Table_1_The Pattern and Distribution of Induced Mutations in J. curcas Using Reduced Representation Sequencing.XLSX

Mutagenesis in combination with Genotyping by Sequencing (GBS) is a powerful tool for introducing variation, studying gene function and identifying causal mutations underlying phenotypes of interest in crop plant genomes. About 400 million paired-end reads were obtained from 82 ethylmethane sulfonate (EMS) induced mutants and 14 wild-type accessions of Jatropha curcas for the detection of Single Nucleotide Polymorphisms (SNPs) and Insertion/Deletions (InDels) by two different approaches (nGBS and ddGBS) on an Illumina HiSeq 2000 sequencer. Using bioinformatics analyses, 1,452 induced SNPs and InDels were identified in coding regions, which were distributed across 995 genes. The predominantly observed mutations were G/C to A/T transitions (64%), while transversions were observed at a lower frequency (36%). Regarding the effect of mutations on gene function, 18% of the mutations were located in intergenic regions. In fact, mutants with the highest number of heterozygous SNPs were found in samples treated with 0.8% EMS for 3 h. Reconstruction of the metabolic pathways showed that in total 16 SNPs were located in six KEGG pathways by nGBS and two pathways by ddGBS. The most highly represented pathways were ether-lipid metabolism and glycerophospholipid metabolism, followed by starch and sucrose metabolism by nGBS and triterpenoid biosynthesis as well as steroid biosynthesis by ddGBS. Furthermore, high genome methylation was observed in J. curcas, which might help to understand the plasticity of the Jatropha genome in response to environmental factors. At last, the results showed that continuously vegetatively propagated tissue is a fast, efficient and accurate method to dissolve chimeras, especially for long-lived plants like J. curcas. Obtained data showed that allelic variations and in silico analyses of gene functions (gene function prediction), which control important traits, could be identified in mutant populations using nGBS and ddGBS. However, the handling of GBS data is more difficult and more challenging than the traditional TILLING strategy in mutated plants, since the Jatropha genome sequence is incomplete, which makes alignment and variant analysis of target sequence reads challenging to perform and interpret. Therefore, providing a complete Jatropha reference genome sequence with high quality should be a priority for any breeding program.

诱变育种结合测序分型(Genotyping by Sequencing, GBS)是一项强大的研究工具，可用于引入遗传变异、解析基因功能，并鉴定作物基因组中目标性状背后的因果突变。本研究依托Illumina HiSeq 2000测序仪，通过两种不同的测序分型策略（nGBS与ddGBS），对82株乙基甲磺酸酯(ethylmethane sulfonate, EMS)诱导的麻疯树(Jatropha curcas)突变体与14份野生型材料进行测序，共获得约4亿条双端测序读段，用于检测单核苷酸多态性(Single Nucleotide Polymorphisms, SNPs)与插入/缺失(Insertion/Deletions, InDels)变异。经生物信息学分析，研究人员在编码区中共鉴定出1452个诱导型SNPs与InDels变异，分布于995个基因中。所观测到的突变类型以G/C到A/T的碱基转换为主，占比64%；碱基颠换的发生频率较低，仅为36%。关于突变对基因功能的影响，18%的变异位点位于基因间区。实际上，在经0.8% EMS处理3小时的样本中，可获得杂合SNPs数量最多的突变体材料。代谢通路重构分析显示，通过nGBS策略共在6条京都基因与基因组百科全书(KEGG)通路中定位到16个SNPs位点，而ddGBS策略则定位到2条通路中的SNPs；其中nGBS鉴定到的富集程度最高的通路为醚脂代谢与甘油磷脂代谢，其次是淀粉与蔗糖代谢，ddGBS鉴定到的富集通路则为三萜类生物合成与类固醇生物合成。此外，研究团队在麻疯树基因组中观测到较高的甲基化水平，这有助于解析麻疯树基因组响应环境因子的可塑性机制。最后，研究结果表明，持续利用无性繁殖组织是一种快速、高效且准确的嵌合体消解方法，对于麻疯树这类长寿命植物尤为适用。本研究获得的数据表明，通过nGBS与ddGBS策略，可在突变体群体中鉴定出控制重要性状的等位变异，并开展基因功能的计算机模拟分析（基因功能预测）。但由于麻疯树参考基因组序列尚不完整，相较于传统的TILLING技术，GBS数据的处理难度与挑战更高，这导致目标序列读段的比对与变异分析工作难以开展且结果解读难度较大。因此，构建高质量的完整麻疯树参考基因组序列，应当成为麻疯树育种相关研究的优先事项。