Rana Md Masud, Aycan Murat, Takamatsu Takeshi, Kaneko Kentaro, Mitsui Toshiaki, Itoh Kimiko
Department of Life and Food Sciences, Graduate School of Science and Technology, Niigata University, Niigata 950-2181, Japan.
Agronomy Division, Bangladesh Rice Research Institute, Gazipur-1701, Bangladesh.
Methods Protoc. 2019 Jun 25;2(2):54. doi: 10.3390/mps2020054.
Next-generation sequencing (NGS) is a revolutionary advancement allowing large-scale discovery of functional molecular markers that has many applications, including plant breeding. High-quality genomic DNA (gDNA) is a prerequisite for successful NGS library preparation and sequencing; however, few reliable protocols to obtain such plant gDNA exist. A previously reported nuclear pellet (NP) method enables extraction of high-yielding gDNA from fresh leaf tissue of maize ( L.), but the quality does not meet the stringent requirements of NGS. In this study, we optimized the NP method for whole-genome sequencing of rice ( L.) through the integration of simple purification steps. The optimized NP method relied on initial nucleus enrichment, cell lysis, extraction, and subsequent gDNA purification buffers. The purification steps used proteinase K, RNase A, phenol/chloroform/isoamyl alcohol (25:24:1), and chloroform/isoamyl alcohol (24:1) treatments for protein digestion and RNA, protein, and phenol removal, respectively. Our data suggest that this optimized NP method allowed extraction of consistently high-yielding and high-quality undegraded gDNA without contamination by protein and RNA. Moreover, the extracted gDNA fulfilled the quality metrics of NGS library preparation for the Illumina HiSeq X Ten platform by the TruSeq DNA PCR-Free Library Prep Kit (Illumina). We provide a reliable step-by-step guide to the extraction of high-quality gDNA from fresh leaf tissues of rice for molecular biologists with limited resources.
下一代测序（NGS）是一项革命性的进展，它能够大规模发现功能性分子标记，具有许多应用，包括植物育种。高质量的基因组DNA（gDNA）是成功进行NGS文库制备和测序的前提条件；然而，现有的获取植物gDNA的可靠方法很少。先前报道的核沉淀（NP）方法能够从玉米（L.）的新鲜叶片组织中提取高产gDNA，但质量不符合NGS的严格要求。在本研究中，我们通过整合简单的纯化步骤，对用于水稻（L.）全基因组测序的NP方法进行了优化。优化后的NP方法依赖于初始细胞核富集、细胞裂解、提取以及随后的gDNA纯化缓冲液。纯化步骤分别使用蛋白酶K、核糖核酸酶A、苯酚/氯仿/异戊醇（25:24:1）和氯仿/异戊醇（24:1）处理来消化蛋白质并去除RNA、蛋白质和苯酚。我们的数据表明，这种优化后的NP方法能够持续提取高产、高质量且未降解的gDNA，且无蛋白质和RNA污染。此外，提取的gDNA满足了TruSeq DNA PCR-Free文库制备试剂盒（Illumina）用于Illumina HiSeq X Ten平台的NGS文库制备的质量指标。我们为资源有限的分子生物学家提供了一份从水稻新鲜叶片组织中提取高质量gDNA的可靠的分步指南。
