实验海洋生物学重点实验室知识库

中华绒螯蟹( Eriocheir sinensis) 是我国重要的经济动物，其生长和发育都具有明显的性别二态性，因此研究中华绒螯蟹性别决定机制对其遗传育种工作有着重要的意义。目前已有研究建立了中华绒螯蟹的高密度遗传图谱并筛选出了性别标记，且与其性别分化相关的多个基因已被鉴别出来。然而，迄今为止，在中华绒螯蟹中仍未出现简单快速且重复性好的鉴定性别的方法，其性别决定与分化机制仍然是未知的。

本研究结合中华绒螯蟹胚胎转录组数据和一对InDel性别标记测定了性别相关基因在中华绒螯蟹早期发育阶段的表达模式。首先，根据转录组注释信息及已发表文献，在中华绒螯蟹胚胎转录组中筛选出31个性别相关基因，并根据其RPKM值聚类成两类：在前三个时期（受精卵期，卵裂期，囊胚期）高表达的基因和在后两个时期（原肠胚期，心跳期）高表达的基因。对这31个基因进行DESeq（different expression gene analysis）分析和荧光定量验证，发现其中有12个基因在前三个时期和后两个时期的表达量呈显著性差异，且12个基因均为在后两个时期高表达的基因。结合其功能预测和相关文献分析，将这12个基因暂时定为与中华绒螯蟹早期性别决定相关的候选基因。然后，利用雌雄基因组比对，筛选出了一条操作简单，重复性好的雌性特异性InDel标记F23&R23，可实现中华绒螯蟹幼体阶段个体的雌雄鉴定；将此特异性标记定位到雌性基因组中的scaffold8227，并在scaffold8227上预测出三条编码蛋白基因：8227-cds1，8227-cds2和8227-cds3，三条编码序列未在已有数据库上比对到信息，但通过荧光定量分析，发现8227-cds1基因在成体雄性中华绒螯蟹各组织中的表达量都高于雌性，而8227-cds3基因在成体雌性中各组织均一表达，而在成体雄性中，精巢组织特异性高表达。最后，利用新开发的雌性特异性标记对中华绒螯蟹幼体期个体进行雌雄鉴定，再在已知雌雄的个体间检测了胚胎转录组筛选出的候选基因。分析其表达模式，发现，EsRpo1，Es-PGDS-like和EsDmrt11E基因在幼体期的表达都具有雌雄显著性差异。而且，被推测与雌性发育相关的基因，如EsRspo1和EsFoxl2基因都在幼体的前两个时期显著高表达；而被推测与雄性发育相关的基因，如Es-PGDS-like和EsDmrt11E基因都在大眼幼体时期显著高表达。另外，在幼体期间还检测了8227-cds3基因的表达量，发现该基因在溞状幼体Ⅴ期之前，其表达量都呈现雌性显著高于雄性，到了溞状幼体Ⅴ期和大眼幼体期，其表达量在雌雄之间逐渐持平。综合以上信息，我们推断，中华绒螯蟹卵巢发生可能在精巢发生之前。

本研究首次筛选到中华绒螯蟹雌性特异的InDel标记，并佐证了中华绒螯蟹为ZZ/ZW性别决定系统；并首次通过性别标记对单个个体进行分析，为进一步调查中华绒螯蟹的性别决定系统提供了丰富的背景资料，也为以后甲壳类性别决定研究提供了新的思路。

Investigation of sex determination system in Eriocheir sinensis is important because of sex-dimorphism in its growth traits. However, little information about the sex-related genes in embryonic and larval development stages were exposed. To obtain more information of sex determination in Chinese mitten crab, we combined the transcriptome in embryonic development stage in Eriocheir sinensis and the female specific marker to analysis the expression pattern of sex-related genes in early development stage in E. sinensis..

Firstly, we performed the transcriptome analysis in embryonic development stage (fertilized egg stage, cleavage stage, blastula stage, gastrula stage and heartbeat stage) of Chinese mitten crab using next-generation sequencing technology. Thirty-one of 32,088 annotated unigenes were identified as sex-related genes including 16 genes involved in primary sex determination in model organisms and 8 genes of SOX family and 7 genes of DMRT gene family. Heatmap based on the RPKM value in five embryonic development stages indicated that these genes were clustered into two branches. Analysis of the differentially expressed 12 genes, including 3 genes of SOX family, 3 genes of DMRT gene family and 6 genes involved in primary sex determination in model organisms, showed significantly difference between the first three periods (fertilized egg stage- cleavage stage- blastula stage) and the last two periods (gastrula stage- heartbeat stage) and all 12 genes were up-regulated after blastula stage. The 12 genes were regarded as the candidate genes of sex determination in E.sinensis. Secondly, we preformed the large-scale genome alignment between female and male in E.sinensis and obtained a female specific InDel marker-F23&R23, F23&R23 was mapped to scaffold8227 in female genome. We found three codon sequence: 8227-cds1, 8227-cds2 and 8227-cds3 and performed the expression pattern of the three genes. Finally, we performed the expression pattern of the candidate sex determination genes in the Chinese mitten crabs which was identified sex by qRT-PCR. In conclution, we inferred that the sex determination might initiated after blastula stage and the ovaries development might initiated earlier than the testes development in E.sinensis.

The study enriched the genetic resources of the E. sinensis and provided a background information for further investigation in sex determination of E. sinensis.