Location: Crops Pathology and Genetics Research2013 Annual Report
1a. Objectives (from AD-416):
Research will be conducted on the development and application of methods and protocols for genotyping of rice using next-generation DNA sequencing and on the development and characterization of genetic mapping and mutant populations of rice. Specific objectives include: 1) next-generation genotyping of rice germplasm; 2) sequencing of expressed rice genes from California varieties; 3) development and screening of rice populations for genetic analysis of agriculturally important traits.
1b. Approach (from AD-416):
Single nucleotide polymorphisms (SNPs) will be identified by sequencing reduce representation libraries of selected rice germplasm in conjunction with computational genomics. SNP markers identified in this manner will be used to map traits of interest where possible. Exome sequencing will be employed to sequence protein coding regions of the genomes with emphasis on proteins implicated in yield and tolerance to abiotic stress. Development of genetic mapping and mutant populations will consist of generation advance through single-seed descent. Screening populations for agronomic traits of interest will be performed in consultation and conjunction with Rice Experiment Station breeders.
3. Progress Report:
The goal of this project is to develop tools and resources for improving rice varieties for California, which contributes directly to objective 2 and 3 of the in-house project. The emphasis is on developing DNA markers to predict the presence of traits such as cold tolerance, disease resistance, and grain quality and to develop rice populations (mapping and mutant). The markers will accelerate breeding of improved varieties. The mapping and mutant populations will facilitate gene discovery and characterization and may provide useful germplasm for future variety development by public breeding programs. Progress has been made on all three objectives: 1) Next-generation genotyping of rice germplasm: We have employed next-generation sequencing to identify and genotype single-nucleotide polymorphism (SNP) markers. These markers allow the highest level of resolution in distinguishing the most closely-related varieties such as those found in the California breeding programs. DNA libraries from about 45 important California varieties have been sequenced, SNPs have been identified, a manuscript describing the results of this study has been published; 2) Sequencing of expressed rice genes from California varieties: Using an approach called exome sequencing, we have characterized a number of California varieties. Sequences representing the expressed regions of the genome (i.e. exome) of rice were identified by our cooperators and used to generate DNA probes for capturing these regions via solution-based hybridization with rice genomic DNA. Target (i.e. captured) sequences were subjected to short read DNA sequencing using an Illumina sequencing platform. These sequences are under analysis. A manuscript describing the rice exome sequencing approach used is in preparation; 3) Development and screening of rice populations for genetic analysis of agriculturally important traits: A recombinant inbred line (RIL) mapping population derived from a cross between California varieties M-203 and M-206 is being advanced to the F7 generation and phenotypic analysis of the RILs for milling quality, seedling vigor, reproductive cold tolerance, and other important traits is underway. Additional genetic mapping populations under development include recombinant inbred lines of the following crosses: M-204/S-301 (F5:6 generation), S-301/M-204 (F5:6), and Calmochi-101/94Y561 (F2:3). Induced mutants (M2 generation) derived from the chemical mutagenesis (3 mM sodium azide) of the California variety M-204 were planted for creation of a reverse genetics resource and will be available for screening via Targeting of Induced Local Lesions in Genomes (TILLING) by sequencing. These M2 plants will also be subjected to heat stress during grain filling in order to identify mutants that may be tolerant to heat-induced grain chalkiness. M3 seeds harvested from sibling M2’s of this M-204 mutant population have been planted for generation advance (i.e. development of fixed mutant lines) and the M4 seeds will be harvested at the end of 2013 (or early 2014). M2 and M3 seeds of the M-204 mutants are currently being screened for enhanced tolerance to abiotic stresses and for altered grain quality traits. Due to the low mutation densities observed from exome sequencing of the M-204 mutant populations derived from gamma-irradiation (350 and 400 Grays) and 1 mM sodium azide mutagenesis, these populations are not being used currently.