Development and implementation of advanced methods for the identification of barley flowering time genes using next generation sequencing

开花时间的变化一直是使作物适应广泛环境的育种计划的主要目标。因此，破译开花时间变异的遗传基础对于新品种的知识育种具有重要意义。温带作物如大麦和小麦的特点是基因组大，主要由重复序列组成。尽管这些复杂的基因组对所有的基因尝试都是一个极端的挑战，但新技术允许以一种快速和经济有效的方式访问这些大型基因组的基因空间。尽管通过下一代测序同时进行遗传作图和突变鉴定的第一份报告进入了科学文献，但高通量鉴定因果突变的持续瓶颈仍然是适当的方法和遗传物质。与传统的基于地图的克隆工作一样，这种高速制图方法需要分离种群作为主要材料。在此，我们提出建立两种新的大麦突变体鉴定策略，这有望极大地加快定位群体的产生。首先，我们建议通过杂合突变体的自交受精来产生等基因定位群体，就像最近提出的那样。由于这些突变株可以在原始筛选的M2科中鉴定，因此建立分离群体确实需要一个单代。其次，等位基因突变的测序有希望在没有任何基于连锁分析的情况下揭示致病基因，因为通常只有一个基因或极少数基因在所有等位基因突变中表现出严重的变化。使用9个不同的等位基因序列，每组中有2到6个等位基因，将揭示这种方法的力量。一旦建立起来，这两种方法都将允许利用过去几十年产生的大麦突变种群的广泛遗传资源。

Variation in flowering time has always been a major target in breeding programs to adapt crops to a wide range of environments. Deciphering the genetic basis of flowering time variation is thus important for knowledge based breeding of new varieties. Temperate crops like barley and wheat are characterized by large genomes mostly comprising of repeats. Even though these complex genomes are an extreme challenge to all genetic attempts, new technologies allow accessing the gene spaces of these large genomes in a fast and cost-efficient way. Albeit the first reports on simultaneous genetic mapping and mutation identification by next generation sequencing found their way into scientific literature, the continuing bottleneck for high throughput identification of causal mutations remains the appropriate methods and genetic material. Like conventional map-based cloning efforts, such high-speed mapping methods require segregating populations as their primary material. Here we propose to establish two new strategies for mutant identification in barley, which carry the promise to tremendously speed up the generation of mapping populations. First, we propose generation of isogenic mapping populations through self-fertilization of heterozygous mutants as recently proposed for rice. As such mutant plants can be identified within the M2 families of the original screens, it does require one a single generation to establish the segregating population. Second, sequencing of allelic mutants carries the promise to reveal causative genes without any linkage-based analysis, as it is usually only one gene or a very small number of genes that feature severe changes within all allelic mutants. Using nine different allelic series with two to six alleles in each group will unravel the power of this approach. Once established, both of these methods will allow for exploiting the extensive genetic resources of barley mutant populations generated over the last decades.