Location: Crops Pathology and Genetics Research2013 Annual Report
1a. Objectives (from AD-416):
The long-term objective of this project is to develop improved rice (Oryza sativa L.) germplasm for use in breeding elite varieties adapted to temperate environments by identifying, characterizing, and manipulating genes that affect crop productivity. In temperate regions, seedling cold tolerance in rice is important for successful stand establishment and plant development, both of which directly impact yield. Over the next five years, two major objectives will be addressed: 1) the molecular genetic basis of rice seedling cold tolerance conferred by the major quantitative trait loci qCTS12 and qCTS4 will be determined and 2) the information obtained from the analysis of qCTS12 and qCTS4 will be used to evaluate currently available germplasm and to develop new germplasm with enhanced cold tolerance. qCTS12 and qCTS4 have been fine mapped to regions containing a small number of genes.
1b. Approach (from AD-416):
During this project, the genes that confer cold tolerance will be identified through transformation and candidate gene analysis experiments. The genes and the proteins they encode will be characterized at the molecular level and their effect under field conditions will be determined. The utility of this of this information for evaluating other rice germplasm and for developing efficient approaches to identifying novel sources of cold tolerance will be examined. New germplasm for breeding will be developed by transferring the qCTS12 and qCTS4 genes through conventional and molecular breeding approaches. In addition to providing tools and resources for germplasm improvement, genetic dissection of qCTS12 and qCTS4 will contribute to our fundamental understanding of cold tolerance in rice. Formerly 5306-21000-016-00D (3/08).
3. Progress Report:
This is the final report for this project which was terminated in April of 2013, and has been replaced by 5306-21000-021-00D, "Generation and Characterization of Novel Genetic Variation in Rice for the Enhancement of Grain Quality and Agronomic Performance". Transgenic rice plants containing OsGSTZ2, the gene believed to be responsible for the seedling cold tolerance previously identified on chromosome 12 (i.e. qCTS12 locus), were analyzed. Although all the plants tested contained the expected gene constructs, none exhibited enhanced cold tolerance. Analysis revealed that the gene constructs were not being expressed in the plants and conclusions as to the role of OsGSTZ2 could not be made. No resources were available to determine why these plants were not expressing the gene. As an alternative approach, a population of mutants was screened to search for mutations in OsGSTZ2 as well as the adjacent and closely-related gene OsGSTZ1. Previous research suggests that OsGSTZ1 is not responsible for the qCTS12 seedling cold tolerance. If mutations that affect (e.g. knock out) the function of these genes can be found then the plants harboring those mutated genes can be tested for their seedling cold tolerance. No mutations in OsGSTZ2 were identified in a screen of 2,000 mutants; however, plants with possible alterations in OsGSTZ1 are being grown for seeds that will be tested to confirm the presence of mutations. If confirmed, seedlings will be tested for changes in cold tolerance. Our expectation is that mutations affecting OsGSTZ1 will not affect cold tolerance. In addition to the candidate qCTS12 genes, a search for mutations in one candidate for seedling cold tolerance identified on chromosome 4 (i.e. qCTS4 locus) was conducted. Several possible mutants with alterations in this candidate gene were identified and are being grown for seed and testing. In FY2012, collaborators in Korea (Chungnam National University) obtained T-DNA insertion mutants in the same qCTS4 candidate and they are currently analyzing these plants. In FY2013, genetic analysis of the ability of seedlings to recover after exposure to constant cold stress was continued. As noted in FY2012, two closely-related lines (6885-2 and 4853-9) derived from crossing a cold tolerant variety (M-202) and a cold-sensitive variety (IR50) exhibited differences with 6885-2 showing more rapid and vigorous recovery although both have qCTS12 and qCTS4. Using a reduced representation sequencing approach called Restriction Enzyme Site Comparative Analysis (RESCAN), these two lines were analyzed and differences in their genomes were detected including one major M-202 introgression on chromosome 6 and a few smaller regions present in 6885-2 but not in 4853-9. These differences were not detected using the traditional approach of microsatellite marker genotyping and this work demonstrated the utility of RESCAN for rapidly identifying regions of interest in the genomes of very-closely-related germplasm.
1. Application of reduced representation sequencing to genotype very closely-related germplasm differing in cold stress recovery. Genotyping by DNA sequencing is of particular value when evaluating very closely-related germplasm although sequencing costs may still be prohibitive. A cost-effective reduced representation sequencing approach to genotype advanced backcross lines derived from the same cross was employed by ARS scientists at Davis, California. Six candidate regions for recovery genes on four chromosomes were detected including one previously identified as containing a quantitative trait locus for seedling cold tolerance on chromosome 6. These regions were not detected using traditional simple sequence repeat marker surveys. This work establishes the utility of this approach and identifies DNA markers for the mapping and incorporation of this trait into breeding materials for the improvement of U.S. rice varieties.
Kim, S., Tai, T. 2013. Identification of SNPs in closely-related temperate japonica rice cultivars using restriction enzyme-phased sequencing. PLoS One. 8(3): e60176. doi:10.1371/journal.pone.0060176.