2012 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.
During FY2012, work on genetic analysis of rice seedling cold tolerance continued. Our studies to date indicate OsGSTZ2, one of two tandemly arranged glutathione transferase zeta genes, underlies qCTS12, a major seedling cold tolerance quantitative trait loci. Transgenic rice plants were obtained for testing the OsGSTZ2 gene from the cold susceptible variety IR50. Work is underway to compare the response to cold stress of these plants and those obtained in FY2011 which have the OsGSTZ2 gene from the cold tolerant variety M-202 as well as plants that have the OsGSTZ1 gene. Unlike OsGSTZ2, the OsGSTZ1 genes from IR50 and M-202 appear to encode identical proteins and should not be the reason for the difference in cold tolerance. In addition to the transgenic work, conventional genetic crossing work continued for generating IR50 plants with the genes from M-202. Offspring from a cross between IR50 and 6885-2 (IR50 with qCTS12 and qCTS4 from M-202) were assessed with DNA markers and by visual rating after cold stress. Plants with one or both of the QTLs were identified. Two of these plants, one with qCTS4 and the other with qCTS12, were analyzed by limited DNA sequencing and seeds were harvested. At the same time, the responses 6885-2, the parental lines (M-202 and IR50), and other rice varieties were characterized using physiological indicators and an additional cold test was developed and employed. This “recovery” test was used to assess the ability of seedlings to recover after exposure to constant cold stress. Some rice lines exhibited severe cold damage but were able to recover. Interestingly, two closely-related lines (6885-2 and 4853-9), both of which have qCTS4 and qCTS12, exhibited differences with 6885-2 showing more rapid and vigorous recovery. A manuscript describing this work is in press. Recently, limited DNA sequencing of these lines revealed one major M-202 introgression on chromosome 6 and a few smaller regions present in 6885-2 but not in 4853-9. Seedlings from the IR50/6885-2 cross were evaluated using the recovery test and genetic analysis of the recovery trait is underway. The second year of field trials of the advanced backcross lines was not successful due to poor field conditions (i.e. unusually low temperatures) that resulted in poor performance of all lines including tolerant checks. Fertility was very low and seeds were of insufficient quantity and quality for FY2012 field trials. The availability of lines from the IR50/6885-2 cross which are being grown for seed and the use of a different field site will facilitate testing of the individual contribution of each locus to seedling cold tolerance under controlled and field conditions in FY2013. Collaborators in Korea (Chungnam National University) have obtained and are currently analyzing insertional mutants in one of the candidate genes for the qCTS4 QTL.
Development and application of rice seedling cold stress recovery assay. Seedling cold tolerance in rice may be evaluated using many conditions and methods, some of which are more relevant to real world situations. The ability of various germplasm accessions and genetic mapping lines to recover from low temperature stress was evaluated by ARS scientists at Davis, CA, and significant differences were observed. This work establishes the foundation for the genetic analysis of this recoverability trait and its incorporation into breeding materials for the improvement of U.S. rice varieties.
Tsai, H., Howell, T., Nitcher, R., Missirian, V., Watson, B., Ngo, K., Lieberman, M., Fass, J., Uauy, C., Tran, R., Ali, K., Filkov, V., Tai, T., Dubcovsky, J., Comai, L. 2011. Discovery of rare mutations in populations: TILLING by sequencing. Plant Physiology. 156:1257-1268.
Monson-Miller, J., Sanchez-Mendez, D.C., Tai, T., Comai, L. 2012. Reference genome-independent assessment of mutation density using restriction enzyme-phased sequencing. Biomed Central (BMC) Genomics. 13:72.
Kim, S., Tai, T. 2011. Identification of genes necessary for wild-type levels of seed phytic acid in Arabidopsis thaliana using a reverse genetics approach. Molecular Genetics and Genomics. 286:119-133.