Development and characterization of a new TILLING population of common bread wheat (Triticum aestivum L.).
Development and characterization of a new TILLING population of common bread wheat (Triticum aestivum L.).
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DOI：
10.1371/journal.pone.0041570
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Hu YG
中科院分区：
文献类型：
--
作者：
Chen L;Huang L;Min D;Phillips A;Wang S;Madgwick PJ;Parry MA;Hu YG
诱变是作物改良的重要工具。然而，由于基因冗余，小麦（Triticum aestivum L.）的六倍体基因组在基于表型筛选识别所需遗传变化方面存在问题。 TILLING（靶向基因组中诱导的局部损伤）是一种强大的反向遗传策略，可以检测诱变群体个体中诱导的点突变，可以解决将序列信息与基因的生物学功能联系起来的主要挑战，还可以识别作物育种的新变异。由于多倍体可耐受高突变密度，小麦特别适合耕作。然而，目前世界上可用的小麦TILLING群体仅有少数，远远不能满足不同生长环境下研究人员和育种者的需求。此外，当前的 TILLING 筛查方案需要昂贵的荧光检测系统，限制了其使用，特别是在发展中国家。我们开发了一种新的 TILLING 资源，其中包含常见小麦品种“晋麦 47”的 2610 个 M2 突变体。在田间的 M2 和 M3 品系中观察到许多形态和农艺性状发生改变的表型。为了简化程序并降低成本，我们使用未标记的引物和非变性聚丙烯酰胺凝胶或琼脂糖凝胶进行突变检测。使用带有 RAPD 和内含子剪接连接 (ISJ) 引物的 PCR 测试了这一新资源的价值，并且还在 300 和 512 个突变系中的三个选定候选基因中进行了 TILLING，结果显示 RAPD/ISJ 分析显示 1/34 kb 的高突变密度，TILLING 分析显示 1/47 kb 的高突变密度。总共在 3 个目标基因中鉴定出 31 个新等位基因，并通过测序得到确认。结果表明，该突变体群体为小麦研究界提供了有用的资源。我们希望这种反向遗传资源的使用将为小麦改良和功能基因组学提供新的等位基因多样性。
Mutagenesis is an important tool in crop improvement. However, the hexaploid genome of wheat (Triticum aestivum L.) presents problems in identifying desirable genetic changes based on phenotypic screening due to gene redundancy. TILLING (Targeting Induced Local Lesions IN Genomes), a powerful reverse genetic strategy that allows the detection of induced point mutations in individuals of the mutagenized populations, can address the major challenge of linking sequence information to the biological function of genes and can also identify novel variation for crop breeding. Wheat is especially well-suited for TILLING due to the high mutation densities tolerated by polyploids. However, only a few wheat TILLING populations are currently available in the world, which is far from satisfying the requirement of researchers and breeders in different growing environments. In addition, current TILLING screening protocols require costly fluorescence detection systems, limiting their use, especially in developing countries. We developed a new TILLING resource comprising 2610 M2 mutants in a common wheat cultivar ‘Jinmai 47’. Numerous phenotypes with altered morphological and agronomic traits were observed from the M2 and M3 lines in the field. To simplify the procedure and decrease costs, we use unlabeled primers and either non-denaturing polyacrylamide gels or agarose gels for mutation detection. The value of this new resource was tested using PCR with RAPD and Intron-spliced junction (ISJ) primers, and also TILLING in three selected candidate genes, in 300 and 512 mutant lines, revealing high mutation densities of 1/34 kb by RAPD/ISJ analysis and 1/47 kb by TILLING. In total, 31 novel alleles were identified in the 3 targeted genes and confirmed by sequencing. The results indicate that this mutant population represents a useful resource for the wheat research community. We hope that the use of this reverse genetics resource will provide novel allelic diversity for wheat improvement and functional genomics.