全基因组重测序筛选弓背青鳉三亚群体性别遗传标记

Screening of Sex Genetic Markers in the Sanya Population of Oryzias curvinotus by Whole-Genome Resequencing

【目的】开发弓背青鳉(Oryzias curvinotus)三亚群体(下称“三亚青鳉”)遗传性别鉴定分子标记,为进一步挖掘其性别决定基因奠定基础。【方法】用染色体商(Chromosome quotient,CQ)法比较雌雄两性个体的全基因组重测序覆盖度,筛选潜在的性别特异性序列区域(CQ<0.1)并设计引物,通过PCR扩增验证引物区分三亚青鳉群体及其子代性别的适用性。【结果】经CQ分析筛选,CQ<0.1的特异性区域279个,设计引物279对。随机挑选的60对引物中,29对标记引物在三亚青鳉群体雄鱼中扩增得一个DNA条带,雌鱼无条带,均可鉴定三亚青鳉遗传性别。从29对引物中随机挑选2对引物(命名为Marker1和Marker2)进一步验证,引物Marker1、Marker2在雄鱼中扩增产物分别为791、556 bp,两对引物对三亚青鳉子代群体的扩增结果与亲本一致。用两对性别特异性标记对三亚群体的F2代全同胞家系94个个体进行扩增,结果发现F2群体自然性别比约1∶1。【结论】性别特异性引物Marker1和Marker2均可对三亚青鳉群体进行遗传性别鉴定,在三亚青鳉野生亲本及其子代中均有稳定性和通用性,可用于弓背青鳉三亚群体遗传性别鉴定。

【Objective】This study aimed to develop molecular markers for genetic sex identification in a Sanya population of Oryzias curvinotus(named SY-medaka),and provide a foundation for exploring its sex-determining genes.【Method】We used chromosome quotient(CQ)to compare the whole-genome resequencing coverage of male and female individuals.Potential sex-specific sequence regions(CQ<0.1)were screened to design primers.The applicability of the primers to distinguish the sex of the wild Sanya population and its offspring was verified by PCR amplification.【Result】CQ analysis screened 279 specific regions with CQ<0.1.Among the 60 pairs of randomly selected primers,29 pairs of marker primers were screened because a DNA amplification band was obtained in the male fish of the wild SY-medaka population,and no band was obtained in the female fish.The primers Marker1 and Marker2 were randomly selected from 29 pairs of primers for further validation.The amplification product of primer Marker1,Marker2 in males were 791,556 bp,respectively.The two pairs of primers were amplified to the offspring population of Sanya medaka and the results were consistent with the parents.In addition,a natural sex ratio of approximately 1:1 was observed by sex-specific markers in 94 individuals of a sub-F2 generation of a full sibling family from the Sanya population.【Conclusion】Both sex-specific primers Marker1 and Marker2 can be used for the genetic identification of the Sanya population of O.curvinotus.They are stable and universal in both wild parent and its offspring.