2005 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? What 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 Pi-ta for identification of new sources of resistance; identifying the interaction components in the Pi-ta gene-mediated signal recognition and transduction pathways for precisely engineering resistance, and analyzing structural and functional relationships of AVR-Pita for predicting the stability of blast resistance in current cultivars, and. 4)identifying differentially expressed genes after rice is infected either with 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 needs to 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. 2.List the milestones (indicators of progress) from your Project Plan. Objective 1. Mapping and genomic analysis of disease and end-use quality genes in rice. Milestone 1 (12 months) Employ a Molecular Geneticist. Conduct molecular characterization of core subset from companion project. Begin identification of R-genes in the Kaybonnet Ipa 1-1/Zhe733 population. (The original milestones were revised by recently hired Dr. Steven Brooks. The milestones are based on his revised "Approach and Research Procedures" for Objective 1.) Milestone 2 (24 months) Continue molecular characterization of core subset. Continue identification of R-genes in the Kaybonnet Ipa 1-1/Zhe733 population. Conduct TILLING for R-gene identification. Attempt to identify SNP markers for AGPase/sucrose synthase. Milestone 2 (24 months) Develop a method for screening rice varieties for sensitivity to Rhizoctonia solani (RS) toxin and its association with disease susceptibility. Evaluate Francis-derived segregation distortion lines by developing F2 mapping populations from crosses with Dragon Eyeball 100 (aromatic). Milestone 3 (36 months) Publish molecular characterization of core subset. Continue TILLING for disease resistance. Develop additional mapping population(s) to identify R-genes. Continue identification marker(s) for starch biosynthesis. Milestone 3 (36 months) Identify toxin sensitivity mutants and genetically map toxin sensitivity gene(s). Produce doubled haploid (DH) populations to evaluate quality traits including aroma. Milestone 4 (48 months) Publish TILLING for disease resistance. Publish marker(s) for R-gene and/or starch biosynthesis. Milestone 4 (48 months) Continue mapping and mutant analysis for identification of candidate gene(s) for RS toxin sensitivity. Determine linkage blocks in DH population associated with cooking quality by linkage analysis. Objective 2. Introgress novel resistance genes from the wild Oryza species into cultivated rice and identify genetic stocks. Milestone 1 (12 months) Complete genotyping of available Oryza spp. accessions. Determine the presence of Pi-ta and Pi-b in the available Oryza spp. accessions. Begin crossing to select parents for RIL population development. Milestone 2 (24 months) Complete and publish confirmation of trisomic lines using BAC clones. Publish molecular information on Oryza spp. Select cross for RIL population. Survey databases for markers associated with NBS-LRR regions. Milestone 3 (36 months) Enter trisomic lines into the genetic stocks collection. Identify useful markers associated with the NBS-LRR regions to use in germplasm development. Milestone 4 (48 months) Publish information obtained from the RIL population(s). Begin to make selections from the RIL population(s) for germplasm release. Objective 3. Characterize Pi-ta alleles and Pi-ta interacting genes associated with rice blast resistance. Milestone 1 (12 months) Complete and publish pathogenicity assays of lesion mimic plants. Complete construction of the two hybrid library and construct verification. Milestone 2 (24 months) Complete and publish the genetic analysis of blast susceptible plants. Complete sequence analysis of Pi-ta alleles in mutant plants. Completion of Y-2H hybrid screening. Milestone 3 (36 months) Complete and publish characterization of interacting genes both in vivo and in vitro. Milestone 4 (48 months) Complete and publish Pi-ta interacting genes. Objective 4. Identify genes that are anhanced or suppressed by rice blast and sheath blight. Milestone 1 (12 months) Complete DNA microarray analysis and sequence of differentially expressed genes from subtracted cDNA library. Complete verification of expression of differentially expressed genes. Milestone 2 (24 months) Complete analysis of candidate genes. Mapping a few promising candidate genes. Milestone 3 (36 months) Complete the sequence analysis of candidate genes in elite US germplasm for identification of DNA markers. Milestone 4 (48 months) Complete DNA markers for marker-assisted selection. 