Genetic Analysis Of Sex Reversal In The Chinese Tongue Sole(Cynoglossus Semilaevis)

The phenomenon of sex reversal is found in insects,amphibians,reptiles and fishes.It is a complex biological phenomenon that can change the sex ratio of the population and make the breeding process more complicated.half-smooth tongue sole?Cynoglossus semilaevis?is one of the most valuable marine flatfish in the Yellow Sea and Bohai Area of China.Since large-scale breeding,the advantages of its meat quality and rapid growth have been favored by consumers and farmers,and the price remains high.There is a significant difference between two sexes in the half-smooth tongue sole,the female growth rate is about 2 to 4 times that of the male.In the early stages of development,genetic females may change into pseudomales resulting a decrease in female rate in the population,which increases the cost and seriously affects the economic benefits of the half-smooth tongue sole.In actual aquaculture production,it is difficult to distinguish the genetic sex of individuals at the beginning of growth by the shape characteristics,which increases the economic cost.In this research,sexual reversal was used as a trait,and a new locus associated with sex reversal by GWAS.In is beneficial to further explore the genetic characteristics and gender determination mechanism of sex reversal in half-smooth tongue sole,and provide theoretical support for sex control and breeding practice.The main findings are as follows:1)Sex reversal was considered as a binomial trait,a new locus associated with sex reversal was identified by GWAS for 171 Z^TW individuals.This SNP located in the third intron of the doublesex and mab-3 related transcription fator1?Dmrt1?gene on the Z chromosome,named Cyn_Z8564889,and has two alleles A and G.genetic females including three genotypes,Z^AAW,Z^GGW,and Z^AGW.It may be due to multiple copies of Dmrt1 on the genome,2)The Cyn_Z6676874 and Cyn_Z8564889 genotypes that 259 genetic female individuals in population 2 were identified by sequencing,respectively.At the same time,the phenotypic sex of these individuals was observed under the microscope using the tissue sectioning technique.The results showed that under the same environmental conditions,Cyn_Z8564889 regulates sex reversal interactively with Cyn_Z6676874,genetic females reverse into phenomenal males when carrying A allele of Cyn_Z6676874,but the genetic females simultaneously carrying the T allele of Cyn_Z6676874 and A allele of Cyn_Z8564889 changing into pseudomales.These two SNPs explained all genetic variance of sex in half-smooth tongue sole.The genetic interaction between SNPs provides flexible options to select candidate parental fish for breeders.Any single or combined SNP examination can be utilized to improve phenotypic female ratios.At the same time,neutral chromosome types of fin and sperm DNA in pseudomales were identified,respectively.It was verified that pseudomales produce no or little W sperm.the interaction and linkage between Cyn_Z6676874 and Cyn_Z8564889 and the absence of W sperm from pseudomales unravel the genetic architecture of sex reversal in half-smooth tongue sole.3)A pair of cleaved amplified polymorphic sequence?CAPS?markers were developed for the sequence characteristics of the Cyn_Z8564889.When the sample size is large,it can be used to rapidly detect the genotype of the SNP,significant savings in inspection time and cost.4)Dmrt1 polymorphisms were detected by cloning sequencing for Z^AGW and sequencing for the exon of Dmrt1,two SNPs in the second exon of Dmrt1 which linking with Cyn_Z8564889 result in amino acid changes,from Val to Ile,Ala to Ser,respectively.suggesting that Dmrt1 is essential in sex reversal.5)The whole CDS of Fbxl17 gene was obtained by cloning and the amino acid sequence was predicted in half-smooth tongue sole.The complete CDS of Fbxl17 gene was 1956 bp encoding 651 amino acid.The deduced amino acid sequencing of Fbxl17had an estimated molecular weight of 70.96 kD and a theoretical pI of 8.06.Amino acid alignment indicated that it has no signal peptide and transmembrane domain,but contained one F-box domain composed of 42 amino acid and 11 leucine-rich repeats.These domains indicate that it is a member of the FBXL subfamily of the F-box protein family.Real-time PCR analysis showed that the expression trends of Fbxl17 show consistency,which in the gonads of individuals of different genders at different stages.The expression level is significantly lower in female individuals than physiological males,over time,the expression of pseudomales tends to be the same as that of males.In this study,based on the actual SNP Cyn_Z6676874,which related to the sexual reversal,a new locus associated with sex reversal was found by GWAS,named Cyn_Z8564889.Both SNP loci are located on the Z chromosome.Only when the T allele of Cyn_Z6676874 and the A allele of Cyn_Z8564889 locus appear at the same individual,the genetic female individual becomes a phenotype male.It is verified that the pseudomale fish do not produce or produce a small amount of W sperm,which explains why the offspring of pseudomales are pseudomales;the verification of different haplotypes of Dmrt1,the missense mutation on the second exon leads to the change of the secondary structure of Dmrt1,the phenomenon speculated that the genetic mechanism of the half-smooth tongue sole reversal has certain theoretical significance and practical value.In addition,the full-length CDS of the Fbxl17 gene of the half-smooth tongue sole was cloned for the first time.