Source: AGRICULTURAL RESEARCH SERVICE submitted to
GENOMIC CHARACTERIZATION OF RICE GERMPLASM
Sponsoring Institution
Agricultural Research Service/USDA
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0408571
Grant No.
(N/A)
Project No.
6225-21220-002-00D
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Jun 10, 2004
Project End Date
Jul 9, 2008
Grant Year
(N/A)
Project Director
EIZENGA G C
Recipient Organization
AGRICULTURAL RESEARCH SERVICE
(N/A)
STUTTGART,AR 72160
Performing Department
(N/A)
Non Technical Summary
(N/A)
Animal Health Component
40%
Research Effort Categories
Basic
60%
Applied
40%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011530104045%
2011530108015%
2121530104030%
2121530108010%
Goals / Objectives
Characterize rice genome in order to develop useful molecular strategies to accelerate the production of improved rice germplasm. Molecular genetics, molecular cytogenetics and molecular plant pathology approaches will be used to address four integrated objectives: 1) mapping and genomic analysis of disease resistance and end-use quality genes in rice for use by US rice researchers, 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 with either the rice blast fungus or sheath blight fungus in order to develop strategies that will improve resistance in enhanced rice germplasm.
Project Methods
Develop molecular markers associated with the economically important traits of a) disease resistance, with emphasis on rice blast and rice sheath blight and b) end-use quality, with emphasis on starch biosynthesis, which will improve identification of these traits in rice germplasm and promote the usefulness of marker-asisted selection in developing improved rice germplasm. Molecular markers will allow more efficient incorporation of Oryza spp. DNA into cultivated rice and can be used to further identify Oryza spp., backcross progenies, RILs, and genetic stocks. Characterize the Pi-ta gene-mediated signal transduction pathways which will aid in the development of both conventional and novel strategies to improve blast resistance for US rice germplasm. Identify differentially expressed genes upon pathogen attack to enhance understanding of the molecular mechanisms of interactions of necrotrpohic fungal pathogen and host. This may lead to the development of molecular markers for marker-assisted selection.

Progress 06/10/04 to 07/09/08

Outputs
Progress Report Objectives (from AD-416) Characterize rice genome in order to develop useful molecular strategies to accelerate the production of improved rice germplasm. Molecular genetics, molecular cytogenetics and molecular plant pathology approaches will be used to address four integrated objectives: 1) mapping and genomic analysis of disease resistance and end-use quality genes in rice for use by US rice researchers, 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 with either the rice blast fungus or sheath blight fungus in order to develop strategies that will improve resistance in enhanced rice germplasm. Approach (from AD-416) Develop molecular markers associated with the economically important traits of a) disease resistance, with emphasis on rice blast and rice sheath blight and b) end-use quality, with emphasis on starch biosynthesis, which will improve identification of these traits in rice germplasm and promote the usefulness of marker-asisted selection in developing improved rice germplasm. Molecular markers will allow more efficient incorporation of Oryza spp. DNA into cultivated rice and can be used to further identify Oryza spp., backcross progenies, RILs, and genetic stocks. Characterize the Pi-ta gene-mediated signal transduction pathways which will aid in the development of both conventional and novel strategies to improve blast resistance for US rice germplasm. Identify differentially expressed genes upon pathogen attack to enhance understanding of the molecular mechanisms of interactions of necrotrpohic fungal pathogen and host. This may lead to the development of molecular markers for marker-assisted selection. Significant Activities that Support Special Target Populations Rice blast disease is one of the most devastating diseases of this crop that feeds over half of the world. We analyzed blast resistance alleles in the USDA worldwide rice germplasm collection and avirulent AVR-Pita alleles of the blast pathogen using isolates from rice fields in major rice growing areas of the world and identified a new locus of the Pi-ta blast resistance gene Ptr(t) that is required for plant resistance. Numerous insertions, deletions, and transposon insertions were identified in the AVR-Pita gene in virulent pathotypes, and 23 AVR-Pita alleles encoding 23 different avriulent proteins were identified in avirulent pathotypes. This demonstrates the tremendous genetic variability found in the blast pathogen that allows it to overcome plant defense genes. A mapping population was developed using indica and japonica cultivars to identify genes associated with resistance to sheath blight disease. A major QTL was identified on chromosome 9 that contributes over 24% of the variation in sheath blight resistance as determined using seedlings in a microchamber or using adult plants in a mist chamber. The sheath blight pathogen is known to produce a phytotoxin that causes necrosis of host plant tissue during infection. Using traditional genetics, sensitivity to the toxin in rice was found to be controlled by two genes in an epistatic manner. A population has been developed to genetically map one of the two genes. False smut (Ustilaginoidea virens) and kernel smut (Neovossia horrida) are poorly studied diseases of rice (Oryza sativa) in the US. The effects of crop management methods on rice smut diseases were evaluated in order to determine G x E interactions and identify resistance in commercial cultivars. Using a long-term crop rotation experiment, we determined the effects of crop rotation, tillage, and fertility on disease incidence. We also used two disease nurseries for cultivar resistance trials, identified management strategies that minimize smut incidence on susceptible cultivars, and have discovered a genetic source of resistance to kernel smut. Two mapping populations derived from crosses between Bengal and two accessions of wild relatives of rice (O. nivara) are under development. Both O. nivara accessions were determined to be resistant to sheath blight, and one accession also showed resistance to blast disease. The progeny from the cross showed a wide range in reaction to sheath blight disease, indicating that it has great promise for mapping resistance genes. A Nipponbare/O. nivara (IRGC 100897) F2 population of 279 plants is being phenotyped for 13 plant characteristics and 6 seed characteristics, and genotyped with approximately 120 SSR markers. The map developed from this population will be compared to the psuedomolecule developed by the Oryza Map Alignment Project from the BAC end sequences of this O. nivara accession and the Nipponbare sequence to discover how the phenotypic traits map across the two species genomes. (NP301, Component 2C)

