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Identification of phytic acid mutants in oilseed rape (Brassica napus) by large-scale screening of mutant populations through amplicon sequencing.
New Phytologist ( IF 9.4 ) Pub Date : 2019-10-25 , DOI: 10.1111/nph.16281
Niharika Sashidhar 1 , Hans-Joachim Harloff 1 , Christian Jung 1
Affiliation  

Brassica napus (oilseed rape) is an important oil crop in temperate regions, which originated from hybridization of Brassica oleracea and Brassica rapa. Owing to its polyploidy, the functional study of single genes is cumbersome. Phytic acid is considered as an antinutritive compound, and we aimed to knock out the underlying synthesis and transporter genes to identify low phytic acid mutants. We implemented a high-throughput next-generation sequencing screening protocol for an ethylmethane sulfonate population of 7680 plants in six gene families (BnMIPS, BnMIK, Bn2-PGK, BnIPK1, BnIPK2, and BnMRP5) with two paralogues 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 oilseed rape in a fast and cost-effective manner for complex metabolic pathways. Furthermore, the mutants identified in Bn2-PGK2 might be a very valuable resource for industrial production of oilseed rape protein for human consumption.

中文翻译:

通过扩增子测序大规模筛选突变种群,鉴定油菜(甘蓝型油菜)中的植酸突变体。

甘蓝型油菜(油菜)是温带地区重要的油料作物,起源于甘蓝型油菜和甘蓝型油菜的杂交。由于其多倍性,单个基因的功能研究繁琐。植酸被认为是一种抗营养化合物,我们的目标是敲除潜在的合成和转运蛋白基因,以鉴定低植酸突变体。我们对六个基因家族(BnMIPS,BnMIK,Bn2-PGK,BnIPK1,BnIPK2和BnMRP5)的7680种植物的甲烷磺酸乙酯种群实施了高通量下一代测序筛选方案,每个基因有两个旁系同源物。总共发现了1487个突变,并且Sanger测序证实了绝大多数(96%)。此外,Bn的双重突变体的表征。2-PGK2显示植酸含量显着降低。我们建议使用三维池与扩增子叠加和下一代测序相结合,以快速且经济高效的方式识别多倍体油菜籽中的突变,以实现复杂的代谢途径。此外,在Bn2-PGK2中鉴定的突变体可能是工业生产供人食用的油菜籽蛋白的非常有价值的资源。
更新日期:2019-12-13
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