1) Overview:Senior Personnel: PI, Hugo K. Dooner, Rutgers U.; coPI, Charles Du, Montclair State U.
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. In this work, 14,400 new Ds-adjacent sites will be sequence-indexed in 3-D pools by Illumina technology and all lines will be deposited in the Maize Genetics Stock Center (Co-op) for free distribution. The objectives are: (1) To characterize the transposition frequency of 78 single Ds-GFP (or Ds*) launching platforms generated by Agrobacterium transformation and map them to the genome. Highly active platforms will be identified and deposited in the Maize Genetics Co-op to complement those already mapped. These platforms will allow visual selection of transpositions from most regions of the genome and, thus, enable researchers to create regional gene knock-out collections. (2) To generate collections of Ds* transpositions each from Ds* launching platforms located on all 10 chromosomes which will serve as the foundation for a Ds*-based maize single-gene knockout resource. (3) To sequence-index these collections by high throughput Illumina sequencing of 3-D DNA pools. To this end, a specific software package was developed: InsertionMapper extracts Ds* transposon junctions (dsgs) from the large amount of next-generation sequencing (NGS) data and maps them to the maize genome. (4) To make all data available in a web-searchable database of insertion site sequences (acdsinsertions.org) cross-referenced to stocks available from the Maize Genetics Co-op.
2) Intellectual merit of the proposed activity
The availability of a mutant line in which a single known gene has been disrupted gives biologists a powerful tool in understanding the action 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. This work will complete the production of a reverse genetics resource based on the transposon Ds that will enable the community to generate and build up a single-gene knockout resource for maize.
3) Broader impacts resulting from the proposed activity
This project will integrate NGS at Rutgers with bioinformatic sequence analysis at Montclair State University (MSU), a predominantly undergraduate institution in NJ. Students in a Genomics class at MSU will annotate all Ds*-adjacent sequences generated by the MySeq Sequencer. The project will provide 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 will be able to work 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 prior collaborations. They have collaborated in previous NSF PGRP-funded projects which led to the development of a bioinformatics tool and the publication of three joint papers. About a third of the Genomics students participating in the project at MSU are members of underrepresented minorities. MSU has 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 will deliver a sequence-indexed reverse genetics resource, considered essential for researchers to fully exploit the maize genome sequence. It will also produce a web-searchable maize transposant database matching sequences of knocked out genes with stocks freely available from the Maize Genetics Co-op . All sequences will be deposited in GenBank and the relevant information from this project will be accessible from MaizeGDB.