摘要
暗纹东方鲀(Takifugu obscurus♀)×红鳍东方鲀(T.rubripes♂)的杂交F_(1)代具备双亲诸多优良性状,市场前景较好。但杂交F_(1)代的形态特征与其亲本难以区分,为河鲀种质资源保护和开发利用带来了困扰,迫切需要开发有效的分子鉴定方法对杂交F_(1)代及其亲本进行精准判别。为实现杂交F_(1)代及其亲本的快速准确鉴定,本研究根据核基因SH3PX3多态性SNP位点,设计荧光PCR扩增引物及探针,优化了荧光PCR参数,建立了暗纹东方鲀、红鳍东方鲀及其杂交F_(1)代的荧光PCR鉴定方法,并对该方法进行了验证。结果显示:杂交F_(1)代的COI序列与母本暗纹东方鲀的序列相似度为100%,在NJ进化树中聚为一支,无法实现杂交F_(1)代和母本的区分;SH3PX3基因荧光PCR体系最佳退火温度为48℃;荧光PCR扩增后,暗纹东方鲀仅FAM通道有Ct值,ΔCt值大于20,红鳍东方鲀FAM信号通道比HEX通道的Ct值高2~5,杂交F_(1)代的FAM通道与HEX通道的Ct值接近,二者之差小于2;基于上述方法对17份暗纹东方鲀、21份红鳍东方鲀和53份杂交F_(1)代样品进行验证,鉴定准确率为100%。本研究建立的荧光PCR鉴定方法不仅具有结果准确、易判读等优点,还避免了测序等繁琐流程,可实现高通量检测,显著提高了检测效率,为河鲀种质资源鉴定与保护、杂交育种和遗传多样性研究提供了技术支持。
Pufferfish,commonly known as"fugu,"belong to the family Tetraodontidae within the class Actinopterygii.With a wide variety of species,the genus Takifugu has the highest number of members and the greatest economic value.Notable representatives include Takifugu obscurus and T.rubripes.These two pufferfish species are characterized by their tender and delicious flesh as well as their high nutritional value.They are widely used in aquaculture,pufferfish toxin pharmaceutical development,and vertebrate genome research,thus holding significant economic,scientific,and medicinal value.T.obscurus is primarily distributed along the coasts of the Yellow,Bohai,and East China Seas in China.It can also enter the middle and lower reaches of rivers such as the Yangtze River and other connected lakes.It is one of China's important freshwater economic aquaculture species.Due to its high fat content and strong edibility,it is highly favored by domestic consumers,especially those who enjoy cooking.However,issues associated with aquaculture,such as small body size,slow growth,and sensitivity to low temperatures,have led to weaker market competitiveness.T.rubripes is mainly found in the Yellow Sea,East China Sea,and Taiwan waters of China.It is widely cultured in northern China,Japan,and South Korea and is suitable only for marine aquaculture.Compared to T.obscurus,T.rubripes has a faster growth rate and larger body size.However,when facing nutritional deficiency,its larvae may engage in cannibalism.After multiple generations of self-crossing,the germplasm of T.rubripes has degraded,with issues such as deformities and diseases severely impacting breeders.Hybrid breeding combines superior traits of parents to produce new hybrid varieties with improved characteristics.To obtain varieties with better traits and meet the rapidly developing needs of the pufferfish industry,aquaculture farmers have utilized the principle of hybrid vigor to increase production and efficiency.They have crossed T.obscurus(♀)with T.rubripes(♂)to produce hybrid F_(1) offspring with good market prospects.The hybrid F_(1) generation inherits the fast growth rate and large body size of the paternal parent(T.rubripes)and the freshwater culture capability of the maternal parent(T.obscurus).Therefore,the hybrid pufferfish not only retains the freshwater culture capability but also achieved improved growth rates and yields.However,the hybrid F_(1) generation has morphological features that blended with those of the parents.T.obscurus,T.rubripes,and their hybrid F_(1) generations are similar in appearance and body size at the larval,juvenile,and adult stages,making it impossible to distinguish them by naked eye observation.Additionally,certain tissues or processed meat products of pufferfish cannot be differentiated based on appearance alone.In recent years,the hybrid offspring,which share similar morphological features with their parents,have been easily mixed into the parental groups used for hybrid breeding.This has led to prominent issues in pufferfish aquaculture,such as mixed germplasm,serious quality degradation,and