2006 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Improvement of rice for yield, grain quality and pest resistance is required to keep the US rice industry competitive in the global marketplace. With rice being the first cereal grain to be sequenced, there is a need to use this information in developing molecular markers/tools associated with the aforementioned traits to accelerate identification of these traits in adapted and unadapted rice (Oryza sativa) germplasm, and related Oryza species. The following approaches are being undertaken in this project to facilitate this effort: . 1)mapping and genomic analysis of disease resistance and end-use quality genes in rice to promote the usefulness of marker-assisted selection in developing improved rice germplasm,. 2)introgressing novel disease resistance genes from the wild Oryza species into cultivated rice for use by US rice breeders and identifying genetic stocks for use by rice researchers,. 3)determining allelic variation of the Pi-ta blast resistance gene for identification of new resistant germplasm sources; identifying the signal recognition and response components of the Pi-ta gene-mediated pathways for precisely engineering resistance, and analyzing structural and functional relationships of AVR-Pita gene of the pathogen for predicting the stability of blast resistance in current cultivars, and. 4)identifying differentially expressed genes after rice is infected with either the rice blast fungus or sheath blight fungus in order to develop strategies that will improve resistance in enhanced rice germplasm. The primary focus of the program is identification of agronomically important genes such as disease resistance and end-use quality genes using molecular techniques. It is expected that new genetic markers will be developed for germplasm characterization and marker-assisted selection. Secondly, identification and utilization of disease resistance genes in rice and related species will accelerate the development of improved rice germplasm using both conventional and novel strategies. Pesticide usage will be decreased and more cost-effective, environmentally benign methods of controlling rice diseases developed. To improve methods of disease control, the molecular basis of disease resistance needs to be understood. Goals of this research project pertain to the Genome Characterization and Genetic Improvement sub-component of National Program 301 - Plant Genetic Resources, Genomics and Genetic Improvement, and to the Host Plant Resistance component of National Program 303 – Plant Diseases. Specific national program objectives that are addressed through this research include: Objective 1.2.7 Identify genes responsible for plant product quality and resistance to disease, pests, and weather losses; Objective 1.2.8 Maintain, characterize, and use genetic resources to optimize, safeguard, and enhance genetic diversity and promote viable and vigorous plant production systems; and Objective 3.2.4 Develop and release to potential users varieties and/or germplasm of agriculturally important plants that are new or provide significantly improved (either through traditional breeding or biotechnology) characteristics enhancing pest or disease resistance. 2.List by year the currently approved milestones (indicators of research progress) Year 1 (FY 2005) Fill Molecular Geneticist vacancy. Conduct molecular characterization of core subset of NSGC germplasm collection. Begin identification of resistance genes in the Kaybonnet lpa 1-1/Zhe733 population. Complete genotyping of available Oryza spp. accessions with microsatellite markers distributed throughout the genome. Determine the presence of Pi-ta and Pi-b blast resistance genes in the available Oryza spp. accessions using cloned DNA markers. Initiate Advanced Backcross (ABC) population development using Oryza spp. for the identification of novel blast resistance genes. Document the apparent disease reaction of lesion mimic plants. Construct rice DNA library in a yeast vector for the study of disease resistance genes. Complete DNA microarray analysis and verification of expression of differentially expressed genes for sheath blight resistance from a subtracted cDNA library. Year 2 (FY 2006) Identification of R-genes in the Kaybonnet 1pa 1-1/Zhe733 population. (Delayed from 2005) Develop a method for screening rice varieties for sensitivity to Rhizoctonia solani (RS) toxin and its association with disease susceptibility. Develop F2 mapping populations from crosses with Dragon Eyeball 100 (aromatic) for the study of segregation distortion. Complete morphological and cytological analysis of trisomic lines and verify using BAC clones. Document the genotypic diversity of Oryza spp. accessions using microsatellite markers. Select among segregating wild species populations for those with the best seed set and blast resistance to continue ABC (advanced backcross) population development. Use public databases to identify putative disease resistance genes having NBS-LRR (nucleotide binding site-leucine rich repeat) regions for use in locating chromosomal regions having novel resistance genes in progeny from wild Oryza species crosses. Complete and publish the genetic analysis of mutants of unknown blast resistance genes that cause host plant susceptibility. Complete the DNA sequence analysis of Pi-ta alleles in mutant susceptible plants. Use cloned Pi-ta disease resistance gene from rice plants and avirulence gene AVR-Pita from