Impacts
(N/A)

Publications

  • Brooks, S.A., Yan, W., Jackson, A.K., Deren, C.W. 2008. A natural mutation in Rc reverts white-rice-pericarp to red and results in a new, dominant, wild-type allele: Rc-g. Theoretical and Applied Genetics. 117(4):575-580.
  • Liu, G., Bernhardt, J., Jia, M.H., Wamishe, Y., Jia, Y. 2007. Molecular characterization of rice recombinant inbred line population derived from a japonica-indica cross. Euphytica. 159:73-82.
  • Wang, Z., Jia, Y., Rutger, J.N., Xia, Y. 2007. Rapid survey for presence of a blast resistance gene Pi-ta in rice cultivars using the dominant DNA markers derived from portions of the Pi-ta gene. Plant Breeding. 126:36-42.
  • Wamishe, Y.A., Jia, Y., Singh, P., Cartwright, R.D., Eizenga, G.C., Lee, F. N. 2007. Studies on isolates of Rhizoctonia solani from Arkansas for management of rice sheath blight. Frontiers of Agriculture in China. 1(47) :361-367.
  • Jia, Y., Lin, H., Wang, Z., Valent, B., Rutger, J.N. 2007. Host active defense responses occur 24 hours after pathogen inoculation in the rice blast system. Chinese Journal of Rice Science. 14(4):302-310.
  • Brooks, S.A. 2007. Sensitivity to a phytotoxin from Rhizoctonia solani correlates with sheath blight susceptibility in rice. Phytopathology. 97:1207-1212.
  • Venu, R.C., Jia, Y., Gowda, M., Jia, M.H., Jantasuriyarat, C., Stahlbert, E., Li, H., Rhineheart, A., Reddy, P., Singh, P., Rutger, J.N., Kudrna, D., Wing, R., Nelson, J.C., Wang, G. 2007. Rl-sage and microarray analysis of the rice defense transcriptome after Rhizoctonia solani infection. Molecular Genetics and Genomics. 278:421-431.
  • Zhou, E., Jia, Y., Singh, P., Correll, J.C., Lee, F.N. 2007. Instability of the Magnaporthe oryzae Avirulence gene AVR-Pita alters virulence. Fungal Genetics and Biology. 44:1024-1034.
  • Li, W., Lei, C., Cheng, Z., Jia, Y., Huang, D., Liu, Z., Wang, J., Shi, K., Zhang, X., Su, N., Guo, X., Zhai, H., Wan, J. 2008. Identification of SSR markers for a broad-spectrum blast resistance gene Pi-20(t) for marker- assisted breeding. Molecular Breeding. 22:141-149.
  • Jia, Y., Marin, R. 2008. Identification of a new locus Ptr(t) required for rice blast resistance gene Pi-ta-mediated resistance. Molecular Plant- Microbe Interactions. 21(4):396-403.
  • Jia, Y., Gealy, D.R., Lin, M., Wu, L., Black, H.L. 2008. Carolina foxtail (Alopecurus carolinianus): Susceptibility and suitability as an alternative host to rice blast disease (Magnaporthe oryzae [formerly M. grisea]. Plant Disease. 92(4):504-507.


