Screening SNP Markers And Mining Genes For Reproductive Traits In Litopenaeus Vannamei

Litopenaeus vannamei is the most productive species of shrimps in China and the world.China’s annual demand for parent shrimp exceeds 1 million pairs,and the demand for shrimp seedlings exceeds 1.5 trillion.However,there are great individual differences in the reproductive capacity of female shrimps in the production of larvae.Although a lot of studies have been carried out on the molecular mechanism of ovarian development,the molecular basis of the difference in reproductive ability of female L.vannamei is still lack of systematic understanding.In order to analyze the molecular basis of egg-laying amount and frequency of female L.vannamei,the high fecundity and low fecundity populations were established,and the selected genomic regions were located by selective sweep analysis.Some key SNPs and functional genes were pick out.The SNPs were verified in two newly constructed populations with obvious different reproductive abilities,and the expression of the selected genes in different ovarian development stages was analyzed.This study will preliminarily reveal the molecular mechanism of the difference in reproductive performance of L.vannamei and provide important reference for breeding new varieties with high reproductive ability.The main results are as follows:1.Single nucleotide polymorphism(SNP)and functional gene screening for fecundity of L.vannamei: based on the records of egg-laying amount and frequency of 604 L.vannamei females,the comprehensive selection index for fecundity of L.vannamei was calculated,30 female shrimps with high fecundity and 30 female shrimps with low fecundity were selected to form DNA mixing pools and carry out genome-wide resequencing.The genomic difference between the two mixing pools was analyzed by selective sweep analysis(Fst > 0.162,πratio >1.2).A total of 64,046 SNPs and 121 candidate genes were obtained from the genomic different regions.The KEGG pathway analysis revealed that some genes were enriched in the pathway associated with ovarian development,including the 5-hydroxytryptamine synapse pathway,Ovarian sterogenesis pathway,and the Arachidonic acid metabolism pathway.The results indicated that the high fecundity of L.vannamei may be related to neurotransmitter transmission,sex hormone synthesis and nutrition accumulation.2.Validation of SNP loci related to fecundity of female shrimp: 30 females of high fecundity and 30 females of low fecundity were selected to form fecundity difference groups.By using time-of-flight mass spectrometry,44 loci were successfully genotyped in the fertile segregation population,and 6 loci were significantly correlated with reproductive ability by chi-square test(P < 0.05),two loci were significantly correlated with reproductive ability(P <0.01).3.The expression patterns of candidate genes related to reproductive ability at different ovarian development stages: Real-time quantitative PCR was used to analyze the expression of LvPLCB4,LvRERE,LvCYP2L1,LvPARD3 and LvTRX2 in 4 different ovary development stages,including proliferative stage,small growth stage,large growth stage and mature stage in high and low fecundity female groups.The results showed that the expression of LvPLCB4,LvCYP2L1,LvPARD3 and LvTRX2 genes was significantly higher in the high fecundity group than that in the low fecundity group,it is suggested that these genes may play a role in promoting the oocyte proliferation and growth during the ovarian development of L.vannamei.However,the mechanism of the function of these genes is still not clear,it is worth further study.