Facile Production and Efficient Indexing of Transposon-tagged Lines Using Next-generation Sequencing Technology for Maize

PI: Hugo K. Dooner (Rutgers University)

CoPIs: Chunguang Du (Montclair State University) and Randall A. Kerstetter (Rutgers University)

Transposable element (TE) insertions cause mutations that help researchers to elucidate gene function. Collections of insertions that have been sequence-indexed (i.e., in which the host DNA adjacent to the insertion is known) are valuable resources in organisms with a sequenced genome and have been identified by the maize community as essential to fully exploit the maize genome sequence just generated. Recently, the feasibility of combining high-throughput DNA sequencing with efficient multi-dimensional pooling strategies to rapidly and simultaneously sequence and index hundreds of new insertions has been demonstrated. This project will develop and use a general method for generating, sequencing, and indexing Ac/Ds transposable element insertions in maize that is rapid, accurate, and cost-effective by taking advantage of next-generation sequencing technology. Specifically, the project will: (1) sequence-index a collection of existing lines that contain a unique transposed Ac element; (2) complete a set of 120 roughly equidistant transgenic Ds elements that serve as launching platforms and carry easily scored markers that will allow simple visual selection of element transposition from any region of the genome and, thus, enable researchers to generate regional gene knock-out collections, (3) sequence-index 10,000 Ds element insertion sites from model platforms using a novel method that should be applicable to any collection of insertions produced in a common genetic background; and (4) develop a web-searchable database of insertion site sequences cross-referenced to lines that will be freely available from the Maize Genetics Stock Center (http://maizecoop.cropsci.uiuc.edu). All relevant information from this project will be accessible from MaizeGDB (http://www.maizegdb.org).

This project addresses a critical need in that it will deliver a sequence-indexed reverse genetics resource, considered essential for researchers to fully exploit the maize genome sequence. It will integrate next-generation sequencing technology at Rutgers with bioinformatic sequence analysis at Montclair State University, a predominantly undergraduate institution in NJ. Students at MSU will assist with the assembly, annotation, and mapping of short reads as putative Ac-adjacent sequences and with the design of primers for validation. The project will also provide research training opportunities for MSU informatics and molecular biology students, many of whom are from underrepresented groups. Students at both Rutgers and MSU will also participate in the project as summer interns in the molecular biology lab and maize genetics nursery.