Progress 10/01/06 to 09/30/07

Outputs
Progress Report Objectives (from AD-416) Characterize rice genome in order to develop useful molecular strategies to accelerate the production of improved rice germplasm. Molecular genetics, molecular cytogenetics and molecular plant pathology approaches will be used to address four integrated objectives: 1) mapping and genomic analysis of disease resistance and end-use quality genes in rice for use by US rice researchers, 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 with either the rice blast fungus or sheath blight fungus in order to develop strategies that will improve resistance in enhanced rice germplasm. Approach (from AD-416) Develop molecular markers associated with the economically important traits of a) disease resistance, with emphasis on rice blast and rice sheath blight and b) end-use quality, with emphasis on starch biosynthesis, which will improve identification of these traits in rice germplasm and promote the usefulness of marker-asisted selection in developing improved rice germplasm. Molecular markers will allow more efficient incorporation of Oryza spp. DNA into cultivated rice and can be used to further identify Oryza spp., backcross progenies, RILs, and genetic stocks. Characterize the Pi-ta gene-mediated signal transduction pathways which will aid in the development of both conventional and novel strategies to improve blast resistance for US rice germplasm. Identify differentially expressed genes upon pathogen attack to enhance understanding of the molecular mechanisms of interactions of necrotrpohic fungal pathogen and host. This may lead to the development of molecular markers for marker-assisted selection. Accomplishments Evolution of disease resistance genes in rice: Evaluating genetic sequence variability in cultivated, wild and weedy species of rice allows scientists to study gene evolution. Scientists at the Dale Bumpers National Rice Research Center at Stuttgart, AR determined the genetic sequence of the Pi-ta gene in 52 accessions derived from six rice species. Results indicate that the Pi-ta gene is an ancient gene which is found in wild, weedy relatives of cultivated rice that has undergone numerous mutations through evolution which have had no apparent affect. However, a recent mutation has resulted in a single amino acid change altering the protein produced by this gene to confer resistance to the fungus which causes blast disease in rice plants. Knowledge of the relationship between the structure and function of this gene will help geneticist to develop rice cultivars with durable resistance to diseases. (NP301 Plant Genetic Resources, Genomics, and Genetic Improvement, Component 2: Crop Informatics, Genomics, and Genetic Analyses, Problem Statement 2C: Genetic Analyses and Mapping of Important Traits) Phytotoxin is identified as a component of susceptibility to sheath blight disease in rice: Although sheath blight is an important disease of rice worldwide, effective methods for screening for resistance in rice germplasm have been lacking. Sensitivity to a phytotoxin derived from Rhizoctonia solani was shown to correlate with disease susceptibility by researchers at the Dale Bumpers National Rice Research Center in Stuttgart, AR. The phytotoxin method increased the accuracy of disease evaluations and will facilitate the identification of chromosomal regions associated with sheath blight resistance. (NP301 Plant Genetic Resources, Genomics, and Genetic Improvement, Component 2: Crop Informatics, Genomics, and Genetic Analyses, Problem Statement 2C: Genetic Analyses and Mapping of Important Traits) Identification of chromosomal locations of agronomic traits in rice: Mapping populations between cultivated rice and its two ancestral species, O. nivara and O. rufipogon, can be used to understand the evolution of important agronomic genes. At the Dale Bumpers National Rice Research Center in Stuttgart, AR a mapping population between the U.S. medium grain cultivar M-202 and an O. nivara accession (IRGC 100195) was evaluated for seedling vigor, and agronomic and grain quality traits. The chromosomal locations of genes (QTL) for these traits were identified and many corresponded to regions identified in other mapping populations. Validation of putative QTL is critical for precisely mapping important genes and eventually determining gene function. (NP301 Plant Genetic Resources, Genomics, and Genetic Improvement, 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) Technology Transfer Number of Non-Peer Reviewed Presentations and Proceedings: 15 Number of Newspaper Articles,Presentations for NonScience Audiences: 8

Impacts
(N/A)

