Development Of Sex-specific Molecular Marker And Identification Of Early Differentially Expressed Genes In Dai-qu Stock Large Yellow Croaker

The molecular mechanisms of sex determination and differentiation in fish are diverse and plastic compared to those in mammals,and cover almost all vertebrate sex determination modes,mainly including genotypic sex determination and environmental sex determination.Studies have shown that the sex chromosomes of most fish do not have heteromorphic,and it is difficult to distinguish them in morphology and molecular level.Therefore,the research on the sex determination region and sex determination gene of fish has always been a hot and difficult topic in this field.On the other hand,the traits of fish in economic aquatic products usually show sex dimorphism,and the development of sex-specific molecular markers is an important tool to realize the realization of unisexual culture and study the molecular mechanism of sex determination.The large yellow croaker,Larimichthys crocea,is an important marine economic fish in China,belonging to the genus Larimichthys in the family Sciaenidae,mainly distributed in the coastal areas of East Asia,and is loved by consumers.The previous studies have divided large yellow croaker into three geographical populations in Dai-qu stock,Min-yuedong stock and Nao-zhou stock from north to south.Min-yuedong stock and Dai-qu stock large yellow croaker are two geographical groups with distinct characteristics,e.g.morphology and growth,survival rate,fatty acid,randomly amplified polymorphic DNA and complete mitochondrial genome,even in the karyotype.At present,the molecular mechanism of sex determination and the development and application of sex molecular markers in Dai-qu stock large yellow croaker have not been reported,and there are relatively few studies on genes related to reproductive development and sex regulation.Based on a well-characterized large yellow croaker sex-determining region on chromosome 22,screened the genomic region between dmrt1 and dmrt3 genes of Dai-qu stock large yellow croaker.147 pairs of primers were designed for screening the sex-specific markers by PCR amplification.A pair of sex-specific molecular markers were successfully obtained between dmrt1 and cfap157.PCR amplification showed that the marker could only amplify one 500 bp band in females,but two bands of 500 bp and 390 bp could be amplified in males.At last,1 pair of primers were successfully screened and identified.A 110 bp specific deletion was found in the male specific band of large yellow croaker.This marker was verified in more than 300 Dai-qu stock large yellow croaker,and the results showed that the identified genetic sex was 100% consistent with phenotypic sex.The average coincidence rate between genetic sex identification and phenotypic sex was only 79.6% in Min-yuedong stock large yellow croaker.In this study,a sex-specific marker was developed and identified,which further verified that the Dai-qu stock large yellow croaker also belonged to XX♀-XY♂ type.Based on the sex-specific molecular markers developed above,the sex identification of juvenile large yellow croaker can be carried out.In this study,trunk samples of juvenile Dai-qu stock large yellow croaker at 20,30,40,55 and 70 days(dph)after hatching were collected,and DNA was extracted for sex identification.Transcriptome resequencing technology was used to sequence the males and females samples at the above different developmental stages,and the results of differential expression m RNA were analyzed.A total of 419.95 G of Clean Data were obtained from the whole transcriptional Data,and the effective Data amount of each sample ranged from 12.56 to 16.09 G,Q30 bases ranged from 89.28 to 92.38%,and the average GC content was 49.51%.Reads were compared to the reference genome to obtain the genome alignment of each sample,with the alignment rate ranging from 93.03 to 94.85%.Differential expression m RNA analysis of male and female gonads of Dai-qu stock large yellow croaker at different developmental stages showed that there were few differentially expressed genes at 20 dph.At 30 dph,differentially expressed genes began to increase.The most differentially expressed genes were found at 40 dph,and the dmrt1 gene began to express.It is speculated that 40 dph is the critical period for sex determination.According to GO and KEGG enrichment analysis,the differentially expressed genes are enriched in "Endocrine System","Endocrine and metabolic Diseases","Reproductive" and other pathways in different developmental stages of male and female gonads.Further analysis of anp32,hipk3,calpain-2 and other genes;The genes differentially expressed in adjacent gonad development were "Steroid biosynthesis","Oocyte meiosis","Progesterone-mediated Oocyte maturation" and "p53signaling Pathway" and "PI3K-Akt signaling Pathway" were enriched,and further analysis revealed ebp,msmo1,lss,plk1,cdk1,bub1,ccnb1 and other genes.In conclusion,this study developed and identified a sex-specific molecular marker of Dai-qu stock large yellow croaker.This marker was used to identify the genetic sex of juvenile Dai-qu stock large yellow crocea,and the differentially expressed genes at the early stage of gonad development were analyzed by transcriptome.It was found that the sex-critical gene dmrt1 was initially expressed in males at 40 dph,indicating that 30-40 dph was the key period for sex determination.This study provides a theoretical basis for the elucidation of the molecular mechanism of sex determination in the Dai-qu stock large yellow crocea,and provides a useful tool for achieving sex-controlled breeding.