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

全基因组插入突变是一种为基因组中几乎所有基因创造功能丧失突变的工具。在玉米中，突变转座子是一种用于产生插入突变的系统。本研究的总体目标是利用高效、高通量的下一代测序方法Mutant-seq （Mu-seq）对转座子插入进行测序，建立一个突变体诱导的玉米突变体序列索引集合。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与基因组学数据库（MaizeGDB）中，并将其用于反向和正向遗传学实验。在初步工作中，我们产生了分离的突变标记f2群体。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建立突变体收集的遗传背景。我们首先选择了代表玉米第一个参考基因组的凹痕库自交系B73。最近几个月，我在实验室建立了Mu-seq协议，并将其应用于576个f2家族的mu标记B73群体。在这个最初的Mu-seq文库中，20081个生发转座子插入影响11457个不同的基因模型。最近，我们将燧石池的法国自交系F7加入了该项目。F7因其在温带气候条件下的稳定生长特性和可获得的基因组序列而成为中欧玉米研究的重要遗传资源。为了建立自交系F7的反向遗传数据库，我们将在建议的项目中对2304个突变标记的f2家族进行Mu-seq实验，并随后将该欧洲突变资源整合到MaizeGDB中。本提案的第二个目标是利用转座子标记的f2群体进行前向遗传筛选，以鉴定根特异性突变，并随后在第二个资助期克隆和表征相关基因。BSR-seq和序列索引Mu-seq文库的结合将促进对特定突变表型的基因鉴定，这些文库为每个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.