4a.What was the single most significant accomplishment this past year? Identification of differentially expressed genes to sheath blight Sheath blight is one of the most damaging diseases in US rice. Two Robust Long–Serial Analysis of Gene Expression (RL-SAGE) libraries were constructed using RNA isolated from the sheath blight inoculated and uninoculated leaves of the rice cultivar Jasmine 85 at the Dale Bumpers National Rice Research Center, Stuttgart, AR and in cooperation with Ohio State University. A total of 44,282 expressed genes were analyzed from early interactions of rice with R. solani. Similar data were also obtained by DNA microarray analysis and by a subtracted cDNA library. Many defense-related genes were highly induced in the inoculated library. This important accomplishment provides a starting point in identifying the candidate genes that are responsible for quantitative sheath blight resistance. 4b.List other significant accomplishments, if any. Novel disease resistance genes in wild species Accessions of rice wild relatives, Oryza spp. are a poorly exploited source of disease resistance genes. At the Dale Bumpers National Rice Research Center, Stuttgart, AR, Oryza spp. accessions were genotyped with DNA markers to determine their relatedness and screened for sheath blight and blast resistance. Associations between the DNA markers and disease resistance identified chromosomal regions that may be the source of novel blast resistance genes. Crosses are being made to further characterize these possible novel resistance genes in selected Oryza spp. accessions. Successful incorporation of novel disease resistance genes from wild rice species into rice could greatly broaden the narrow genetic base for disease resistance that presently exists in U.S. rice germplasm. Induction of blast susceptible mutants of Katy Katy blast susceptible mutants are essential to isolate novel blast resistance genes. At the Dale Bumpers National Rice Research Center, Stuttgart, AR, five blast susceptible mutants of Katy were verified from a fast neutron mutagenized population. Two of the blast susceptible mutants contain the Pi-ta gene. These mutants are important for studying molecular mechanisms of the Pi-ta-mediated disease resistance. 4c.List any significant activities that support special target populations. Participated in the final evaluation meeting held on July 7, 2005 of "Scientific Instrumentation and Curriculum Development for Teaching Biotechnology at University of Arkansas at Pine Bluff (UAPB)", a teaching project funded by the USDA/CSREES 1890 Capacity Building Grants program as members of the Project Evaluation Committee. A written report was submitted after the meeting. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. Since this is the first year of the project, the major accomplishments over the life of the project are the same as those in Question 4. 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? Possible putative Katy mutant lines were selected from a total of 5000 mutated lines and increased an additional generation. Seed of the selected lines was distributed to several rice research scientists for use in their research programs. Provided expression data of 22,000 rice genes to the appropriate USDA-NRI funded RiceCAP (Rice Coordinated Agriculture Project) and other genetic stocks to US scientists for their research in rice. Trained several students and scientists on disease testing and molecular marker development and utilization. Participated in the annual University of Arkansas Rice Field Day through poster presentations on molecular marker research with rice germplasm and using molecular techniques to identify and understand the rice diseases, blast and sheath blight. Presentations were made at the Marker Assisted Breeding Workshop held at the DB NRRC, June 14-16, 2005 as part of the USDA-NRI funded RiceCAP to provide "An Overview of DNA Marker Technology As It Applies to Rice Improvement". Jia, Y. 2004. Release of rice genetic stock lesion mimic mutant 1. USDA-ARS Germplasm Release. 