Location: Crops Pathology and Genetics Research2008 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
During FY2008, progress was made on four milestones 1) develop/obtain M-202 BAC library, 2) sequence and expression analysis of candidate seedling cold tolerance genes, 3) screening of mutant lines for altered cold tolerance, and 4) development and characterization of near-isogenic rice lines for cold tolerance assessment. Tissue from the cold tolerant rice M-202 was harvested and sent to the Lucigen Corporation which was contracted to construct a genomic DNA BAC library of M-202. Construction of this library (average insert size: 110 kb) has been completed and the library will be delivered in August 2008. This library will be used to complete additional Year 1 milestones (identify BACs spanning qCTS12 and qCTS4, construct clones for transformation, initiate rice transformation). DNA sequence and expression analysis of candidate seedling cold tolerance genes is ongoing. Sequence information for the OsGSTZ1 gene from M-202 and the cold susceptible IR50 has been obtained. There are several differences between M-202 and IR50; however, none appear to affect the encoded protein. This suggests that if OsGSTZ1 is responsible for the difference in cold tolerance it is not due to functional differences in the proteins from M-202 and IR50. Sequencing of the other major qCTS12 candidate gene, OsGSTZ2, was initiated. Some differences in the DNA sequence between M-202 and IR50 have been identified. Two of these sequence differences are predicted to result in two amino acid differences between the OsGSTZ2 proteins from M-202 and IR50. It is unknown whether these differences have any functional significance. DNA sequencing of ten cultivars exhibiting similar or greater cold tolerance than M-202 and ten cultivars exhibiting similar or greater cold susceptibility than IR50 was initiated to examine whether specific differences in the candidate tolerance genes is correlated with the response of these cultivars to low temperature. Quantitative RT-PCR analysis was initiated to study the expression of qCTS12 and qCTS4 candidate genes in seedling leaf tissue during exposure to low temperatures although no results are available at this time. Over 1,000 mutant lines of the cold tolerant rice Nipponbare were screened for altered seedling response to qCTS12 low temperature conditions. A total of 13 mutant lines exhibiting either more sensitivity (6 lines) or more tolerance (7 lines) than normal Nipponbare were identified following initial screening and re-testing. Screening of Nipponbare mutant lines using qCTS4 low temperature conditions is currently underway and should be completed by October 2008. Work continued on the production of near-isogenic lines for use in studying the effects of the qCTS4 and qCTS12 loci in natural field environments. BC4F4 seeds derived from self fertilization of BC4F3 plants that were confirmed to contain various chromosomal segments of the qCTS4 region were planted for seed production. DNA marker analysis is underway to confirm the genetic profile of these plants at the qCTS4 locus and to examine the qCTS12 locus. Seeds collected from these plants will be used in field studies in FY2009. This addresses National Program 301, Component 2.
1. Identification of rice mutants with altered responses to low temperatures. Rice seedlings are sensitive to low temperatures (=15 to 20°C) and exposure may result in a variety of responses including tissue damage/death, wilting, stunting, and loss of chlorophyll. Among the two major types of rice (japonica and indica), japonica varieties are more cold tolerant. We have screened seedlings from over 1,000 mutant lines of the japonica variety Nipponbare and identified 13 lines which appear to be altered in their response to low temperatures (6 with less and 7 with more tolerance than normal Nipponbare). These mutants can be used to isolate genes involved in seedling cold tolerance and as breeding materials for developing improved rice varieties. This accomplishment addresses NP 301 Component 2: Crop Informatics, Genomics, and Genetic Analyses, Problem Statement 2C: Genetic Analyses and Mapping of Important Traits.
5. Significant Activities that Support Special Target Populations