1种快速鉴定金钱鱼遗传性别的方法

A rapid method for genetic sex identification in the spotted scat(Scatophagus argus)

金钱鱼(Scatophagus argus)是我国东南沿海名优养殖鱼类,具有XY性别决定系统,Dmrt1是其性别决定候选基因。金钱鱼生长具有性别二态性,雌鱼生长快于雄鱼。目前缺乏快速鉴定金钱鱼遗传性别的分子标记,阻碍了其性别控制育种技术的建立。本研究以公布的金钱鱼基因组数据,在Dmrt1附近设计多对标记引物,并通过PCR扩增验证标记的性别特异性。其中,标记引物Dmrt1-Marker-4-F/R在雌鱼中仅扩增出一条593bpX染色体条带,而在雄鱼中能扩增出593 bp和693 bp两条条带,分别来自X和Y染色体,表明该标记为共显性标记。利用该标记检测我国南海沿岸3个不同地理群体213尾金钱鱼的遗传性别与表型性别完全一致。此外,快速DNA提取试剂盒提取的片段较短DNA样品也可用于该对标记引物准确鉴定遗传性别。本研究建立了一种快速、准确、经济可靠的金钱鱼遗传性别鉴定方法,将旨为促进金钱鱼性别控制育种技术的建立,并为金钱鱼性别决定与分化机制研究提供依据。

The spotted scat(Scatophagus argus)is a valuable fish species in the southeast coastal areas of China.The female of this species grows at a comparatively more rapid pace than its male counterparts;as such,mono-female fingerlings may be used to improve production.In a previous study,the spotted scat was shown to possess an XX-XY sex-determination system.The truncate Dmrt1b and normal Dmrt1 are located on the X and Y chromosome,respectively;the male-specific Dmrt1 is the candidate sex determination gene.However,the lack of a rapid and accurate sex-specific marker has hampered the establishment of sex control during the breeding process,and investigations of sex determination and differentiation mechanisms in spotted scat.This study designed three pairs of sex-specific primers on Dmrt1/Dmrt1b according to published male and female genomic data on spotted scat using next-generation sequencing technology.The sex-specificity of these primers was validated using the polymerase chain reaction(PCR)-amplification in three male and female fish.All three markers were able to amplify the Y chromosome-specific band or the X and Y chromosome-specific bands.One pair of these primers,the Dmrt1-Marker-4-F/R,are a pair of co-dominant sex-specific primers located on the 3’untranslated region of Dmrt1.A single 593 bp-X chromosome-specific fragment was amplified in female fish using the Dmrt1-Marker-4-F/R primers,while the 593 bp-X and 693 bp-Y were specific fragments amplified in male fish.The Dmrt1-Marker-4-F/R were subsequently tested in three different geographic populations of spotted scat(213tails in total)from the South China Sea;the results of the genetic sex were consistent with their phenotypic sex.This sex-specific maker confirmed that the spotted scat possesses an XX-XY sex-determination system.It also implied that developing sex-specific markers in the sex determination gene or candidate sex determination gene region is very effective.In addition,this pair of primers was able to amplify shorter length deoxyribonucleic acid(DNA)fragments extracted by a rapid DNA extraction kit.This study establishes a time-effective and straightforward method for rapid genetic sex identification in spotted scat,accelerating the development of sex control breeding in spotted scat.However,the XX fish lack Dmrt1,critical for testicular development and male reproduction;this means it is impossible to reverse the sex of the XX female to a functional neo-male and as such,a genetically all-female fingerling is currently unavailable.As the phenotypic XX-female is irreversible,it becomes a new bottleneck for the sex control breeding of spotted scat.The growth comparison experiment demonstrated that some XY male fish were observed to be growing at a more rapid rate than some of the XX females;the XY spotted scat is sexually reversible.This means that selective breeding and sex control technologies may be combined to produce fast-growing all-male spotted scat in future.