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). Cole, Nancy. 2005. Teams seek rice cures-U.S., China share seeds, germplasm vs. disease. Arkansas Democrat Gazette, May 22, 2005. McCarty, Larry. 2005. Geneticist hopes to aid rice growers in fighting disease. The Stuttgart Daily Leader, March 11, 2005. Review Publications Jia, Y., Singh, P., Winston, E.M., Wamashe, Y., Correll, J., Lee, F.N., Moldenhauaer, K., Gibbons, J., Rutger, J.N. 2005. Development of molecular strategies to control major rice fungal diseases in the US [abstract]. Rice Technical Working Group Meeting Proceedings. Abstract p. 109. Jia, Y., Wang, Z., Fjellstrom, R.G., Moldenhauer, K., Flowers, C.B., Rutger, J.N. 2005. Rice pi-ta gene confers resistance to two major pathotypes of the rice blast fungus in the U.S. [abstract]. Rice Technical Working Group Meeting Proceedings. Abstract p. 84. Jia, Y., Winston, E.M., Singh, P., Zhou, E., Wamishe, Y., Jia, M.H., Correll, J., Rutger, J.N. 2005. Molecular coevolution of rice resistance gene pi-ta and the corresponding Magnaporthe grisea avirulance gene avr-pita [abstract]. In: Plant and Animal Genome Conference Proceedings. p. 74. Jia, Y., Winston, E.M., Singh, P., Zhou, E., Wamishe, Y., Jia, M.H., Correll, J. 2004. Molecular mechanisms of durable rice blast resistance [abstract]. In: Proceedings, 2nd International Rice Functional Genomics Conference, Tucson, Arizona. Abstract p. 72. Jia, Y., Zhou, E., Lin, M.J., Rutger, J.N. 2004. Development and characterization of rice deletion mutants for functional genomics [abstract]. In: Proceedings, 2nd International Rice Functional Genomics Conference, Tucson, Arizona. Abstract p. 178. Jia, Y. 2004. Registration of Katy lesion mimic mutant. Crop Science. 45:1675. Lee, F.N., Cartwright, R.D., Jia, Y., Correll, J.C. 2005. Magnaporthe grisea race shift for virulence to the major R gene, Pita, in Arkansas. Proceedings Southern Region American Phytopathology Society. Abstract p. 116. Lee, F.N., Cartwright, R.D., Jia, Y., Correll, J.C., Moldenhauer, K.A., Gibbons, J.W., Boyett, V., Zhou, E., Boza, E., Seyran, E. 2005. A preliminary characterization of the rice blast fungus on 'Banks' rice. In: Norman, R.J., Meullenet, J.-F., Moldenhauer, K.A.K., editors. B.R. Wells Rice Research Studies 2004, Arkansas Agricultural Experiment Station Research Series 529. p. 103-110. Available: http://www.uark.edu/depts/agripub/Publications/researchseries/ Johnson, V.A., Redus, M., Gibbons, J.W., Moldenhauer, K.A., Jiang, J., Jia, Y. 2005. Marker assisted selection for the rice blast resistance gene pi-ta: development and use of an improved co-dominant analysis method [abstract]. Rice Technical Working Group Meeting Proceedings. Abstract p. 62. Eizenga, G.C., Lee, F.N., Jia, Y., Yan, W. 2004. DNA markers identify blast resistance genes and genotype newly introduced rice germplasm [abstract] Agronomy Abstracts. 2004 CDROM. Eizenga, G.C., Padolino, T.H., Azam, M., Brar, D.S., Cheema, A.A., Ismachin, A., Ismail, A., Koh, H.J., Senghaphan, R., Shu, Q., Tuan, V.D., Wu, D., Zhu, X., Maluszynski, M. 2005. Evaluation of rice mutants in multi-location trials conducted in the Southeast Asian region [abstract]. Rice Technical Working Group Meeting Proceedings. Abstract p. 71-72. Eizenga, G.C., Xiang, G., Jia, Y., Lee, F. 2005. Identification of disease resistance in the Oryza spp. and following its introgression into cultivated rice with DNA markers. Rice Technical Working Group Meeting Proceedings. Abstract p. 115. Eizenga, G.C., Ho, Q.P. 2005. Introduction and identification of IR36 rice trisomic lines. Rice Technical Working Group Meeting Proceedings. Abstract p. 69-70. Eizenga, G.C., Agrama, H., Jia, Y., Lee, F.N. 2005. Genotyping of selected rice wild relatives (Oryza spp.) and their progenies [abstract]. Plant and Animal Genome Conference. p. 147. Eizenga, G.C., Agrama, H.A., Lee, F.N., Jia, Y. 2005. Continued evaluation of blast resistance genes in rice wild relatives (Oryza spp.) and unique rice (O. sativa) accessions utilizing DNA markers. In: Norman, R.J., Meullenet, J.-F., Moldenhauer, K.A.K., editors. B.R. Wells Rice Research Studies 2004, Arkansas Agricultural Experiment Station Research Series 529. p. 30-37. Available: http://www.uark.edu/depts/agripub/Publications/researchseries/