Preliminary Analysis Of The Genetic Basis Of Sex Determination In Spinyhead Croaker(Collichthys Lucidus)

The mechanism of sex determination is a hotspot in fish genetic breeding and reproductive biology.In order to further understand the knowledge of fish sex determination,it is necessary to expand the research objects,especially the fish with multiple sex system,which are still vacant at present.In this study,sex-determining region(SDR)of Collichthys lucidus(X1X1X2X2/X1X2Y)was located and characterized,and molecular markers that could identify the hereditary gender of the southern population was developed,and the candidate sex-determining genes was preliminarily screened and characterized.This study on the genetic basis of sex determination in a species with a multiple sex system lays a foundation for understanding the influence of chromosome fusion on the sex determination region and the sex determination mechanism.The results were as shown:(1)Based on the female reference genome,SDR was located on Chr.1 4.3 Mb～4.9 Mb(0.6 Mb)and Chr.7 26.8 Mb～28.7 Mb(1.9 Mb)by GWAS and sex dimorphism genotype analysis.(2)The SDR region was preliminary characterized by statistical GC,gene and repeat distribution,and sequencing depth and depth ratio.The results showed that the Chr.1 before 23 Mb was significantly different from that of autosomes,with the content of all kinds of repetitive sequences which is higher and the content of protein coding sequence which is lower than that of autosomes,with the average sequencing depth which is about 1.5 times that of autosomes.The Chr.1 after 23 Mb and chr.7 showed no significant difference from autosomal features.In Chr.1 SDR,Contents of repetitive sequences except TR,other especially LINE and LTR are higher than those of autosomal.However,contents of protein coding sequences and sequencing depth were closer to the autosomes.In Chr.7 SDR,Content of TR is similar to that of chr.1 SDR,while the content of LTR was higher than that of autosomes.Other than that,no other obvious differences and autosomal characteristics were found.(3)A 68 bp Y chromosome specific deletion(27,630,158 bp-27,630,225 bp)was found by Chr.7 SDR male and female sequencing depth.Primer pair was design in the upstream and downstream of the predicted missing fragments.And the genomic DNA of male and female was used as the template for PCR verification.The female presented with one band(460 bp)and the male presented with two bands(460 bp and 392 bp).The primers were used to detect Ningde population and Jiulong River Estuary population,and the detection rates of females were 100%and males were 97%and 96%,respectively.The primers were used to identify the genetic gender of Zhoushan population,and the bands of males and females showed no difference,all of which showed a single band(460bp).(4)According to the female reference genome,83 genes were found in SDR.And 80 protein coding genes was annotated.According to the location of the sex-determining region,combined with the results of SNP annotation and gonad transcriptome differential expression analysis,7 male-determined candidate genes and 5 females-determined candidate genes were finally screened.The gender dimorphism variation and gene function of the first three genes with the largest FPKM value in the candidate sex determination genes were preliminarily characterized(male upregulated expression:hsf2bp,hyal5,iqub;female up-regulated expression drd4,zp3,ftsj1).No sexdetermining genes reported in other animals were found in SDR of spinyhead croaker.