摘要
为分辨斑节对虾(Penaeus monodon)发育早期的性别,利用改良的插入/缺失(InDel)标记追踪了从受精卵、无节幼体、溞状幼体、糠虾幼体、仔虾后1 d(PL1)、仔虾后30 d(PL30)及亚成虾个体的性别,建立了测序和荧光定量PCR熔解曲线的检测方法。结果表明,InDel标记可扩增获得雌雄特异性序列,用测序法判定亚成虾性别的结果与外部观察结果一致,准确率达100%,对受精卵、无节幼体、溞状幼体及糠虾幼体均可得到雌雄特异性序列;进而建立了基于SYBR Green实时荧光定量PCR(Quantitative real-time polymerase chain reaction,q RT-PCR)熔解曲线的快速鉴定斑节对虾性别的方法,其中雄性特异序列的熔解温度(Melting temperature,Tm)为(79.10±0.10)℃,雌性为(78.45±0.20)℃,通过特异的熔解曲线可准确区分PL30、亚成虾和无节幼体Ⅵ期个体,准确率可达96.3%以上。
In order to distinguish the sex of Penaeus monodon at embryonic and early stages of postembryonic development,we collected the individuals of fertilized eggs,Nauplii larvae,Zoea larvae,Mysis larvae,PL1(1 day post-larvae),PL30(30 day post-larvae)and subadults,tracked them by an improved Insertion/Deletion(InDel)marker,then established the detection methods of sequencing and fluorescence quantitative PCR.The results indicate that a sex-specific sequences could be obtained by this method.The result of sex discriminating of subadult shrimps by sequencing method(accuracy rate of 100%)was consistent with that by external observation method.Sex-specific sequences could be amplified from the samples of fertilized eggs,Nauplii larvae,Zoea larvae,Mysis larvae and post-larvae.Furthermore,we explored a rapid discriminating method based on the melting curve of qRT-PCR(Quantitative real-time polymerase chain reaction)with SYBR Green,and the value of melting temperature(Tm)of sex-specific sequences was(79.10±0.10)℃for male and(78.45±0.20)℃for female.The sex of individuals of PL30,subadults and Nauplii lavae at Stage VI could be distinguished by the specific melting curve accurately,with an accuracy rate over 96.3%.
作者
黄智康
江世贵
周发林
黄建华
杨其彬
姜松
李运东
杨丽诗
HUANG Zhikang;JIANG Shigui;ZHOU Falin;HUANG Jianhua;YANG Qibin;JIANG Song;LI Yundong;YANG Lishi(College of Fisheries and Life Science,Shanghai Ocean University,Shanghai 210306,China;South China Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences/Key Laboratory of South China Sea Fishery Resources Exploitation&Utilization,Ministry of Agriculture and Rural Affairs,Guangzhou 510300,China;Shenzhen Experimental Station of South China Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Shenzhen 518121,China)
出处
《南方水产科学》
CAS
CSCD
北大核心
2020年第3期113-118,共6页
South China Fisheries Science
基金
广东省现代农业产业技术体系创新团队建设专项(2019KJ149)
现代农业(虾)产业技术体系建设专项资金(CARS-48)
中国水产科学研究院南海水产研究所中央级公益性科研院所基本科研业务费专项资金资助(2019TS09)。
作者简介
黄智康(1993—),男,硕士研究生,研究方向为水产动物遗传育种与繁育。E-mail:hzhzk1993@163.com;通信作者:杨丽诗(1981—),女,博士,副研究员,从事水产遗传育种与生物技术研究。E-mail:yangls2016@163.com。
