Identification And Application Of Sex Markers And Sex-related Gene In Pseudomale Cynoglossus Semilaevis

Cynoglossus semilaevis,as a kind of marine fish with high market value,is an economic species with high breeding popularity in China's coastal areas,especially in the northern coastal areas.In the past 20 years of domestication,the scale of cultivation of Cynoglossus semilaevis has been steadily expanding.At the same time,Cynoglossus semilaevis is one of the species with the highest degree of sex dimorphism in flatfishes.The females have absolute growth advantages over males.In fact,due to the extremely low market value of male fish,only a small proportion of male fish will be selectively reserved for breeding in production,and the rest will be disposed as soon as possible.Therefore,it is one of the common breeding directions for the vast number of researchers and breeding technicians to cultivate full-female or highfemale Cynoglossus semilaevis.In the practice of fish production,we know that the pseudo males of Cynoglossus semilaevis are very common.The pseudo males,as a "neutral" fish with inconsistent physiological sex and genetic sex,may be the reason for the imbalance of sex ratio of Cynoglossus semilaevis population.Previous studies have found that offspring produced by pseudo males tend to become pseudo males just like their fathers,which breaks 1:1 sex ratio of Cynoglossus semilaevis population and leads to a higher male proportion of the physiological sex in the offspring.At a rough estimate,the proportion even reaches 80%.This has a great influence on the breeding of Cynoglossus semilaevis since it leads to the decrease of production efficiency year by year.Therefore,controlling the sex ratio of Cynoglossus semilaevis by selecting the breeding parents is widely carried out in recent years.The identification of pseudo males has got rid of the primary stage of visual observation.The developed sex-specific molecular markers can effectively determine the genetic sex of Cynoglossus semilaevis.The sex molecular marker of Cynoglossus semilaevis is also advancing with the progress of molecular biological technology.From the early AFLP marker to microsatellite marker,then to SNP and Indel marker,the detection efficiency are continuously improving.As to application scenario,it has moved from the laboratory to the breeding workshop in production.On the basis of summarizing previous studies,this paper enriches and optimizes the achievements on the excavation of sex-specific markers of Cynoglossus semilaevis by using current molecular biology methods,especially genomics tools,and strives to improve the traditional methods and establish a set of reasonable detection system that is more suitable for front-line in production.At the same time,the mechanism behind the sex marker of Cynoglossus semilaevis is preliminarily discussed during the marker excavating,which provides some new ideas and theoretical support for deconstructing the sex determination mechanism of Cynoglossus semilaevis,especially the mechanism of trans-generational inheritance of pseudo males.The specific work accomplished in this paper is as follows:1.Based on the genomic information of Cynoglossus semilaevis,two Indel markers and two SNP molecular markers were developed and verified.The Indel markers have higher resolution and lower agarose concentration than the previous markers,thus it can save time and reduce labor cost.We further transformed and optimized an indel marker which has been verified with expanded sample size.By combining the recombinant enzyme isothermal amplification system RAA and the lateral chromatography LFD system,we have optimized the traditional PCR and agarose electrophoresis detection scenarios and developed RAA-LFD detection test strip for sex identification of Cynoglossus semilaevis.This is also the first rapid test strip for sex identification of fish.The reagent strip can realize amplification detection and visual presentation of target gene fragments under isothermal conditions(usually 38?)for 30 min using sample DNA as a template.2.Genotyping by sequencing was carried out for sex trait related-SNP developing between different sex groups of Cynoglossus semilaevis.GWAS analysis was carried out for discovering multiple sex-related SNPs and a sex-related gene.Eight SNPs were captured on Z chromosome of Cynoglossus semilaevis for sex identification,and they could be used alone or in combination to carry out single-point and multi-point evaluation respectively.The multi-site reaction system will provide better accuracy.Among 8 SNPs,we found a sex-related gene prdm6 of Cynoglossus semilaevis,which is a histone methylated transferase gene.It is differentially expressed in gonads of Cynoglossus semilaevis with different sex.The expression in males is significantly higher than that in pseudo males and females,suggesting that histone methylation may be related to different sex of Cynoglossus semilaevis.3.It's the first time that seminal plasma exosomes were isolated and identified from half smooth tongue sole,which is also nonreported in all kinds of fish semen.The existence of exosomes was proved by transmission electron microscope observation,particle size tracing analysis and western blot.At the same time,the differences in small RNAs profile of seminal plasma exosomes from male and pseudomales Cynoglossus semilaevis were analyzed respectively through high-throughput next generation sequencing.Finally,4 mi RNAs and 6 pi RNAs were developed as ideal biomarkers,which can effectively distinguish male and pseudomale Cynoglossus semilaevis.Further,through the prediction and analysis of the target genes,it was found that the target genes of the four micro RNAs are all related to ovarian development and maintenance,which laid a solid foundation for the follow-up discussion on the regulation of sex of Cynoglossus semilaevis by small RNA.The signature PIRNAs: PIR-MMU-6643660,PIR-MMU-32360528,PIR-MMU-72274,PIR-MMU-31018127,PIR-MMU-29271668 and PIR-XTR-979116 are all highly expressed in male donors of Cynoglossus semilaevis,while very low in pseudomales.Among the 6 pi RNAs,the first 4 pi RNAs are related to sexual development and methylation from target gene prediction,while the latter 2 signals are only related to sexual development.At present,the functions of six pi RNA in all species including fish are not clear,which indicates that the regulation mechanism of these pi RNAs in the interaction with targets should be further studied.