王中铎,何传猛,姚泽彬,李金蓬,陈子阳,邓爱萍,赖卓欣,李银芳,董忠典,郭昱嵩

• 1:广东海洋大学水产学院
• 2:广东省水产动物病害防控与健康养殖重点实验室

摘要(Abstract)：

【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。

关键词(KeyWords)： 弓背青鳉;三亚群体;性别特异性标记;重测序;染色体商区域;自然性别比

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金（41806195;31972794）;; 粤桂联合基金-面上项目（2020A1515410009）

作者(Author): 王中铎,何传猛,姚泽彬,李金蓬,陈子阳,邓爱萍,赖卓欣,李银芳,董忠典,郭昱嵩

参考文献(References)：

• [1] CAPEL B. Vertebrate sex determination:evolutionary plasticity of a fundamental switch[J]. Nature Reviews Genetics, 2017,18(11):675-689.
• [2] NAGAHAMA Y, CHAKRABORTY T, PAUL-PRASANTH B, et al. Sex determination, gonadal sex differentiation, and plasticity in vertebrate species[J]. Physiological Reviews,2021, 101(3):1237-1308.
• [3] MOORE E C, ROBERTS R B. Polygenic sex determination[J]. Current Biology, 2013, 23(12):R510-R512.
• [4] KONDO M, NANDA I, SCHMID M, et al. Sex determination and sex chromosome evolution:insights from medaka[J].Sexual Development:Genetics, Molecular Biology, Evolution,Endocrinology, Embryology, and Pathology of Sex Determination and Differentiation, 2009, 3(2/3):88-98.
• [5] AIDA T T. On the inheritance of color in a fresh-water fish,Aplocheilus latipes Temmick and Schlegel, with special reference to sex-linked inheritance[J]. Genetics, 1921, 6(6):554-573.
• [6] HAMAGUCHI S, TOYAZAKI Y, SHINOMIYA A, et al.The XX-XY sex-determination system in Oryzias luzonensis and O. mekongensis revealed by the sex ratio of the progeny of sex-reversed fish[J]. Zoological Science, 2004, 21(10):1015-1018.
• [7] TAKEHANA Y, NARUSE K, HAMAGUCHI S, et al.Evolution of ZZ/ZW and XX/XY sex-determination systems in the closely related medaka species, Oryzias hubbsi and O. dancena[J]. Chromosoma, 2007, 116(5):463-470.
• [8] TAKEHANA Y, DEMIYAH D, NARUSE K, et al. Evolution of different Y chromosomes in two medaka species, Oryzias dancena and O. latipes[J]. Genetics, 2007, 175(3):1335-1340.
• [9]施文瑞,王磊,陈军平,等.鱼类性别特异性DNA分子标记研究进展[J].水产科学, 2018, 37(6):862-866.
• [10] DONG Z D, CHEN Y, LI H, et al. Liver comparative transcriptome analysis reveals the mechanism of the Hainan medaka, Oryzias curvinotus, to adapt to salinity[J]. Journal of the World Aquaculture Society, 2022, 54(3):749-763.
• [11]董忠典,宦章,黎学友,等.炔诺酮短期暴露对海洋青鳉仔鱼的毒性效应[J].广东海洋大学学报, 2022, 42(6):65-73.
• [12]宋露露.海洋酸化对海洋青鳉鱼性腺分化的影响[D].上海:上海海洋大学, 2017.
• [13]王昊.海洋酸化条件下纳米CuO颗粒对海水青鳉的毒性效应[D].上海:上海海洋大学, 2019.
• [14]周运迪,吴星星,赵海萍,等.青鳉foxl2基因敲除突变体的构建与表型分析[J].广东海洋大学学报, 2019, 39(2):20-30.
• [15]张海瑞,王中铎,黄顺楷,等.弓背青鳉的胚胎发育及自发荧光观察[J].广东海洋大学学报, 2019, 39(2):38-44.
• [16] HAYAKAWA H, LE Q D, KINOSHITA M, et al. Genetic similarity of the Hainan medaka populations collected from hyper-and hypo-osmotic environments in northern Vietnam[J]. Ocean Science Journal, 2015, 50(2):231-235.
• [17] WANG Z D, LONG S S, LIAO J A, et al. Complete mitogenome of Hainan medaka Oryzias curvinotus(Teleostei:Beloniformes)and transcriptional differences between male and female liver[J]. Mitochondrial DNA Part B, 2017,2(1):157-158.
• [18] MATSUDA M, SAKAIZUMI M. Evolution of the sexdetermining gene in the teleostean genus Oryzias[J]. General and Comparative Endocrinology, 2016, 239:80-88.
