Development Of Molecular Markers For Gender Identification In Large-mouth Bass And Localization Of Genes Related To Gonad Development

The largemouth bass is an important freshwater aquaculture fish in China,and it exhibits certain sex differences in growth rate.It is difficult to distinguish the sex of individuals from their appearance outside the breeding season.Furthermore,our understanding of the mechanism of sex determination and gonadal development in largemouth bass is still limited.Therefore,this study first used the largemouth bass genome resequencing data to locate the sex determination region and develop gender-specific markers,and then fine-mapped the sex determination region.We also used genome-wide association analysis to map the chromosome interval associated with the gonadosomatic index of largemouth bass and to mine candidate functional genes for gonadal development.The main research results are as follows:1.Preliminary localization of the sex determination region of largemouth bass and development of gender-specific molecular markersThrough analyzing the resequencing data of 688 largemouth bass using genome-wide association analysis and genetic differentiation(Fst),we preliminarily located the sex determination region on chromosome 10 of largemouth bass.We detected the SNP and Indel loci with genotype concordance(the male is heterozygous XY and female is homozygous XX)larger than 95% in both males and females.The results showed that these loci were distributed in the interval of 35.03-35.29 Mb.We defined this 260-Kb region as the sex determination region(SDR)of largemouth bass and annotated eight protein-coding genes within this interval.Based on three deletions longer than 10 bp,we developed three sex markers(MFS-1,MFS-2,and MFS-3)and verified their accuracy using five different populations of largemouth bass,with an accuracy of96.6% for these three markers.2.Fine mapping and candidate gene mining of the sex determination regionMultiple genes were found within the sex determination interval identified in this study,but no commonly-known sex determination genes in fish were detected.To identify the main sexdetermining gene in largemouth black bass,we conducted a joint analysis with sequencing data from individuals from different populations,including Sanming,Putian,Foshan,Nanchang,Guangdong,and northern subspecies(a total of 33 individuals).Based on the distribution of sexbiased SNPs,we narrowed down the sex determination interval to 127.68Kb(35.04-35.17 Mb)and identified 6 candidate functional genes.The most significant SNP in the GWAS analysis was located in the fbxl19 gene.3.Mapping of genes related to sex gland development in largemouth black bassA whole-genome association analysis was performed on 688 largemouth black bass individuals with sex gland index as the phenotype.Seven SNPs significantly associated with male gonadosomatic index(MGSI)were found on chromosome 7,and two SNPs significantly associated with female gonadosomatic index(FGSI)were found at suggestive levels on chromosome 1.Candidate functional genes were identified within 200 Kb upstream and downstream of these SNPs.Fourteen candidate MGSI genes were annotated,among which the gimap8 gene had six copies within this interval and the most significant SNP in the MGSI GWAS analysis was located in this gene.There were nine genes possibly related to FGSI,among which the mak gene exhibited high tissue specificity and played an important role in spermatogenesis,and the ropn1 l gene played an important role in sperm activation by maintaining the integrity and mobility of the fibrous sheath.In summary,this study mapped the sex-determining interval of the largemouth bass to a region of about 130 Kb,identified related candidate genes,and successfully developed a more widely applicable molecular marker for sex identification.At the same time,this study preliminarily located the chromosome region related to the sex gland index of the largemouth bass.The results of this study not only provide a theoretical basis for the discovery of sex-determining genes and the study of mechanisms related to sex gland development in largemouth bass,but also will contribute to the development of mono-sex breeding of largemouth bass.