摘要
本研究以菲律宾蛤仔(Ruditapes philippinarum)的D形幼虫和壳顶幼虫为研究对象,建立快速、高效的单个幼虫基因组DNA提取方法,并成功应用于菲律宾蛤仔的遗传学鉴定和单倍型分析。此方法的原理在于通过物理和化学手段破坏细胞结构,释放DNA,并通过热变性和快速冷却步骤实现DNA的收集。进行实验时,先将分离的单个幼虫转移到PCR管中,加入10μL PCR缓冲液,100℃加热5 min,迅速转移到冰盒中,并加入0.5μL蛋白酶K,并振荡混匀,在PCR仪55℃条件消化60 min,100℃加热10 min后,4℃离心并保存DNA。经超微量紫外可见光分光光度仪检测、琼脂糖凝胶电泳分离和DNA序列比对,评价幼虫DNA的质量。结果显示,以幼虫DNA为模板,设计用于扩增16Sr RNA和COX1基因的引物,扩增获得PCR产物的电泳条带清晰,测序验证准确,表明16Sr RNA和COX1均能对菲律宾蛤仔幼虫进行遗传鉴定和单倍型分析,COX1共获得单倍型14个,单倍型多样性为0.937;16Sr RNA共得到单倍型13个,单倍型多样性为0.705。研究结果证实,该方法可从几百微米大小的贝类幼虫中提取高质量的基因组DNA,为贝类幼虫的物种鉴定和遗传学分析提供了一种准确、高效和可靠的方法,在贝类幼虫生态学和遗传育种领域具有广阔的应用前景。
Ruditapes philippinarum—a marine bivalve mollusc known for its adaptability across a range of temperatures,salinities,and habitats—thrives primarily in tidal flats and shallow marine areas.It is an ideal candidate for high-density cultivation in tidal flats,making it one of the four major cultivated shellfish in China.Genetic exchanges are prevalent among populations in adjacent marine areas,and artificial translocation and cultivation of this species in China have affected the genetic resources of local wild populations,notably altering the genetic structure and ecological balance of these groups.Thus,there is an urgent need for an effective genetic identification method to assess their genetic diversity and germplasm resource status.Efficient extraction of larval DNA is essential for genetic research on shellfish;however,methods for extracting and identifying trace amounts of DNA have not been widely reported.This study addresses these limitations by establishing an efficient method suitable for individual larvae of the marine shellfish,R.philippinarum,and other bivalve mollusks.This method can extract high-quality genomic DNA from larvae as small as a few hundred micrometers,with the aim of providing an accurate and efficient DNA extraction method for species identification and genetic analysis of marine shellfish larvae.Here,D-shaped and umbo larvae were selected for DNA extraction.A rapid and efficient method for extracting genomic DNA from a single larva was developed and successfully applied for genetic identification and haplotype analysis.This method involved the use of physical and chemical means to disrupt the cell structure,and release and collect DNA through thermal denaturation and rapid cooling.A single larva was transferred to a PCR tube containing 10μL of PCR buffer,followed by heating at 100℃ for 5 min and quick transfer to an ice bath.After adding protease K(0.5μL)for digestion at 55℃for 60 min,the tube was heated again at 100℃for 10 min and then centrifuged at 4℃to collect the DNA.The larval DNA quality was evaluated using a NanoDrop UV-Vis Spectrophotometer,agarose gel electrophoresis,and DNA sequencing.The results demonstrated that PCR products amplified by 16SrRNA and COX1 showed a single clear band of target products,corroborated by their precise sequences.This suggests that the extracted larval DNA can be effectively used for genetic identification and haplotype analysis using these markers.Haplotype analysis revealed 14 COX1 haplotypes with a diversity of 0.937,and 13 haplotypes detected by 16SrRNA with a diversity of 0.705.These findings indicate that the PCR buffer DNA extraction method can yield high-quality genomic DNA from larvae several hundred micrometers in size.This method offers an accurate,efficient,and reliable approach for species identification and genetic analysis of shellfish larvae,with broad potential applications in genetic research and molecular breeding.
作者
张磊
许星鸿
刘志鸿
吴彪
周丽青
李转转
马培振
孙秀俊
ZHANG Lei;XU Xinghong;LIU Zhihong;WU Biao;ZHOU Liqing;LI Zhuanzhuan;MA Peizhen;SUN Xiujun(Marine Science and Fisheries College,Jiangsu Ocean University,Lianyungang 222005,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)
出处
《渔业科学进展》
北大核心
2025年第4期192-200,共9页
Progress in Fishery Sciences
基金
国家重点研发计划(2023YFD2401705)
国家自然科学基金(32273107)
崂山实验室科技创新项目(LSKJ202203803)
中国水产科学研究院基本科研业务费(2023TD30)共同资助。
