Departamento de Genética Molecular de Plantas, Centro Nacional de Biotecnología (CNB), Consejo Superior de Investigaciones Científicas (CSIC), Madrid-28049, Spain.
BMC Plant Biol. 2009 Dec 14;9:147. doi: 10.1186/1471-2229-9-147.
Arabidopsis thaliana is the main model species for plant molecular genetics studies and world-wide efforts are devoted to identify the function of all its genes. To this end, reverse genetics by TILLING (Targeting Induced Local Lesions IN Genomes) in a permanent collection of chemically induced mutants is providing a unique resource in Columbia genetic background. In this work, we aim to extend TILLING resources available in A. thaliana by developing a new population of ethyl methanesulphonate (EMS) induced mutants in the second commonest reference strain. In addition, we pursue to saturate the number of EMS induced mutations that can be tolerated by viable and fertile plants.
By mutagenizing with different EMS concentrations we have developed a permanent collection of 3712 M2/M3 independent mutant lines in the reference strain Landsberg erecta (Ler) of A. thaliana. This population has been named as the Arabidopsis TILLer collection. The frequency of mutations per line was maximized by using M1 plants with low but sufficient seed fertility. Application of TILLING to search for mutants in 14 genes identified 21 to 46 mutations per gene, which correspond to a total of 450 mutations. Missense mutations were found for all genes while truncations were selected for all except one. We estimated that, on average, these lines carry one mutation every 89 kb, Ler population providing a total of more than five million induced mutations. It is estimated that TILLer collection shows a two to three fold higher EMS mutation density per individual than previously reported A. thaliana population.
Analysis of TILLer collection demonstrates its usefulness for large scale TILLING reverse genetics in another reference genetic background of A. thaliana. Comparisons with TILLING populations in other organisms indicate that this new A. thaliana collection carries the highest chemically induced mutation density per individual known in diploid species.
拟南芥是植物分子遗传学研究的主要模式物种，全世界都在努力鉴定其所有基因的功能。为此，通过在化学诱导突变体的永久库中进行 TILLING（靶向诱导基因组局部突变）的反向遗传学，在哥伦比亚遗传背景下提供了一个独特的资源。在这项工作中，我们旨在通过在第二常见的参考菌株中开发新的乙基甲磺酸（EMS）诱导突变体群体来扩展拟南芥中可用的 TILLING 资源。此外，我们还致力于饱和可容忍有活力和可育植物的 EMS 诱导突变的数量。
通过用不同的 EMS 浓度诱变，我们在拟南芥的参考菌株 Landsberg erecta（Ler）中开发了一个包含 3712 个 M2/M3 独立突变体系的永久库。这个群体被命名为拟南芥 TILLer 集合。通过使用具有低但足够种子育性的 M1 植物，最大限度地提高了每条线的突变频率。应用 TILLING 搜索 14 个基因的突变体，每个基因发现了 21 到 46 个突变，总共发现了 450 个突变。所有基因都发现了错义突变，除了一个基因外，所有基因都选择了截断突变。我们估计，这些系平均每 89kb 携带一个突变，Ler 群体提供了超过五百万个诱导突变。据估计，TILLer 集合显示出比以前报道的拟南芥群体个体中 EMS 突变密度高两到三倍。
TILLer 集合的分析表明，它在另一个拟南芥参考遗传背景下进行大规模 TILLING 反向遗传学是有用的。与其他生物体中的 TILLING 群体的比较表明，这种新的拟南芥集合携带了二倍体物种中已知的个体中最高的化学诱导突变密度。
