A mutation screening platform for rapeseed (Brassica napus L.) and the detection of sinapine biosynthesis mutants
A mutation screening platform for rapeseed (Brassica napus L.) and the detection of sinapine biosynthesis mutants
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DOI：
10.1007/s00122-011-1760-z
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发表时间：
2012-03-01
影响因子：
5.4
通讯作者：
Jung, Christian
中科院分区：
文献类型：
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作者：
Harloff, Hans-Joachim;Lemcke, Susanne;Jung, Christian
本研究以甘蓝型油菜（Brassica napus L.）使用EMS（甲基磺酸乙酯）作为诱变剂。这些群体分别来自春季型品系YN 01 -429和冬季型栽培品种Express 617，包括5，361和3，488株M-2植物。基于二维8x合并策略建立了高通量筛选方案。选择芥子气生物合成途径的基因用于确定突变频率和为油菜育种创造新的遗传变异。油菜籽粕是一种富含蛋白质的蛋白质源，其蛋白质含量约为40%。然而，它作为动物饲料或人类食物的用途受到抗营养化合物如芥子气的限制。应用靶向诱导基因组局部损伤（TILLING）策略鉴定芥子气生物合成途径主要基因的突变。我们构建了两个芥子气合成基因BnaX.SGT和BnaX. REF 1的几个TILLING扩增子的基因座特异性引物，覆盖了80-90%的编码序列。两个群体的筛选分别显示BnaX.SGT序列（135个错义突变和13个无义突变）和BnaX. REF 1序列（162个错义突变、3个无义突变和8个剪接位点突变）中的229和341个突变。这些突变体为低硫油菜的育种提供了新的资源。错义和无义突变的频率对应于靶密码子的频率。Express 617群体的突变频率范围为1/12至1/22 kb，YN 01 -429群体的突变频率范围为1/27至1/60 kb。我们的TILLING资源是公开可用的。由于高突变频率与89合并策略的组合，可以以具有成本效益的方式常规地鉴定突变体。然而，必须仔细设计引物以从多倍体油菜籽基因组扩增单个序列。
We developed two mutant populations of oilseed rape (Brassica napus L.) using EMS (ethylmethanesulfonate) as a mutagen. The populations were derived from the spring type line YN01-429 and the winter type cultivar Express 617 encompassing 5,361 and 3,488 M-2 plants, respectively. A high-throughput screening protocol was established based on a two-dimensional 8x pooling strategy. Genes of the sinapine biosynthesis pathway were chosen for determining the mutation frequencies and for creating novel genetic variation for rapeseed breeding. The extraction meal of oilseed rape is a rich protein source containing about 40% protein. Its use as an animal feed or human food, however, is limited by anti-nutritive compounds like sinapine. The targeting induced local lesions in genomes (TILLING) strategy was applied to identify mutations of major genes of the sinapine biosynthesis pathway. We constructed locus-specific primers for several TILLING amplicons of two sinapine synthesis genes, BnaX.SGT and BnaX.REF1, covering 80-90% of the coding sequences. Screening of both populations revealed 229 and 341 mutations within the BnaX.SGT sequences (135missense and 13 nonsense mutations) and the BnaX.REF1 sequences (162 missense, 3 nonsense, 8 splice site mutations), respectively. These mutants provide a new resource for breeding low-sinapine oilseed rape. The frequencies of missense and nonsense mutations corresponded to the frequencies of the target codons. Mutation frequencies ranged from 1/12 to 1/22 kb for the Express 617 population and from 1/27 to 1/60 kb for the YN01-429 population. Our TILLING resource is publicly available. Due to the high mutation frequencies in combination with an 89 pooling strategy, mutants can be routinely identified in a cost-efficient manner. However, primers have to be carefully designed to amplify single sequences from the polyploid rapeseed genome.