大黄鱼性别特异SNP标记的开发与验证

大黄鱼是我国养殖量最大的海水经济鱼类,其雌鱼生长显著快于雄鱼,但两性的外部形态差异不明显,也没有异形性染色体,依靠传统方法无法对其活体准确进行生理性别和遗传性别的判别与鉴定,需要开发性别特异的分子标记。本研究从2尾雌鱼和2尾雄鱼、以及分别由50雌鱼与50尾雄鱼组成的2个混合样品的基因组重测序数据比较中筛选与性别显著关联的SNP位点,对其中11个位点分别设计引物在15尾雌鱼和15尾雄鱼中扩增出PCR产物进行Sanger测序验证,鉴定出1个与性别完全连锁的位点(SNP6,15尾雌鱼均为纯合、15尾雄鱼均为杂合)。然后,设计等位基因特异性PCR引物,其中包括2条雌性与雄性通用引物和1条雄性特异引物,在闽—粤东族与岱衢族大黄鱼合计近2 200个个体中进行扩增,结果在全部雌鱼中都只扩增出1个348 bp的条带,而在全部雄鱼中还扩增出1个194 bp的Y染色体特异条带,检出率达到100%。研究表明,大黄鱼属于XX♀-XY♂类型的性别决定。本研究鉴定出一个雄性特异SNP标记,并建立了一种新的大黄鱼遗传性别鉴定技术,为大黄鱼单性育种、基因组选择育种和性别决定分子机制研究提供了重要的技术手段。

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.