The Study Of The Formation Mechanism Of Intersex Phenotype Of Lanzhou Large-Tailed Sheep Using Whole-Genome Resequencing Technology

Intersex,also known as hermaphrodism,is a phenomenon that dioecious animals exhibit intersexual characteristics of both sexes which is caused by gender differentiation or abnormal development.This phenomenon is commonly observed in goat populations,and there are also several reports on pig,horse,roe deer and sheep.The intersexual individual is accompanied by abnormal development of gonads or genital organs and disordered hormone secretion.Thus they cannot produce eggs and sperms,being unable to reproduce offspring,with high death rates,which do the harm on animal husbandry and production.Lanzhou large-tailed sheep is one of the excellent local sheep breeds,which is endangered resources in China.The occurrence of intersexual individuals has seriously hindered the preservation and propagation work on Lanzhou large-tailed sheep,but the mechanism of the formation of intersex is not clear yet;therefore,to explore the formation of intersex will be beneficial to the development of animal husbandry of the sheep industry.This study intended to performed genome wide re-sequencing on the only two intersex and two normal Lanzhou large-tailed sheep to uncover the formation mechanism of intersexual Lanzhou large-tailed sheep.In addition,considering that both Lanzhou large-tailed sheep and Tan sheep belong to the Mongolian pedigree among the three sheep pedigree（Mongolian,Kazakh and Tibetan）in China,and the genetic relationship between the two breeds were close,so the re-sequencing data of four Tan sheep were used as normal control group.Then the sequences alignment,PCR amplification and other methods were applied to study the sequence of sheep which is the homologous sequence of PIS in goat.Furthermore,the genetic differentiation factor and the whole genome copy number variation of the intersexual sheep group and normal sheep group were calculated and analyzed based on the whole genome SNP information to find out the genes or regions that play important roles in the development of intersex in sheep.This findings would provide important basis for timely detecting and eliminating the intersexual individual,as well as the preservation and expansion of Lanzhou large-tailed sheep,further reducing the loss caused by intersexual individuals in animal husbandry and production.The results were listed as follows:（1）The results of the bioinformatics analysis showed that the average sequencing depth of Lanzhou Large-tailed sheep and Tan sheep were 17.3×and 19×,respectively.After quality control and comparison,we found the mapping ratios to the sheep reference genome were all above 94%,and we got 198.5 G effective data finally.Using the Blat software for alignment,we found a region of 8787 bp in length（chr1:247747059-247755846）on the sheep was similar to 8773 bp in the 11.7 kb deletion in the goat that result in the interal syndrome（PIS）in goats,and the query coverage is 100%.The reads statistical results of resequencing data showed that all the 8 sheep individuals had reads mapped to this region.Also,PCR amplification and agarose gel electrophoresis showed that all the 8 individuals of Large-tailed sheep（Contains the four sequenced individuals）had amplification products in 4 amplified regions.These results indicated that neither the intersexual nor normal individuals were not in the absence of this region.（2）Based on the calculate of the genetic differentiation factor(F_ST)of intersexual Lanzhou large-tailed sheep and normal sheep（normal Lanzhou large-tailed sheep and Tan sheep）,as well as intersexua and normal Lanzhou large-tailed sheep,and the gene annotation and functional enrichment analysis on selected areas,some genes associated with androgen biosynthesis and follicle stimulating hormone entry were uncovered in the selected region,such as SRD5A2,SRD5A3,PAWR.（3）This study found out some copy number variation regions which had significant different copy numbers between normal and intersexual groups by calculating the genome-wide copy number of 8 sheep and passing a series of filter conditions.Then the candidate copy number variable regions were further analyzed by gene annotation and functional enrichment analysis.As a result,4 candidate copy number variation regions were revealed,chr9:71660801-71662800,chr1:50776001-50778000,chr4:58119201-58121600,and chr16:778801-780800.These 4 regions are closer to or have overlap with the ZFPM2,LHX8,IMMP2L,and SLIT3 genes,respectively,and these 4 genes mainly play important roles in the process of female gonad development and female sex differentiation.Based on the above results,this study found that the deletion of the sequence of 8787 bp（chr1:247747059-247755846）in Lanzhou large-tailed sheep was not the cause of the intersex phenotype;The variations in SRD5A2,SRD5A3,and PAWR genes affecting the expression levels or protein activity,the synthesis and secretion of androgens in vivo,and the ability to maintain the complete development of the testes and their accessory reproductive organs,finally might cause the intersex.The ZFPM2,LHX8,IMMP2L,and SLIT3 genes are closely related to female gonadal development,so the copy number variation occurring in or near these genes might affect the expression of the gene and affect the formation and development of female gonads.The results obtained from the analysis of the possible mechanisms of intersex formation in Lanzhou large-tailed sheep in this study will provide a theoretical reference for the preservation and propagation of Lanzhou large-tailed sheep,and provide the basis.for timely eliminating intersexual individuals and reduce the economic benefit in animal husbandry and production.