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 (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.
The availability of a mutant line in which a single gene has been disrupted gives biologists a powerful tool in understanding the function of that gene. Thus, sequence-indexed collections of single insertions are critical resources for elucidating gene function in organisms with sequenced genomes and are deemed essential by the community to fully exploit the maize genome sequence. It has recently become feasible to combine high-throughput sequencing with multi-dimensional pooling strategies to sequence and index hundreds of new insertions at a time. This work is providing a reverse genetics resource to the scientific community based on the well-studied transposons Ac and Ds. Specifically, the work has succeeded in placing 340 germinal native transposon insertion sites in one of the ten maize pseudomolecules and indexing them to a particular stock. Project members at Rutgers and Montclair State U., including undergraduate students at MSU assisted with the annotation and mapping of putative Ac- or Ds-adjacent sequences and with the design of primers for validation. At the same time, the work produced a set of transgenic Ds* launching platforms carrying an easily scored marker that will allow simple visual selection of element transposition from any region of the genome and, thus, enable researchers to generate their own regional gene knock-out collections. A total of 160 lines carrying a transpositionally active, marked Ds* element were generated and the location of 82 active platforms was mapped. The distribution of platforms is fairly random across the genome and all 20 maize chromosome arms are represented among the mapped platforms. In addition to providing stocks with Ds launching platforms, the work produced a large set of lines carrying a single trDs* element in the genome: 50,000 of these lines were identified. By high throughput sequencing of 3-D pools of Ds transposants, 2200 trDs* target sites were mapped to the reference genome using the project-generated, publicly available pipeline InsertionMapper: all are listed in the project's website and more than 1200 of them are already available from the Maize Genetics Stock Center (Co-op) in Illinois. The rest will be available as soon as the necessary numbers of seeds is produced for the Co-op to list them. In addition to developing a software package to map the insertions to the genome, the bioinformaticians in the project developed a project website (acdsinsertions.org) with multiple search capabilities, including BLAST. The information found in that website has been put together by lab workers in the. Dooner lab at and undergraduate students in Dr. Du’s genomics classes at Montclair State University. This project integrated high throughput sequencing at Rutgers with bioinformatic sequence analysis at Montclair State University, a predominantly undergraduate institution in NJ. MSU students annotated all Ds*-adjacent sequences generated by the high throughput DNA sequencer. The project provided informatics and molecular biology students at that institution with the opportunity to participate directly in maize research and fulfill their independent research requirement for graduation. Students at both Rutgers and MSU worked in the project as summer interns in the molecular biology lab and maize genetics nursery of the PI. The PI and coPI are members of underrepresented groups and have a record of fruitful continuous collaborations dating back 10 years. They have collaborated in NSF PGRP-funded projects which have led to the development of three bioinformatics tools and the publication of seven joint papers. About a third of the Genomics students participating in the project at MSU were members of underrepresented minorities. MSU added the research opportunity from this project to its outreach campaign to attract and retain minority high school graduates. This project is relevant to U.S. agriculture in that it deals with maize, the most important American crop today. It addresses a critical need in that it delivers a sequence-indexed reverse genetics resource, considered essential for researchers to fully exploit the maize genome sequence. All data has been made available through a web-searchable database of insertion site sequences (www.acdsinsertions.org/) that are cross-referenced to seed stocks available from the Maize Genetics Cooperation Stock Center (http://maizecoop.cropsci.uiuc.edu). All relevant information from this project is accessible through www.acdsinsertions.org/ and through MaizeGDB (www.maizegdb.org).
