| Sex determination and differentiation is an important and fundamental scientific question in biology.In vertebrates and model invertebrates,sex determination genes and their downstream sex differentiation factors have been already identified,which provide important foundation for clarifying sex determination and differentiation mechanisms.However,knowledge about sex determination and differentiation mechanism in crustaceans is still very limited,although crustacean represents a very large taxonomy in animal kingdom.Like other animals,crustaceans exhibit apparent sexual dimorphism.So understanding their sex determination and differentiation mechanisms will not only enrich theoretical knowledge on sex determination in crustacean,but also contribute to the development of mono-sex culture for economic species.Detection of sex-linked markers and sex determination genes is vital for studying sex determination mechanisms.In this study,we analyzed the characteristics of the sex determination region of the Pacific white shrimp Litopenaeus vannamei,identified sex-linked markers and established a molecular method for sex discrimination,developed sex-specific transcriptomes of embryos and larvae,which provides the bases for sex determination mechanism study.Furthermore,combining the analyses both at genomic and transcriptome level,one candidate sex determination gene was found,which will be very helpful for further understanding the sex determination mechanism in shrimp.The progresses are as follows:1.Characteristic analysis on the sex determination region and detection of sex-linked markers.Based on the existing sex-linked Marker19299 and GWAS analysis,structure characteristics of the sex determination region of L.vannamei were analysed.Two contigs(contig 122401 and contig 4497889)were obtained after mapping the sex Marker19299 to the genomic sequences of L.vanamei.A fragment of2630 bp was obtained through amplification in female DNA based on the sequences of these two contigs,which is located at the sex determination region.Further analysis showed that apparent sex-bias amplification existed in contig 122401,the female DNA contained more copies of contig 122401 sequence than that of males.Contig4497889 displayed sequence difference with an extra 9 bp insertion and a 205 bp repeat in females compared to males,which means there are significant difference in the sex determination region between genders.Based on the above characteristics in the sex determination region,a fast detection method to distinguish genetic sex in L.vannamei was developed.This method was carried out by PCR amplification and gel electrophoresis,which was more efficient at a low-cost.Besides,based on the whole genome sequencing data,five sex-linked markers were obtained which located in four scaffolds.These markers were heterozygote in females and homozygote in males,which further supporting the ZZ/ZW sex determination system in L.vannamei.These data might provide a new insight into the sex determination mechanism in shrimp.2.Construction of sex-specific transcriptomes of embryos and larvae in L.vannamei.A DNA/RNA co-extraction method was explored.After sex detection using the extracted DNA and the sex probe,RNA samples of the same gender were mixed for micro-transcriptome sequencing.Based on this technique,the sex-specific transcriptome of Zoea I stage,which is an important sex development stage,was established.Based on the transcriptome of Zoea I stage,a total of 60 DEGs between females and males were obtained.They might participate in the sex determination and differentiation process at early development stages.Paralogous genes of some known sex-related genes in model organisms showed no sex-biased difference at Zoea I stage.Candidate sex-linked SNPs were detected by BSR analysis and validated in six populations of L.vannamei.SNPs in Unigene0020898 and Unigene0020336 were fully linked to genders,suggesting that these two genes are located in the sex-determining region.Subsequently,using the co-extraction method and the micro-transcriptome construction technique,seven sex-specific transcriptomes from limb bud embryo stage to post larvae stage of L.vannamei were established.This is the first report on sex-specific transcriptomes of early developmental stage in L.vannamei.It will promote the researches on sex determination and differentiation process in shrimp.3.Characteristic analysis and expression patterns of Fem-1 family in L.vannamei.In the sex-specific transcriptome of Zoea Ⅲ stage,we found a homologous gene of C.elegans Fem-1 named Lv Fem-1c,it shows siginificantly higher expression level in females than in males.Besides,another two genes Lv Fem-1a and Lv Fem-1b were obtained.These Lv Fem-1 genes include eight to nine ANK domains,which showed the highest similarity with Es Fem-1 from the crab E.sinensis.Three Lv Fem-1genes showed different expression patterns in gonads of juvenile,subadults and adults of males and females.These data indicated that Lv Fem-1 family may play an important function in the sex development of L.vannamei.4.Functional analysis of the candidate sex determination and differentiation genes in L.vannamei.For the genes obtained in the sex-linked region,only c131213 displays expression difference in gonads.It was ovary specific,which was expressed throughout the development of ovary.This indicates that c131213 might play important rolesin ovary development.In the sex specific transcriptomes at early development stages,Unigene0018354 was obtained.It begins to express only in females from limb bud embryo stage,which suggested that the stage could be an important stage for sex determination and sex differentiation in L.vannamei.What’s more,the female-specific expression pattern of Unigene0018354 is maintained from limb bud embryo to adults.Therefore,we suggested that Unigene0018354 might be an important candidate gene for sex determination or sex differentiation.Its function is under further study. |
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