Location: Crops Pathology and Genetics Research2013 Annual Report
1a. Objectives (from AD-416):
Objective 1: Evaluate and apply chemical and physical agents to generate populations of rice mutants for forward and reverse genetic analyses. Sub-objective 1A: Evaluate chemical and gamma-irradiation protocols for seed mutagenesis. Sub-objective 1B: Identify alternative seed mutagenesis approaches to increasing mutation density. Sub-objective 1C: Determine the GECN in rice by reduced representation sequencing. Objective 2: Develop rice induced mutant resources (genotypic and phenotypic data, seeds) as a public resource for functional genomics research and as novel germplasm for rice breeding. Sub-objective 2A: Phenotype M2 individuals and their corresponding self progeny (M3 generation) using a standard panel of traits/descriptors and established rice mutant ontology. Sub-objective 2B: Sequence the exome and catalog mutations of M2 individuals. Sub-objective 2C: Produce fixed mutant lines for quantitative trait phenotyping (e.g., field sites, multiple locations, replications, and users, destructive phenotyping/selective screening). Objective 3: Identify, characterize, and make available mutant phenotypes/mutations that affect rice grain quality and agronomic traits as tools for functional studies and/or varietal improvement. Sub-objective 3A: Identify grain quality and agronomic mutants using phenotypic evaluation. Sub-objective 3B: Identify useful mutant phenotypes by screening populations for mutations in genes involved in biosynthesis and/or accumulation of cooking, eating, and nutritional quality components (i.e. reverse genetics). Sub-objective 3C: Simultaneous mapping of mutations and germplasm enhancement.
1b. Approach (from AD-416):
Goal 1A: The mutation density of ~4 mutants per Mb using seed mutagenesis will be improved through modification of current protocols. Goal 1B: Two approaches will be taken: 1) mutagenesis of tetraploid rice; and 2) seed mutagenesis at mutagen doses that elicit high or complete pollen sterility followed by crossing with wild-type pollen. Goal 1C: The GECN in rice will vary depending on the mutagen, the dose, and the inflorescence (primary vs. secondary tillers). Goal 2A: Phenotypic data from a systematic evaluation of M2 and M3 mutants will add value to the mutant resource and provide users with additional criteria for selecting mutant lines for further characterization and/or incorporation into variety development programs. Goal 2B: Generate in silico mutation profiles of 1,500 to 2,000 independent M2 rice mutants by exome sequencing existing and newly created mutant populations. Goal 2C: Many valuable mutations/mutant phenotypes may be missed in early generations (i.e. M2 or M3). Fixed lines (=M6 generation) will provide researchers a long-term resource for evaluation of traits under field conditions, that require multiple replications (e.g. locations and years), or that are destructive in nature. Goal 3A: Mutations that improve grain quality (milling, cooking, eating, and nutritional) or agronomic performance of rice or that result in novel phenotypes of commercial value may be identified through phenotypic evaluation (i.e. forward genetics). Goal 3B: Through a combination of TILLING and targeted exome sequencing, mutations in genes of interest will be identified and the corresponding mutants will be characterized. Goal 3C: Develop a functional genomics/variety development pipeline by applying next-gen sequencing-based methods in combination with accelerated mapping population development to identify mutant phenotypes, map and clone the underlying genes, and generate enhanced germplasm for breeding programs.
3. Progress Report:
This new project was implemented on 04/17/2013 and replaces project 5306-21000-017-00D. The overall goal is to employ classical mutagenesis to induce genetic variation in rice that will be exploited for functional genomics and variety improvement. In FY2013, research activities addressing all major project objectives were carried out. For objectives 1 and 3, rice seeds of two varieties (Kitaake and 29Lu1) representing the major subgroups of rice, japonica (grown primarily in the U.S. and other temperate regions) and indica (more widely grown), were treated with various chemical and physical agents known to induce changes in DNA. The effectiveness of the treatments in generating changes for both applications will be evaluated throughout this project. This involves assessment of physical and chemical traits (i.e. phenotyping) and analysis of DNA of individuals to identify specific changes at the molecular level (i.e. induced genetic variation) that are responsible for altered phenotypes. To address phenotyping, existing populations of individuals derived from mutagenesis (i.e. mutant populations) were planted and various traits of agricultural importance are being evaluated. This initial work is intended to identify potential problems (and solutions) when the populations developed from the mutagenesis of Kitaake and 29Lu1 are phenotyped in FY2014. This phenotyping work addresses objectives 2 and 3. To assess the effect of mutagenesis on DNA, an approach called exome sequencing is being employed. The exome is the part of the genome that encodes proteins (i.e. expressed genome) and consists of DNA sequences called exons. The complete genome sequence of rice is available and the exons of the about 30,000 genes can be identified computationally to design DNA probes for isolating or capturing exons from the DNA of different individuals. These exons are then sequenced and compared with DNA from untreated (wild-type) individuals to identify changes (i.e. mutations). A cooperative study to evaluate the utility of exome sequencing to characterize mutations in rice mutants was conducted and the results support this approach. The exome sequencing work addresses objectives 1 and 3. Specific progress in Year 1 includes: 1) Phase 1 treatments of Kitaake and 29Lu1 were perfromed and seeds were planted; 2) Phase 2 treatments of Kitaake, 29Lu1, and tetraploid germplasm were performed and seeds were planted; 3) Phenotyping of a small set of M2 from established populations of mutants is underway. Protocols for trait evaluation are being tested; 4) Exome sequencing of existing Nipponbare (n=72) and M-204 (n=13) mutants has been performed with UC Davis cooperators. A manuscript has been submitted for publication; 5) M3 seeds from 3-4,000 M2 lines of both Kitaake (sodium azide) and M-204 (sodium azide) have been planted for generation advance to M4. A smaller set of Nipponbare M3 seeds (about 110-120 lines) with visible mutant phenotypes has also been planted; and, 6) UC Davis cooperators have designed new DNA probes for hybridization to rice exome sequences based the initial study. These new reagents will be used for exome sequencing of mutants in FY2014.
1. Global detection of induced mutations in rice. Efficient methods to characterize populations of rice mutants are needed to increase their utility for basic and applied research. A method called exome sequencing was tested on a small set of mutants derived from chemical treatment. Using rice genome information and computational tools, DNA sequences were designed and synthesized to capture coding regions of the rice genome (i.e. the exons) from multiple mutants. Following sequencing of the captured exons, computational analysis was performed to detect mutations likely to affect gene/protein function. Exome sequencing of a large population of rice mutants with linked seed resources will facilitate creation of a mutation database that will allow researchers and breeders to efficiently identify mutants of interest and acquire seeds for downstream applications.