Title
DBI-0822383. TRPGR:Efficient identification of induced mutations in crop species by ultra-high throughput DNA sequencing.
Objectives
- Improve the efficiency of mutation discovery (UCD)
- Develop protocols that combine oligo capture of barcoded superpooled samples with Solexa sequencing for efficient mutation detection (FHCRC)
- Devise the optimal target number and pooling for mutation discovery, and the informatic pipeline for analysis (UCD)
- Define and optimize approaches for TILLING polyploid genomes (UCD)
- Target an outreach program to underrepresented students and to crop plant scientists
Abstract
P.I.: Luca Comai (University of California at Davis); co-P.I.: Jorge Dubcovsky (University of California at Davis); co-P.I. Steve Henikoff (Fred Hutchinson Cancer Research Center, Seattle); key personnel: Robert Tran, Dawei Lin (University of California at Davis). The growing information on gene sequence and function stimulates interest in pathways expected to have large effects on important agronomic and quality traits. Nevertheless, while more and more genes are valuable inactivation targets, approaches for crop gene modification that can flexibly target 50 to 3000 genes per crop species are limited. This project will apply ultra-high throughput sequencing to TILLING (Targeting Induced Local Lesions IN Genomes), a general method for the discovery of induced mutations in genes of interest. Working with rice and wheat TILLING lines, it will employ PCR-mediated amplification from pooled genomic templates to target dozens to hundreds of genes. The research will test and implement several technical and computational methods to resolve real mutations from errors in highly complex templates. To target hundreds to thousands of genes, the project will use sequence barcoding to create DNA pools of short adapter-ligated random fragments from hundreds or thousands of existing TILLING lines of Arabidopsis, and later rice and wheat. Aliquots from an amplified pool will be subjected to a capture procedure using custom programmable arrays or long oligos attached to either microarrays or beads, and the eluted fragments subjected to Solexa sequencing. The project will build an economical and effective pipeline that will be sustainable as a user-supported service, superseding more laborious and expensive nucleotide mismatch-detection technology. Rice and wheat span the range of critical characteristics and can therefore serve as models for all crops. They differ in ploidy, diploid versus allopolyploid, sequence information, high versus low, genome size, small versus large, and mutation density, moderate versus very high. While developing a sequencing approach to TILLING, the project will use already existing and tested populations to target a set of genes in each crop that will be useful to the community of scientist and breeders. Thus, this research will facilitate breeding of improved crop varieties by providing facile isolation and analysis of variants. This research will provide methods for efficient TILLING of crop plants. By doing so, it will stimulate both basic discovery as well as targeted trait improvement. For example, in wheat independent inactivation of homologous gene sets followed by breeding can address a host of important agronomic and quality traits. Because discovery of mutations by direct sequencing will allow researchers to choose ab initio which mutant to characterize, in silico collections of induced mutations will provide powerful resources for breeding communities. The developed methods will not only be useful for TILLING but also to discover natural variation, or to identify allelic changes underlying quantitative trait loci. The connected outreach program combines the undergraduate and professional training. It is based on three approaches: an internship program that will pair students and teachers from underrepresented populations with laboratory-to-field experimental genomic pipeline, a database and methods web site, and a yearly workshop for training crop scientists. Data and methods will be accessible to the public through the project web site (http://tilling.ucdavis.edu) and established genomics databases (http://www.gramene.org/; http://www.arabidopsis.org/).
Comai Lab 