Identification of phytic acid mutants in oilseed rape (Brassica napus) by large scale screening of mutant populations through amplicon sequencing.
Identification of phytic acid mutants in oilseed rape (Brassica napus) by large scale screening of mutant populations through amplicon sequencing.
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DOI：
10.1111/nph.16281
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发表时间：
2020-03
期刊：
影响因子：
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通讯作者：
N. Sashidhar;H. Harloff;C. Jung
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文献类型：
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作者：
N. Sashidhar;H. Harloff;C. Jung
甘蓝型油菜（Brassica napus）是温带地区重要的油料作物，起源于B杂交。oleracea和B.拉帕。由于其多倍性，单基因的功能研究是繁琐的。植酸被认为是一种抗营养化合物，我们的目的是敲除潜在的合成和转运基因，以确定低植酸突变体。我们对6个基因家族（BnMIPS、BnMIK、Bn 2-PGK、BnIPK 1、BnIPK 2和BnMRP 5）的7680株植物的EMS群体实施了高通量NGS筛选方案，每个基因具有两个旁系同源物。共发现1487个突变，绝大多数（96%）通过桑格测序得到证实。此外，Bn.2-PGK 2的双突变体的表征显示植酸含量显著降低。我们建议使用三维池与扩增子叠加和下一代测序相结合，以快速和具有成本效益的方式识别多倍体油菜籽中的突变，以用于复杂的代谢途径。Bn 2-PGK 2突变体可能为菜籽蛋白的工业化生产提供重要的资源。
Brassica napus (oilseed rape) is an important oil crop in temperate regions, which originated from hybridization of B. oleracea and B. rapa. Due to its polyploidy, the functional study of single genes is cumbersome. Phytic acid is considered as an anti-nutritive compound and we aimed to knockout the underlying synthesis and transporter genes to identify low phytic acid mutants. We implemented a high throughput NGS screening protocol for an EMS population of 7680 plants in 6 gene families (BnMIPS, BnMIK, Bn2-PGK, BnIPK1, BnIPK2 and BnMRP5) with two paralogs for each gene. A total of 1487 mutations were revealed and the vast majority (96%) were confirmed by Sanger sequencing. Furthermore, the characterization of double mutants of Bn.2-PGK2 showed a significant reduction of phytic acid contents. We propose to use three-dimensional pooling combined with amplicon stacking and next generation sequencing to identify mutations in polyploid rapeseed in a fast and cost-effective manner for complex metabolic pathways. Furthermore, the identified mutants in Bn2-PGK2 might be a very valuable resource for industrial production of rapeseed protein for human consumption.