| P.clarkii is one of the most common freshwater shrimp species in China,the annual output is 1.638 million tons in 2018,which greatly promotes economic development and meat consumption.In order to explore the law of reproduction and development of P.clarkii in Guangxi and provide basis for artificial breeding,the ovarian sections and transcriptome sequencing of P.clarkii in Guangxi was investigated in this study.The main results of the present study are as follows:1.The body indexes of P.clarkii were measured.From May to August,the average body length of P.clarkii was 7.40,9.01,10.01 and 10.12 cm,respectively.The average body weight of P.clarkii was 20.77,28.96,31.95 and34.15 g,respectively.It was found that the growth rate of P.clarkii was the fastest from April to June,and slowed down from July to August.2.According to the ovarian color and section observation,the ovary development of P.clarkii was divided into four different stages,including previtellogenic stage(stage Ⅰ),early vitellogenic stage(stage Ⅱ),middle vitellogenic stage(stage Ⅲ),and mature stage(stage Ⅳ).The results showed that the ovarian development of P.clarkii was not synchronous,and there had different morphological oocytes in the same ovary at one period.July to August was the rapid development period of ovary every year,and a large number of female crayfishes hold eggs in the middle of September.3.The transcriptome sequencing,assembly and functional annotation of P.clarkii ovary were performed,a total of 649,136,402 original sequences were measured,and a total of 628,188,346 clean reads were obtained after quality control.There were 216,444 Unigenes with a length greater than 200 bp after splicing.There were 8,872(4.1%),23,683(10.94%),10,520(4.86%),14,913(6.89%),25,706(11.88%),8,504(3.93%),12,005(5.55%),19,773(9.14%)and3,560(1.64%)annotated Unigenes in NT,NR,KOG,Swiss-Prot,Tr EMBL,PFAM,CDD,GO and KEGG libraries,respectively,then the statistical analysis of annotation was carried out.4.The transcriptome data of four ovarian stages were compared in pairs to obtain five groups of comparative data,including Ⅱ vs Ⅰ,Ⅲ vs Ⅰ,Ⅲ vs Ⅱ,Ⅳ vs Ⅲ and Ⅳ vs Ⅰ,then the differentially expressed genes(DEGs)and their functional annotations were analyzed.Finally,five genes related to ovarian development were specifically analyzed,including Forkhead box L2(Fox L2),cell division cycle 2(cdc2),cyclin B,M-phase phosphoprotein 6(MPP6)and vitellogenin receptor(Vgr),and analyzed their expression differences at different stages.Among them,the expression of Fox L2 and Vgr continued to increase with the development of ovary;conversely,cyclin B and MPP6 continued to decrease;cdc2 increased from stage I to stage Ⅲ and then decreased dramatically in stage Ⅳ.5.RT-q PCR was used to verify the expression trends of nine genes with significant differences,including Lectin C,KAZAL,Astacin,PGHS,vitellogenin,SERPIN,VA5,CRISP and HGP2.The results showed that the expression trend of nine genes was consistent with the results of transcriptome sequencing,indicating that the transcriptome data was reliable.Furthermore,the expression of VA5,CRISP and HGP2 increased with ovarian development of P.clarkii,which was involved in the ovarian development process.This study would provide molecular data support for further study on the regulation mechanism of ovarian development of P.clarkii. |
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