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

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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.