Genomic Study Of Sex Determination In Half-smooth Tongue Sole (Cynoglossus Semilaevis)

Half-smooth tongue sole (Cynoglossus semilaevis) is a commercially valuableflatfish that is widely distributed in Chinese coastal waters. It displays obviouslysexual dimorphic differences between male and female individuals, concentrated onthe growth rate that females grow2-4times faster than males. The existence ofskewed male sex ratio (70%) in domestic stock resulting from sex reversal make aserious impediment to large-scale promotion of tongue sole aquaculture industry.Thus it particularly significant to achieve all-female breeding for tongue sole. Notsimilar to what is seen in most studied teleosts, classical karyotype analysis andartificial gynogenesis testing reveal that the tongue sole employ the femaleheterogamety sex determination system (ZW/ZZ) with a special heteromorphic Wchromosome. Therefore, the tongue sole, with the complex sex determination systemgoverned by the interaction between genetic determination and influence oftemperature, together with the available genome reference sequence withdistinguishable sex chromosomes, is an excellent model to better understand themechanism of sex determination and differentiation. In this thesis, a different level ofgenomic analysis focus on the sex determination and differentiation of tongue solewere carried out, including the construction of BAC libraries and characterization ofpositive clone of sex-related gene, construction of high-density SNP genetic map andgenome-wide association study of sex, and whole genome bisulfite sequencing forthe sex reversal fish. The main results are as follows:Two bacterial artificial chromosome (BAC) libraries for tongue sole, with large,high-quality inserts and deep coverage, were constructed in the BamHI and HindIIIsites of the vector pECBAC1. The two libraries contain a total of55,296BAC cloneswith average insert size of approximately156.4kb arrayed in144384-well microtiterplates and correspond to13.36haploid genome equivalents. The combined librarieshave a greater than99%probability of containing any single-copy sequence. Screening high-density arrays of the libraries generated76and152positive cloneswith average of15.2and30per probe for sex-related genes and female-specificmarkers. Five positive BAC clones containing sex-related genes and female-specificmarkers were sequenced using ABI3730conventional sequencing, generatedassembly sequences with length of127kb (Sox9),145kb (Dmrt1),165kb (Dmrt1),107kb (Cyp19a1a) and135kb (CseF382). A full-length BAC sequence with ninegenes including Cyp19a1a gene was further analyzed on the structural characteristicssuch as repeat sequence, GC content, transposable elements, etc. A comparativeanalysis on the Cyp19a1a gene was carried out among medaka, zebrafish, three-spinestickleback, Fugu rubripes and Tetraodon, suggesting that the conservation ofCyp19a1a gene structure and function, mainly including the same pattern of exonsand introns, five conserved functional domains, the common upstream regulatoryelements and highly expressed in ovarian specificity for all the teleost Cyp19a1agene.A high-density SNP genetic linkage map of tongue sole was constructed byRAD-Seq based on next-generation sequencing technology. Firstly, the whole genomeresequencing of tongue sole parents was performed on HiSeq2000, generated113Mand101M reads, corresponding the genome coverage of92.8%and94.9%,respectively. After a rigorous selection, a total of407,084,397,300and89,989SNPloci covering3,553,3,509and1,925scaffolds with the total length of452Mb,453Mb and448Mb, respectively, were identified for the male heterozygous loci (1:1), thefemale heterozygous loci (1:1) and the parents heterozygous loci (1:2:1).216individuals of the F1generation were sequenced with depth of0.4×, generated thereads distributing from2,275,870to11,639,889for an average of6,224,947. Finally,the number of RAD-tag from699,271to1,134,343with an average of913,371for216individual of F1was attained. A total of13,116SNP loci including the femaleheterozygous loci (5,610), the male heterozygous loci (6,428) and the parentsheterozygous loci (1,078) was identified by separation ratio test (P=0.05). Ahigh-density genetic map with22linkage group corresoponding20autosomes and2sex chromosomes was constructed using a well-proportioned3,528SNP loci, covering a total of1999.535cM, with an average interval0.568cM between markers.A genome-wide linkage analysis of sex trait was performed on the high-density SNPgenetic linkage map, and found that the LOD score of all the W chromosome markerswas significantly greater than2.5with the negative additive effects, indicating thatthe W chromosome allele is associated with sex determination of tongue sole.A single base-resolution methylation profiling of the tongue sole was depicted bythe whole genome bisulfite sequencing (WGBS), revealed the epigenetic mechanismof sex reversal in tongue sole. Total effective data16.81Gb,21.15Gb and18.63Gbwere generated for normal female, normal male and pseudo-male using IlluminaHiSeq2000. The mCs accounting for8.67%,8.86%and8.76%of all cytosine in thegenome for three samples, respectively. More than97%of methylated sites occur inCpG regions, and the majority of mCs have a high level of methylation. On both sidesof the TSS region in tongue sole genes has a lower level of methylation while thecoding regions, introns and3’UTR has a high level of methylation. All of transposableelements (DNA, LINE, SINE, LTR, etc.) showed a similar methylation level with thewhole genome. In addition, small RNAs exhibited different methylation levels with ahigh level of methylation in the downstream region of snRNA and a relative lowermethylation level for rRNA and tRNA, and miRNA presents a similar methylationlevel with the whole genome. According to location of mCs, the tongue sole geneswere divided into methylated genes and non-methylated genes. A total of20,237,20,183and20,199methylated genes, accounting for97.03%,96.77%and96.85%ofall the genes were identified for the normal female, normal male and pseudo-male,respectively. The methylation level of whole genome in normal male and pseudo-maleis relative higher than female. The differential methylated regions (DMRs) was foundto be14.87Mb and11.16Mb by comparison of normal females against male andneomale, respectively, while the DMRs between normal males and neomale is only0.25Mb. Differentially methylated genes origining from DMRs such as Cyp19a1a,Sox9a, Foxl2, Gsdf were further characterized in this thesis.