expression patterns in Kulan hybrids and Guanzhong donkeys,Showed that the KITLG gene is an important candidate for influencing brown hair color in Kulan hybrids.In this study,It is of great significance to provide scientific basis for the protection,development and utilization of genetic resources of Kulan hybrids.