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首页> 外文期刊>BMC Genomics >Construction of a high-density mutant library in soybean and development of a mutant retrieval method using amplicon sequencing
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Construction of a high-density mutant library in soybean and development of a mutant retrieval method using amplicon sequencing

机译：大豆中高密度突变体文库的构建及利用扩增子测序的突变体检索方法的发展

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Functions of most genes predicted in the soybean genome have not been clarified. A mutant library with a high mutation density would be helpful for functional studies and for identification of novel alleles useful for breeding. Development of cost-effective and high-throughput protocols using next generation sequencing (NGS) technologies is expected to simplify the retrieval of mutants with mutations in genes of interest. To increase the mutation density, seeds of the Japanese elite soybean cultivar Enrei were treated with the chemical mutagen ethyl methanesulfonate (EMS); M2 seeds produced by M1 plants were treated with EMS once again. The resultant library, which consisted of DNA and seeds from 1536 plants, revealed large morphological and physiological variations. Based on whole-genome re-sequencing analysis of 12 mutant lines, the average number of base changes was 12,796 per line. On average, 691 and 35 per line were missense and nonsense mutations, respectively. Two screening strategies for high resolution melting (HRM) analysis and indexed amplicon sequencing were designed to retrieve the mutants; the mutations were confirmed by Sanger sequencing as the final step. In comparison with HRM screening of several genes, indexed amplicon sequencing allows one to scan a longer sequence range and skip screening steps and to know the sequence information of mutation because it uses systematic DNA pooling and the index of NGS reads, which simplifies the discovery of mutants with amino acid substitutions. A soybean mutant library with a high mutation density was developed. A high mutation density (1 mutation/74?kb) was achieved by repeating the EMS treatment. The mutation density of our library is sufficiently high to obtain a plant in which a gene is nonsense mutated. Thus, our mutant library and the indexed amplicon sequencing will be useful for functional studies of soybean genes and have a potential to yield useful mutant alleles for soybean breeding.
机译：大豆基因组中预测的大多数基因的功能尚未阐明。具有高突变密度的突变体文库将有助于功能研究和鉴定对育种有用的新等位基因。使用下一代测序（NGS）技术开发具有成本效益的高通量方案，有望简化对目标基因突变的突变体的检索。为了增加突变密度，日本诱变大豆品种Enrei的种子用化学诱变剂甲磺酸乙酯（EMS）处理;用EMS再次处理由M1植物产生的M2种子。所得的文库由1536种植物的DNA和种子组成，显示出较大的形态和生理变异。根据12个突变株的全基因组重新测序分析，平均碱基变化数为每株12,796。平均每行691和35行分别是错义和无义突变。设计了两种用于高分辨率熔解（HRM）分析和索引扩增子测序的筛选策略来检索突变体。最后，通过Sanger测序确认了突变。与几种基因的HRM筛选相比，索引扩增子测序允许人们扫描更长的序列范围并跳过筛选步骤，并且知道突变的序列信息，因为它使用了系统的DNA合并和NGS读取的索引，从而简化了对HRM的发现。具有氨基酸取代的突变体。开发了具有高突变密度的大豆突变体文库。通过重复EMS处理，可实现较高的突变密度（1个突变/ 74？kb）。我们文库的突变密度足够高，可以得到其中无义突变的植物。因此，我们的突变体文库和索引的扩增子测序将对大豆基因的功能研究有用，并有可能产生用于大豆育种的有用突变等位基因。

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