Location: Crops Pathology and Genetics Research2009 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
Progress was made on milestones 1)identification of BACs, 2)construction of clones for transformation, 3)sequence & expression analysis of candidate seedling cold tolerance genes, 4)screening of mutant lines for altered cold tolerance, and 5)development & characterization of near-isogenic rice lines for cold tolerance assessment. The M-202 BAC library obtained in FY2008 was screened for clones spanning qCTS12 & qCTS4. Technical difficulties were encountered in screening the library and a backup strategy was employed. A BAC clone spanning the qCTS4 locus from the cold tolerant cultivar Nipponbare was obtained from the Clemson Univ. Genome Institute. Based on the analysis of qCTS12, a candidate gene approach was taken and a spanning BAC is no longer being pursued. Substantial progress has been made in construction of a number of transformation clones to analyze qCTS12 and qCTS4 candidate genes. For qCTS12, cDNA and RNAi constructs of the two most likely candidates (OsGSTZ1 and OsGSTZ2) have been or will be completed by the end of FY2009. For qCTS4, genomic constructs derived from the spanning BAC will be completed by the end of FY2009 for use in functional complementation tests. Transformation experiments were delayed due to the rice planting moratorium; however, it is expected that some transformations will be initiated by the end of FY2009. As indicated in the FY2008 report, DNA sequence and expression analysis of candidate seedling cold tolerance genes is ongoing. Sequence analysis of the qCTS12 candidate genes indicates that only OsGSTZ2 exhibits differences between M-202 (tolerant) and IR50 (susceptible) that are predicted to result in non-synonymous amino acid changes. Two such changes were observed(isoleucine to valine at position 99 and aspargine to valine at position 184). Sequencing of 10 tolerant and 10 susceptible rice accessions did not reveal a complete correlation between either of these differences and the response to cold. Quantitative RT-PCR analysis indicates that after 24 hours at low temperature OsGSTZ2 is induced about 2-3 fold higher in M-202 than in IR50 while OsGSTZ1 was not induced. The sequencing and expression data suggest that OsGSTZ2 is most likely to be qCTS12; however, transformation results are needed to confirm this and rule out a role for OsGSTZ1. Analysis of qCTS4 candidates has not been completed. In FY2008, putative mutants exhibiting altered responses to qCTS12 low temperature conditions were identified. These responses have not been reproduced in FY2009. Further examination of these putative mutants was delayed by the planting moratorium. Screening for mutants using qCTS4 low temperature conditions in FY2008 was not successful and was discontinued. With regard to near-isogenic lines, BC4F5 seeds were harvested from IR50 lines containing the qCTS4 & qCTS12 regions of M-202. DNA analysis of these lines indicates that some contain only qCTS4 regions while others contain both qCTS4 & qCTS12. Crosses to develop IR50 lines containing only qCTS12 were planned, but not performed due to the planting moratorium and will be completed in FY2010. The near-isogenic lines will be assessed in FY2010.