the blast pathogen to identify interacting proteins that are part of the plant-pathogen disease resistance response. Determine the putative function of candidate genes for sheath blight resistance identified in DNA microarrays, SAGE, and subtracted cDNA libraries. Develop a mapping population to genetically map a few promising genes for sheath blight resistance. Year 3 (FY 2007) Two backcross populations (BC1F2) will be developed for map-based cloning of the two genes controlling insensitivity (resistance) to the host selective (RS) toxin produced by Rhizoctonia solani which causes sheath blight disease in rice. Extensively phenotype one of the BC1F2 populations for RS toxin reaction and isolate DNA to begin molecular mapping of one of the toxin insensitivity genes. Using a sheath blight resistant parent, Jasmine 85, two mapping populations will be advanced to the F3 and F4 generations for eventual use in identifying disease resistance genes other than those associated with the host selective toxin. Develop a method to assay resistance to kernel smut in rice in order to identify germplasm possessing genetic sources of resistance. Register confirmed trisomic lines and make publicly available through the Genetic Stocks – Oryza (GSOR) collection. Continue to develop the ABC populations utilizing disease resistant Oryza spp. accessions. Begin developing chromosomal segment substitution lines (CSSLs) as genetic stocks useful for functional genomics studies Begin fine mapping of possible regions where novel blast and/or sheath blight resistance genes are located in Oryza spp. progeny. Verify the presence of specific proteins identified from the interaction of plant blast resistance genes (Pi-ta) and a pathogen avirulence gene (AVR-Pita) and determine their chromosomal locations through searching public databases. Identify chromosomal regions associated with partial resistance to blast using several crosses among US cultivars. Identify the chromosomal location and map candidate sheath blight resistance genes in a rice mapping population. Perform sequence analysis of candidate genes for sheath blight resistance in elite US germplasm for identification of DNA markers for use in breeding. Year 4 (FY 2008) Population segregation for RS toxin gene 1 will be verified using phenotypic and genotypic data. RS toxin sensitivity gene 1 will be tagged with molecular markers and a genetic map of the locus constructed. A second BC1F2 population will be extensively phenotyped for RS toxin reaction and DNA samples will be prepared to begin molecular mapping of gene 2. Using a sheath blight resistant parent, Jasmine 85, two mapping populations will be advanced to the F4 and F5 generations for eventual use in identifying disease resistance genes other than those associated with the host selective toxin. Kernel smut resistant rice germplasm will be identified and crosses will be made for mapping the resistance genes. Ascertain chromosomal regions and putative disease resistance genes associated with blast and sheath blight resistance in ABC populations having one wild Oryza spp. parent. Identify introgressed lines having novel disease resistance genes as promising germplasm releases for use by US rice breeders in cultivar development programs. Complete and publish the function of Pi-ta interacting genes. Produce some 20,000 mutants in the cultivar Katy to be used as a genetic tool to determine gene function of various economically important traits. Identify chromosomal regions associated with novel resistance to two important races of blast disease using Raminade Strain 3 as a source of resistance. Confirm candidate genes for sheath blight resistance in association study using elite US germplasm and identify tightly linked microsatellite markers that can be used by rice breeders in marker-assisted selection. 4a.List the single most significant research accomplishment during FY 2006. Molecular mechanism explains why rice cultivars lose resistance to rice blast disease: Rice cultivars often lose resistance to blast disease after being deployed for a few years. At the Dale Bumpers National Rice Research Center, Stuttgart, AR, a virus-like mobile piece of DNA called a transposon, was found in the blast pathogen taken from a blast infected field. This transposon was found to disrupt the blast resistance gene Pi-ta which made the cultivar resistant. This finding shows that transposons can contribute to the instability of cultivar disease resistance and is one mechanism of making blast resistance genes ineffective in rice. This demonstrates the importance of developing cultivars with several blast resistance genes so that the resistance is more durable. (NP303, Host Plant Resistance component) 4b.List other significant research accomplishment(s), if any. A new technique for evaluating resistance to sheath blight disease in rice: Identification of genetic sources of resistance is critical to manage this disease which is important to United States rice production. Current methods of evaluation require relatively large amounts of seed and thus screening is postponed until the later stages of breeding. At the Dale Bumpers National Rice Research Center, Stuttgart, AR, a method that determines rice cultivar sensitivity to the host-selective toxin produced by Rhizoctonia solani, the pathogen that causes sheath blight disease, was developed. This method fills a critical need for fast and reliable data for evaluating for sheath blight resistance. (NP301, Genome Characterization and Genetic Improvement component) 4c.List significant activities that support special target populations. Developed and awarded a USDA NRI research grant on genetic engineering of isoflavones in rice (2005-2008) in collaboration with University of Arkansas at Pine Bluff, an 1890 land grant university. 