Development of a sequence-indexed collection of transposon tagged maize mutants as a novel public resource and its application in root developmental biology

全基因组插入诱变是一种为基因组中几乎所有基因创建功能丧失突变的工具。在玉米中，mutator转座子是一种用于产生插入突变的系统，本提案的总体目标是通过使用高效的高通量下一代测序方法Mu-seq（Mu-seq）对转座子插入进行测序来建立mutator诱导的玉米突变体的序列索引集合。然后，我们将把这个新的序列索引转座子插入位点库整合到玉米遗传学和基因组学数据库（MaizeGDB）中，并将其用于反向和正向遗传学实验。这一进程仍在继续，以进一步扩大我们的F2种群。我们选择了两个自交系作为遗传背景，建立我们的突变体收集。我们首先选择了代表玉米第一个参考基因组的自交系B73。最近几个月，我在我们的实验室建立了Mu-seq协议，并将其应用于Mu标记的B73群体的576个F2家族。在这个最初的Mu-seq文库中，20，081个germ转座子插入影响了11，457个不同的基因模型。最近，我们将弗林特池的法国近交系F7加入到该项目中。F7是中欧玉米研究的重要遗传资源，因为其在温带气候条件下的稳定生长特性和其基因组序列的可用性。为了开发近交系F7的反向遗传学数据库，我们将对2，本项目的第二个目标是利用转座子标记的F2代群体进行正向遗传筛选，以鉴定根-茎-叶的突变体。在第二个资助期内，我们会研究特定的突变，并随后克隆和鉴定相关的基因。BSR-seq和序列索引Mu-seq文库的组合将有助于鉴定特定突变表型的基因，Mu-seq文库为每个BSR-seq作图间隔提供有限数量的候选突变。

Genome-wide insertional mutagenesis is a tool to create loss-of-function mutations for virtually all genes in a genome. In maize, mutator transposons are one system used to generate insertional mutations.The overall objective of this proposal is to establish a sequence indexed collection of mutator induced maize mutants by sequencing transposon insertions with an efficient high-throughput next generation sequencing method designated Mutant-seq (Mu-seq). We will then integrate this novel repository of sequence-indexed transposon insertion sites into the Maize Genetics and Genomics Database (MaizeGDB) and use it for reverse and forward genetics experiments.In preliminary work, we generated segregating mutator-tagged F2-populations. This process is still ongoing to further expand our F2-populations. We selected two inbred lines as genetic background for the establishment of our mutant collection. We first selected the inbred line B73 of the dent pool, which represents the first reference genome of maize. In recent months, I established the Mu-seq protocol in our laboratory and applied it to 576 F2-families of the Mu-tagged B73 population. In this initial Mu-seq library 20,081 germinal transposon insertions affect 11,457 different gene models.Recently, we added the French inbred line F7 of the flint pool to the project. F7 is an important genetic resource for central European maize research due to its stable growth properties under temperate climatic conditions and the availability of its genome sequence. To develop a reverse genetics database of the inbred line F7, we will perform Mu-seq experiments on 2,304 mutator-tagged F2-families in the suggested project and subsequently integrate this European mutant resource into MaizeGDB.The second goal of this proposal is to use the transposon tagged F2-population for forward genetic screens to identify root-specific mutations and to subsequently clone and characterize the related genes in the second funding period. The identification of genes that underlie specific mutant phenotypes will be facilitated by the combination of BSR-seq and the availability of sequence indexed Mu-seq libraries, which provide a limited number of candidate mutations for each BSR-seq mapping interval.