Abstract
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 (Zea mays 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 (Oryza sativa 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.
Highlights
Next-generation sequencing (NGS) is an advancement in molecular biology that allows the detection of thousands of nucleotide variations and the development of functional molecular markers in various plant species regardless of their genome size [1]
Twenty-day-old rice seedlings were used for the extraction of genomic DNA (gDNA)
Our protocol resulted in a mean yield of 10.40 μg gDNA/g fresh tissue (Table 1), significantly higher than the CTAB-based and QIAGEN DNeasy Plant Mini Kit extraction protocols
Summary
Next-generation sequencing (NGS) is an advancement in molecular biology that allows the detection of thousands of nucleotide variations and the development of functional molecular markers in various plant species regardless of their genome size [1]. These markers have been used successfully in plant breeding to improve economically important agronomic traits from multiple crop species. Library preparation, sequencing runs, sequence validity, and the number of reads in these technologies depend mostly on the purity and quality of the genomic DNA (gDNA) [2,3]. The isolation of high-purity gDNA improves the accuracy and cost efficiency of high-throughput sequencing
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