Hattori Ricardo Shohei, Kumazawa Keiichiro, Nakamoto Masatoshi, Nakano Yuki, Yamaguchi Toshiya, Kitano Takeshi, Yamamoto Eiichi, Fuji Kanako, Sakamoto Takashi
Department of Marine Biosciences, Tokyo University of Marine Science and Technology, Tokyo, Japan.
Nansei Field Station, National Research and Development Agency, Japan Fisheries Research and Education Agency, Mie, Japan.
Front Genet. 2022 Sep 16;13:1007548. doi: 10.3389/fgene.2022.1007548. eCollection 2022.
Japanese flounder () is an important marine fish species of both fisheries and aquaculture in Northeast Asia. The commercial interest for all-female progenies due to several sex-related traits has prompted basic research on the mechanisms of sex determination in this species. By conducting a linkage analysis of the sex-determining locus, we initially identified 12 microsatellite markers linked to sex in 11 scaffolds, whose localization was restricted to a specific region of linkage group 9. Sequence analysis of this region identified 181 genes based on the UniProt database annotations. Among them, the gene was considered a potential candidate for sex determination because this gene is known to have taken over the role of sex determination in many teleosts. An in-depth sequence analysis of both the coding and non-coding regions of in XX and XY individuals detected nine SNPs linked with maleness. However, because these substitutions were synonymous, the upstream and downstream regions of were also investigated and a male-specific variant with deletions in the promoter region was detected. This truncated Y-specific variant was named , and the shared by both sexes was named . The association analysis using both females and males of the genotypic sex inferred by the presence/absence of found complete association with phenotypic sex and genotype. Gene expression analysis in larvae derived from a single-pair progeny by quantitative real-time PCR detected transcripts in the larval trunks between 20 and 100 days after hatching only in XY larvae. Localization of by hybridization was detected in presumptive Sertoli cells of XY gonads. Expression of was almost undetectable in both XX and XY genotypes. Loss of Amh function by CRISPR-Cas9 induced male-to-female sex reversal, indicating that this gene was necessary for the masculinization of XY individuals. In conclusion, the complete linkage of with males, its early expression in XY gonads before testicular differentiation, and the induction of sex reversal by loss-of-function mutation support the view that is the sex-determining gene in this species.
牙鲆是东北亚地区渔业和水产养殖中重要的海洋鱼类品种。由于一些与性别相关的特性，全雌后代的商业价值引发了对该物种性别决定机制的基础研究。通过对性别决定位点进行连锁分析，我们最初在11个支架中鉴定出12个与性别相关的微卫星标记，其定位局限于连锁群9的特定区域。基于UniProt数据库注释，对该区域进行序列分析鉴定出181个基因。其中，该基因被认为是性别决定的潜在候选基因，因为已知该基因在许多硬骨鱼类中承担了性别决定的作用。对XX和XY个体中该基因的编码区和非编码区进行深入序列分析，检测到9个与雄性相关的单核苷酸多态性（SNP）。然而，由于这些替换是同义的，因此还对该基因的上游和下游区域进行了研究，并检测到一个启动子区域存在缺失的雄性特异性变体。这个截短的Y特异性变体被命名为，两性共有的变体被命名为。利用通过是否存在推断出的基因型性别的雌性和雄性进行关联分析，发现与表型性别和基因型完全关联。通过定量实时PCR对单对后代幼虫进行基因表达分析，仅在XY幼虫孵化后20至100天的幼虫躯干中检测到转录本。通过杂交对进行定位，在XY性腺的假定支持细胞中检测到。在XX和XY基因型中几乎都检测不到的表达。通过CRISPR-Cas9敲除功能导致雄性向雌性性逆转，表明该基因是XY个体雄性化所必需的。总之，与雄性的完全连锁、其在XY性腺中睾丸分化前的早期表达以及功能缺失突变诱导性逆转，支持了是该物种性别决定基因的观点。
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