uncontrolled hybridization.The escape of hybrid individuals can also affect the gene pool of wild populations,which is not conducive for protecting germplasm resources.Accurate species identification is not only essential for distinguishing pufferfish species but also promotes the rational development of fishery resources,ecological surveys,and biodiversity conservation.Therefore,there is an urgent need for a method to distinguish between T.obscurus,T.rubripes,and their hybrid F_(1) generation.In our previous research,we identified a single nucleotide polymorphism(SNP)site in the SH3PX3 nuclear gene,which combined with mitochondrial genes,can be used to identify T.obscurus,T.rubripes,and their hybrid F_(1) generation.Direct sequencing of SNP sites can differentiate between the three.However,when faced with a large number of samples,the sequencing cost is relatively high,and the vast and complex data generated during sequencing impose higher demands on researchers'data analysis capabilities.In recent years,the TaqMan probe method based on fluorescence PCR has been widely used for gene expression,mutation,and polymorphism research because of its high sensitivity,speed,and specificity.Compared to ordinary TaqMan probes,TaqMan-MGB probes can accurately distinguish single-base differences and are commonly used for SNP genotyping.In the present study,we designed fluorescence PCR amplification primers and probes based on the polymorphic SNP site of the SH3PX3 nuclear gene,optimized the fluorescence PCR parameters,and established a fluorescence PCR identification method for T.obscurus,T.rubripes,and their hybrid F_(1) generation.This method was validated,with the results showing that:(1)The COI sequence of the hybrid F_(1) generation was 100%identical to that of the maternal parent T.obscurus,and they clustered together in the NJ phylogenetic tree,making it impossible to distinguish between the hybrid F_(1) and the maternal parent;(2)The optimal annealing temperature for the SH3PX3 gene fluorescence PCR system was 48℃;(3)After fluorescence PCR amplification,only the FAM channel of T.obscurus has a Ct value,and theΔCt is greater than 20,T.rubripes had a Ct value in the FAM channel that was 2 to 5 higher than that in the HEX channel,and the Ct values of the FAM and HEX channels in the hybrid F_(1) were close,with a difference of less than 2;(4)Based on the above method,17 samples of T.obscurus,21 samples of T.rubripes,and 53 samples of hybrid F_(1) were verified,with an identification accuracy rate of 100%.The fluorescence PCR identification method established in this study not only provides accurate results and easy interpretation,but also avoids cumbersome processes such as sequencing.It can achieve high-throughput detection and significantly improve detection efficiency.This method offers technical support for the identification and protection of pufferfish germplasm resources,hybrid breeding,and genetic diversity research.
作者
赵昕
车帅
王焕
孙侦龙
尤颖哲
柳淑芳
庄志猛
ZHAO Xin;CHE Shuai;WANG Huan;SUN Zhenlong;YOU Yingzhe;LIU Shufang;ZHUANG Zhimeng(Marine Fishery College,Zhejiang Ocean University,Zhoushan 316022,China;State Key Laboratory of Mariculture Biobreeding and Sustainable Goods,Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Qingdao 266071,China;Laboratory for Marine Fisheries Science and Food Production Processes,Qingdao Marine Science and Technology Center,Qingdao 266237,China;Jiangsu Zhongyang Group Co.,Ltd.,Nantong 226600,China;Zhangzhou Fisheries Technical Extension Station,Zhangzhou 363000,China)
出处
《渔业科学进展》
北大核心
2026年第1期37-47,共11页
Progress in Fishery Sciences
基金
国家重点研发计划(2019YFC1604700)
国家现代农业产业技术体系(CARS-47)
中央级公益性科研院所基本科研业务费专项资金(2024JC0101
20603022024023)共同资助。
