一种高通量提取桃DNA方法的建立与应用

doi:10.3864/j.issn.0578-1752.2018.13.016

摘要/Abstract

摘要： 【目的】DNA制备是大规模基因型筛选和分子标记辅助选种的重要前提，本研究采用一种1.2 mL八联排管代替单个离心管，探索一种操作简便、节约时间、成本低的桃DNA快速提取方法，以满足高通量遗传研究的需求，提高工作效率。【方法】以普通生长型桃（standard type，ST）‘中油桃8号’为母本，温度敏感半矮生型桃（temperature-sensitive semi-dwarf in Prunus persica，PpTssd）‘09-1-112’为父本，杂交获得F₁代分离群体500株实生苗为载体，包括温度敏感半矮生型246株，普通生长型254株，建立一种高通量、低成本桃DNA提取方法。利用1.2 mL八联排管结合八通道移液器，简化提取步骤，提取桃幼嫩叶片中的基因组DNA；通过紫外分光光度计和琼脂糖凝胶电泳对所提取的DNA浓度、纯度和完整性进行检测。基于高分辨率熔解曲线（high resolution melting，HRM），采用96孔板对温度敏感半矮生型桃和普通生长型桃共500个F₁分离后代单株进行PCR扩增和基因分型，区分TSSDtssd基因型与tssdtssd基因型。基于双亲表型与基因型一致，开发InDel位点，PCR扩增后，采用SDS-PAGE在F₁群体中进行验证，确定利用分离的DNA是否正确区分不同基因型。【结果】分离的DNA经紫外分光光度计检测，浓度范围约为25—200 ng·μL^-1，OD_260nm/OD_280nm约为1.81—1.98，DNA纯度较高；琼脂糖凝胶电泳条带清晰、单一，DNA完整度较高。参考桃基因组（Version 2.0），根据双亲深度测序数据，开发获得SNP标记SNP_Pp03_3758620，应用于高分辨率熔解曲线基因分型，发现温度敏感半矮生型和普通生长型呈现明显不同的峰型，证明提取的DNA模板可应用于HRM基因分型。基于双亲基因型与表型一致，开发InDel标记InDel_Pp03_3829009，聚丙烯酰胺凝胶电泳的验证结果显示PCR扩增具有较高的强度，获得与目的片段大小一致的特异性条带，且在两种不同生长型单株中具有明显的多态性，表明PCR扩增稳定，提取的DNA可用于基于InDel标记的多态性分析。使用该提取方法，每人每天可以完成1 000个样品的DNA提取，成本较低，且不会对幼苗早期生长造成影响。【结论】建立了一种简便、有效、低成本的桃基因组DNA提取方法，可以满足基因分型、品种鉴定及遗传分析等分子生物学研究，实现了大批量不同样本基因组DNA的同时提取，具有较高应用价值。

关键词: 桃, 高通量, DNA提取方法

Abstract: 【Objective】 Preparation of large quantity and high-quality DNA is an important prerequisite for large-scale genotypic screening and molecular marker-assisted of plant breeding. The objective of this study is to present a low-cost, high-throughput peach (Prunus persica L. Batsch) genomic DNA extraction method, meet the needs of high-throughput genetic researches and improve the working efficiency.【Method】The population were obtained from a cross between female parent ‘CN8’ (standard type, ST) and male parent ‘09-1-112’ (temperature-sensitive semi-dwarf in Prunus persica, PpTssd type) to establish a high-throughout protocol for peach DNA extraction. The F₁segregating population were generated to assess the phenotype characteristics, resulting in observed 1﹕1 (254 standard type and 246 semi-dwarf type individuals). Subsequently, DNA extraction was carried out on the young leaves of two parents and 500 progenies by procedure using 1.2 mL thin-wall 8 strip polypropylene PCR tubes instead of a single centrifuge tube. After extraction, the quality of DNA samples was examined with ultraviolet spectrophotometry and 1% agarose gel electrophoresis, respectively. Referencing the peach genome (version 2.0) and using re-sequencing data, single nucleotide polymorphism (SNP) markers were developed and the HRM analysis was employed on F₁ population to conduct SNP genotyping. Ultimately, the extracted DNA samples were validated by using an InDel marker to verify the genotype of 500 individuals.【Result】The concentrations of DNA were in a range between 25 to 200 ng·μL^-1 and the UV absorbance ratios values (1.81-1.98) to determine DNA quality were acceptable and with high-purity. The result of agarose gel electrophoresis proved that DNA bands were clear, single with a high degree of DNA integrity. Referencing the peach genome and using whole genome re-sequencing data of two parents, SNP_Pp03_3758620 was developed in female and male parents, and the HRM analysis was employed to conduct SNP genotyping and divided temperature-sensitive semi-dwarf and standard type individuals into two groups, respectively, which proved DNA templates extracted from this DNA isolation procedure could be employed for HRM genotyping. Based on the genotype and phenotype of two parents, InDel_Pp03_3829009 was developed and the results of polyacrylamide gel electrophoresis showed that PCR amplification products showed desired fragment size, and were polymorphic in PpTssd type and ST type with bright and clear target fragment. It concluded that the extracted DNA samples could be used for indel analysis. Using this method, 1 000 samples of DNA could be extracted per day with low cost and no effect on the early growth of seedlings.【Conclusion】A simple, effective and low-cost method for extracting genomic DNA from peach was established, which can be used for molecular biology, such as genotyping, variety identification and genetic analysis. Simultaneous extraction of genomic DNA from large quantities of different samples was realized. It has high application value.

Key words: peach (Prunus persica), high-throughout, DNA extractionmethod

张南南，牛良，崔国朝，潘磊，曾文芳，王志强，鲁振华. 一种高通量提取桃DNA方法的建立与应用[J]. 中国农业科学, 2018, 51(13): 2614-2621.

ZHANG NanNan, NIU Liang, CUI GuoChao, PAN Lei, ZENG WenFang, WANG ZhiQiang, LU ZhenHua. Establishment and application of a high-throughout protocol for Peach (Prunus persica) DNA extraction[J]. Scientia Agricultura Sinica, 2018, 51(13): 2614-2621.

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