Development and validation of sex - specific SNP markers in Larimichthys crocea

Larimichthys crocea is the largest cultured marine fish species in annual production in China. It shows obvious sex-related dimorphism in growth, where females grow much faster than males. It is difficult to distinguish the gender of the L. crocea through morphological characters, and also it has no evolutionarily differential sex chromosome. Currently, examination of the gonads after dissection is the most reliable way to distinguish the phenotypic sex in this fish. Given that the traditional methods is unsuitable for the identification of phenotypic and genetic sex, it is then necessary to develop sex-specific molecular markers, which is indispensable in sex-control breeding and understanding the mechanism of sex determination in this species. In order to develop sex-specific markers, sequencing data of short-insert libraries of 2 females and 2 males (30× coverage) and a pool of 50 females and a pool of 50 males (50× coverage) were utilized in this study. In brief, the raw sequencing reads were cleaned by removing Illumina sequencing adapters, low-quality sequences. The cleaned reads were aligned to our custom genome assembly (ENA accession no.: PRJEB24300) of the large yellow croaker using BWA, and the genome-wide SNPs were further called. We detected that 11 SNPs (hereafter referred as SNP 1~11) demonstrated obvious difference in allele frequency between males and females through comparative genomic approach. We further genotyped the 11 SNPs in 15 females and 15 males by PCR and Sanger sequencing, and found SNP 5 and SNP 6 were homozygous in all females and heterozygous in all males, perfectly segregated between sexes. We then devised an allelic-specific PCR (AS-PCR) based on SNP 6 for amplifying one band (348 bp) in females and two bands (348 bp and 194 bp) in males, and genotyped near 2 200 L. crocea sampled from Fujian, Guangdong and Zhejiang province. The genotyping result showed that all females were with the 348 bp band and all males were with the extra 194 bp band besides the 348 bp band in the AS-PCR followed by electrophoresis assay, suggesting 100% accuracy of the method in sex identification. 　　In conclusion, this study has detected and validated a male-specific (Y chromosome) SNP marker, and developed a simple method to identify the genetic sex for the large yellow croaker through the AS-PCR followed by electrophoresis assay. The finding provides an indispensible technical basis for sex-control breeding practice, genomic selection and the research on the molecular mechanism of sex determination in the L. crocea.