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| 菲律宾蛤仔幼虫DNA的高效提取及评价 |
| Efficient Extraction and Evaluation of Manila Clam （Ruditapes philippinarum ）Larval DNA |
| 投稿时间：2024-07-05 修订日期：2024-08-26 |
| DOI： |
| 中文关键词: 基因组DNA D形幼虫 遗传分析 COX1 16Sr RNA 菲律宾蛤仔 |
| 英文关键词: Genomic DNA D-shaped larvae Genetic identification COX1 16SrRNA Ruditapes philippinarum |
| 基金项目:*国家重点研发计划项目(2023YFD2401705)；国家自然科学基金项目（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项目（NO.2023TD30） |
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| 中文摘要: |
| 本研究以菲律宾蛤仔的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。先将单个幼虫转移到PCR管中,加入10μL的PCR缓冲液,经过100℃加热5分钟,迅速转移到冰盒中,并加入0.5 μL蛋白酶K,并振荡混匀,在 PCR仪55℃条件消化60分钟,100℃加热10min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,16SrRNA和COX1为引物,扩增获得PCR产物的电泳条带清晰,测序验证准确,表明16SrRNA和COX1都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0.9365；16SrRNA共得到单倍型13个,单倍型多样性为0.7045。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。 |
| 英文摘要: |
| The Manila clam (Ruditapes philippinarum) is a marine bivalve mollusk with a broad temperature, salinity, and distribution range, primarily inhabiting tidal flats and shallow marine areas. It is suitable for high-density artificial cultivation in tidal flats and is one of the four major cultivated shellfish in China. However, there is extensive genetic exchange among R. philippinarum in adjacent marine areas. The artificial translocation and cultivation of R. philippinarum in China have had a certain impact on the genetic resources of local wild populations, profoundly affecting the genetic structure and ecological balance of geographical populations. Therefore, there is an urgent need for an effective genetic identification method to assess the genetic diversity and germplasm resource status of the R. philippinarum. Efficient extraction of larval DNA is a prerequisite for shellfish genetic research, but methods for the extraction and identification of trace amounts of DNA have not yet been reported. To address the limitations of current DNA extraction methods for marine shellfish larvae, this study has established an efficient DNA extraction method suitable for individual larvae of the R. philippinarum and other bivalve mollusks, which can extract high-quality genomic DNA from shellfish larvae the size of a few hundred micrometers. This study aims to provide an accurate and efficient DNA extraction method for species identification and genetic analysis of marine shellfish larvae. In this study, the D-shaped larvae and umbo larvae of R. philippinarum were selected as the research objects to perform DNA extraction from clam larvae. In this study, a rapid and efficient method for genomic DNA extraction from a single larva was established and successfully applied to genetic identification and haplotype analysis in R. philippinarum. First, a single larva was transferred to one PCR tube holding 10 μL PCR buffer. After being heated at 100 ℃ for 5 minutes, the PCR tube was quickly transferred to the ice box. The protease K (0.5 μL) was added to the tube for digestion at 55 ℃ for 60 minutes. Subsequently, the tube was heated at 100 ℃ for 10 minutes, and then centrifuged at 4 ℃ to collect DNA. The quality of larval DNA was evaluated by NanoDrop UV-Vis Spectrophotometer, agarose gel electrophoresis and DNA sequencing. The results showed that PCR products amplified by 16SrRNA and COX1 were both showing single clear band of target products, supported by their accurate sequences. It is therefore suggested that the obtained larval DNA can be successfully applied for genetic identification and haplotype analysis by using 16SrRNA and COX1 in R. philippinarum. According to haplotype analysis results, 14 haplotypes were obtained from COX1, having haplotype diversity of 0.9365, while 13 haplotypes were detected by 16SrRNA, showing haplotype diversity of 0.7045. The present results indicate that the DNA extraction method by PCR buffer can produce high-quality genomic DNA from shellfish larvae with several-hundred micrometer sizes. This provides an accurate, efficient and reliable method for species identification and genetic analysis of shellfish larvae, having broad application prospects in the field of genetic studies and molecular breeding in shellfish. |
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