高效非损伤性扇贝DNA提取方法的建立及效果评价

Development and Evaluation of a Rapid,Cost-Effective and Non-Destructive DNA Extraction Method in Scallops

本研究建立了一种高效、经济的非损伤性扇贝DNA提取方法,在不影响扇贝个体生存状态的前提下,采用擦拭法和室温裂解相结合的方式在20 min内即可获得个体基因组DNA。以虾夷扇贝(Patinopecten yessoensis)、栉孔扇贝(Chlamys farreri)和海湾扇贝(Argopecten irradians)为实验对象,研究了不同擦拭材料(棉签、滤纸)和不同擦拭部位(外套膜、鳃丝、内脏团)的核酸提取效果,评估了本方法在贝类基因分型领域应用的可行性。研究表明,用棉签、滤纸2种材料擦拭扇贝的鳃丝、内脏团或外套膜组织均能获得主带清晰完整的基因组DNA,且不影响扇贝的存活状态。在3种扇贝中采用棉签擦拭法的DNA得率均高于滤纸法,以这2种方式获得的DNA为模板进行SSR和SNP标记分析,能获得均一稳定的扩增条带,SSR标记分型结果准确可靠。本研究建立了简便、快速进行扇贝基因型分析的非损伤性DNA提取方法,为贝类全基因组选育和珍稀贝类资源的种质保护开发提供了新的技术手段。

In this study,we established a rapid,cost-effective and non-destructive DNA isolation method which requires two major steps(swabbing and lysis)to collect genomic DNA in less than 20 minutes.Evaluation of the concentration and quality of genomic DNA obtained by using two swabbing materials(cotton and filter paper)for three sampling tissues(gill,visceral and mantle)were performed in Yesso scallop(Patinopecten yessoensis),Zhikong scallop(Chlamys farreri),and Bay scallop(Argopecten irradians).Our results showed that the quality and quantity of genomic DNA obtained with all the six methods were in good performance in three scallop species.No obviously negative effects were observed on the survival or growth of all the treated scallops.Cotton swab sampling generally yielded a higher quantity of DNA than filter paper swab sampling in three species of scallops.Microsatellites and single nucleotide polymorphism(SNP)markers were well amplified using the genomic DNA extracted by cotton swabbing and filter paper swabbing sampling and SSRs could be accurately genotyped.In summary,a rapid,cost-effective non-destructive DNA isolation method is developed for genotyping in scallops,which can be applied to the genetic analysis of endangered or declining shellfish and facilitate the implementation of genomic selection in shellfish.