5.Describe the major accomplishments to date and their predicted or actual impact. Research accomplished on this project contributes to the goals of National Program 301, Component 2. Crop informatics, genomics, and genetic analyses. Problem Statement 2B: Structural comparison and analysis of crop genomes, and Problem Statement 2C: Genetic analyses and mapping of important traits. During the life of this project, two new methods for evaluating resistance to sheath blight disease have been developed, disease response genes have been identified, and a pathogen transposon has been identified that overcomes plant defense mechanisms. Several genomics tools were developed including blast mutants, a gene expression library, trisomic lines, and disease-resistant Oryza spp. that will aid scientists studying the molecular mechanism of disease resistance and developing molecular markers. Ultimately this will enable rice cultivars to be developed with improved disease resistance. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Methods for developing blast resistant cultivars using molecular marker assisted selection were presented to breeders, geneticists and pathologists. New knowledge on the evolutionary relationship between a rice blast resistance gene and a fungal avirulence gene was presented to geneticists, pathologists, physiologists, molecular biologists, industry representatives and students. Two new screening methods for screening for response to the sheath blight pathogen were presented to pathologists and geneticists interested in developing sheath blight resistant germplasm. The use of molecular techniques to identify and understand rice diseases, blast and sheath blight, were presented to rice researchers, farmers, stakeholders, and industry representatives. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Core, J. 2006. Collection helps fight destructive rice disease. Agricultural Research, July 2006. p. 18. Singh, P., Jia, Y., Boza, E.J., Correll, J.C., Lee, F.N. 2006. Development of a molecular marker from the rice blast avirulence gene AVR-Pita for surveillance of durable rice blast resistance conferred by Pi-ta in Arkansas. In: Norman, R.J., Meullenet, J.-F., Moldenhauer, K.A.K., editors. B.R. Wells Rice Research Studies 2005 Arkansas Agricultural Experiment Station Research Series 540. p. 152-159. Available: http://www.uark.edu/depts/agripub/Publications/researchseries/ Zhou, E., Jia, Y., Correll, J.C., Lee, F.N. 2006. Molecular mechanisms of the instability of avirulence gene avr-pita in rice blast fungus magnaporthe oryzae. In: Norman, R.J., Meullenet, J.-F., Moldenhauer, K.A.K., editors. B.R. Wells Rice Research Studies 2005, Arkansas Agricultural Experiment Station Research Series 540. p. 160-167. Available: http://www.uark.edu/depts/agripub/Publications/researchseries/. Pinson, S.R., Fjellstrom, R.G., Shank, A.R., Oard, J., Groth, D., Jia, Y., Jia, M.H. 2006 Incorporating foreign sheath blight resistance genes into U.S rice germplasm. Texas Rice, Highlighting Research 2006. p. VI-VII Review Publications Jia, Y., Lin, H., Wang, Z., Lin, M.J., Valent, B., Rutger, J.N. 2006. Host active defense responses occur within 24 hours post-inoculation in the rice blast system [abstract]. XII International Congress on Molecular Plant-Microbe Interactions. p. 91. Jia, Y., Xi, J., Rutger, J.N. 2006. Development and characterization of Katy deletion mutant populations. Plant Mutation Reports. 1(1):43-47. Jia, Y., Zhou, E., Winston, E., Singh, P., Wang, Z., Correll, J., Lee, F., Jia, M.H. 2005. Translational genomics: a case study of rice Pi-ta resistance gene. In: Proceedings of International Conference on Plant Molecular Breeding, October 27-30, 2005. p. 3. Cheng, C., De Los Reyes, B.G., Zhang, Y., Ressom, H., Jia, Y., Yun, S.J. 2006. Genomic analysis of the early responses of developing rice seedlings to cold stress [abstract]. In: Proceedings of the XIV Annual International Plant & Animal Genome Conference, Janaury 14-18, 2006, San Diego, California. p. 734. Agrama, H.A., Eizenga, G.C. 2006. Evaluation of linkage disequilibrium in rice and its wild relatives [abstract]. In: Proceedings of the XIV Annual International Plant & Animal Genome Conference, January 14-18, 2006, San Diego, California. p. 14. Eizenga, G.C., Agrama, H.A., Lee, F.N. 2005. Identifying novel R-genes in rice wild relatives with microsatellite markers [abstract]. American Society of Agronomy Abstracts, November 6-9, 2005, Salt Lake City, Utah. 2005 CDROM. Eizenga, G.C., Agrama, H.A., Lee, F.N., Yan, W., Jia, Y. 2005. Understanding the genetic diversity in order to identify R-genes in Oryza spp and O. sativa. In: Proceedings 5th International Rice Genetics Symposium, November 19-23, 2005, Manila, Philippines. 2005 CDROM. Wang, Z., Redus, M., Jia, Y. 2005. Establishment of codominant markers for rice blast resistance gene Pi-ta. Chinese Journal of Rice Science. 19:483-488. Boyett, V.A., Gibbons, J.W., Moldenhauer, K.A., Jia, Y., McClung, A.M., Fjellstrom, R.G. 2006. Advances in marker-assisted selection for rice blast resistance. Rice Technical Working Group Meeting Proceedings, February 29-March1, 2006, Houston, TX. CDROM.