• [19]黄泽胤,陈月碧,黎学友,等.盐度胁迫对弓背青鳉水通道蛋白基因转录的影响[J].生物技术, 2022, 32(4):415-420.
• [20]董忠典,黎学友,廖健,等.雌、雄弓背青鳉(Oryzias curvinotus)肝脏转录组比较分析[J].海洋与湖沼, 2020,51(5):1203-1213.
• [21] DONG Z D, LI X Y, YAO Z B, et al. Oryzias curvinotus in Sanya does not contain the male sex-determining gene dmy[J]. Animals, 2021, 11(5):1327.
• [22]汪淳.弓背青鳉三个地理群体表型比较与性别决定基因初探[D].湛江:广东海洋大学, 2020.
• [23]董忠典,龙水生,黄承勤,等.一种快速鉴定弓背青鳉遗传性别的方法[J].广东海洋大学学报, 2018, 38(3):25-29.
• [24] LI H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM[EB/OL]. 2013:arXiv:1303.3997. https://arxiv.org/abs/1303.3997
• [25] LIU H Y, PANG M X, YU X M, et al. Sex-specific markers developed by next-generation sequencing confirmed an XX/XY sex determination system in bighead carp(Hypophthalmichthys nobilis)and silver carp(Hypophthalmichthys molitrix)[J]. DNA Research, 2018, 25(3):257-264.
• [26] SUN Y L, JIANG D N, ZENG S, et al. Screening and characterization of sex-linked DNA markers and markerassisted selection in the Nile tilapia(Oreochromis niloticus)[J]. Aquaculture, 2014, 433:19-27.
• [27] XU P, ZHANG X F, WANG X M, et al. Genome sequence and genetic diversity of the common carp, Cyprinus carpio[J]. Nature Genetics, 2014, 46(11):1212-1219.
• [28] JIAN J B, YANG L D, GAN X N, et al. Whole genome sequencing of silver carp(Hypophthalmichthys molitrix)and bighead carp(Hypophthalmichthys nobilis)provide novel insights into their evolution and speciation[J].Molecular Ecology Resources, 2021, 21(3):912-923.
• [29] YAO Z B, LONG S S, WANG C, et al. Population genetic characteristics of Hainan medaka with whole-genome resequencing[J]. Frontiers in Genetics, 2022, 13:946006.
• [30] HAN C, ZHU Q Y, LU H M, et al. Screening and characterization of sex-specific markers developed by a simple NGS method in mandarin fish(Siniperca chuatsi)[J].Aquaculture, 2020, 527:735495.
• [31] WEN M, ZHANG Y X, WANG S Y, et al. Characterization of sex locus and sex-specific sequences in the mandarin fishes[J]. Aquaculture, 2022, 561:738650.
• [32] HAN C, ZHU Q Y, ZHOU X N, et al. A PCR-based genetic sex identification method in spotted mandarin fish(Siniperca scherzeri)and big eye mandarin fish(Siniperca kneri)[J]. Aquaculture Reports, 2020, 18:100552.
• [33] SURACHAT K, DEACHAMAG P, WONGLAPSUWAN M. The first de novo genome assembly and sex marker identification of Pluang Chomphu fish(Tor tambra)from Southern Thailand[J]. Computational and Structural Biotechnology Journal, 2022, 20:1470-1480.
• [34]张俊杰,赵瑞阳,蒋丽,等.白斑狗鱼雌、雄基因组混池重测序研究[J].淡水渔业, 2020, 50(3):17-25.
• [35]黄洋,黄远青,邓秋敏,等. 1种快速鉴定金钱鱼遗传性别的方法[J].中国水产科学, 2022, 29(4):515-524.
• [36] HATTORI R S, MURAI Y, OURA M, et al. A Y-linked antiMüllerian hormone duplication takes over a critical role in sex determination[J]. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(8):2955-2959.
• [37] CUI Z K, LIU Y, WANG W W, et al. Genome editing reveals dmrt1 as an essential male sex-determining gene in Chinese tongue sole(Cynoglossus semilaevis)[J]. Scientific Reports,2017, 7:42213.
• [38] CHEN S L, ZHANG G J, SHAO C W, et al. Whole-genome sequence of a flatfish provides insights into ZW sex chromosome evolution and adaptation to a benthic lifestyle[J]. Nature Genetics, 2014, 46(3):253-260.