Publications

  • Olsen, K.M., Caicedo, A.L., Jia, Y. 2007. The evolutionary genomics of weedy red rice in the U.S.A. Journal of Integrative Plant Biology. 69:811- 816.
  • Jia, Y., Coarrea-Victoria, F., McClung, A.M., Zhu, L., Liu, G., Wamishe, Y. , Xie, J., Marchetti, M.A., Pinson, S.R., Rutger, J.N., Correll, J.C. 2006. Rapid determination of rice cultivar responses to the sheath blight pathogen Rhizoctonia solani using a micro-chamber screening method. Plant Disease. 91:485-489.
  • Wang, Z., Redus, M., Jia, Y. 2005. Establishment of codominant marker for rice blast resistance gene pi-ta. Chinese Journal Rice Science. 19(6):483- 488.
  • Chen, C., Yun, K., Ressom, H., Mohanty, B., Bajic, V.B., Jia, Y., Yun, S., De Los Reyes, B.G. 2007. Reactive oxygen species trigger a regulatory module involved in the early responses of rice seedlings to cold stress. Biomed Central (BMC) Genomics. 8:175.


Progress 10/01/05 to 09/30/06

Outputs
Progress 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

Impacts
(N/A)

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. Development of the codominant marker of rice blast resistance gene Pi-ta. Chinese Journal Rice Science. 19(6):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.


Progress 10/01/04 to 09/30/05

Outputs
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) <Revised> 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) <Revised> 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) <Revised> 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. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Objective 1, 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. Milestone Substantially Met 2. Objective 2, 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 advanced backcross (ABC) population development. Milestone Fully Met 3. Objective 3, Milestone 1 (12 months): Complete and publish pathogenicity assays of lesion mimic plants. Complete construction of the two hybrid library and construct verification. Milestone Fully Met 4. Objective 4, 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 Fully Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? Objective 1 These are the revised milestones developed by recently hired Steven Brooks. The milestones are based on his revised "Approach and Research Procedures" for Objective 1. Milestone 2 (FY 2006) <Revised> 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). - Development of the RS toxin screening method facilitates screening breeding lines for sheath blight, and provides the necessary techniques to pursue further research objectives. F2 mapping populations from reciprocal crosses of Francis- derived lines and Dragon Eyeball 100 will be used to identify regions of the genome with distorter loci. Milestone 3 (FY 2007) <Revised> Identify toxin sensitivity mutants and genetically map toxin sensitivity gene(s). Produce doubled haploid (DH) populations to evaluate quality traits including aroma. - Two populations will be used in parallel to identify genetic loci conferring sensitivity to RS toxin. (1) Screening a gamma-radiated M2 population of the variety Cypress (toxin sensitive) for toxin insensitive mutants. (2) Phenotyping a segregating F2 mapping population derived from a cross between Cypress and Jasmine 85 (toxin insensitive). A microsatellite map of F2-reciprocal mapping populations will be used to identify the source of aberrant segregation. If the observed aberration is caused by true segregation distortion, additional crosses will be made to begin evaluating cooking quality traits in DH populations (Francis-derived lines by Koshikari, Basmati, and Khao Dawk Mali). Milestone 4 (FY 2008) <Revised> 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. - Toxin insensitive mutants identified in M2-Cypress population will be confirmed in the M3. True breeding lines will be selected for toxin sensitivity gene identification by microarray analysis, genetic mapping and complementation tests. Toxin sensitivity genes will be identified by linkage analysis in F2 mapping population and targeted for fine scale mapping. DH populations will be scored for association between quality traits and microsatellite markers to identify linkage blocks associated with cooking quality. Objective 2 Milestone 2 (FY2006) Complete and publish confirmation of trisomic lines using BAC clones. Publish molecular information on Oryza spp. Select cross for ABC (advanced backcross) population. Survey databases for markers associated with NBS-LRR (nucleotide binding site-leucine rich repeat) regions. - Verify the available trisomic lines with BAC clones will identify the lines more accurately, then these lines can be used to facilitate incorporation of desirable genes. ABC populations will be developed to identify novel disease resistance. ABC rather than RIL (recombinant inbred lines) populations will be developed so that novel resistance genes can be identified more easily. Identification of NBS- LRR regions will aid in the identification of novel disease resistance genes. Milestone 3 (FY2007) Enter trisomic lines into the genetic stocks collection. Identify useful markers associated with the NBS-LRR regions to use in germplasm development. - Trisomic lines will be available for distribution through the GSOR (Genetic Stocks Oryza) collection. Identification of NBS-LRR regions and associated microsatellite markers will aid in identification of novel blast and/or sheath blight resistance genes. Milestone 4 (FY2008) Publish information obtained from the ABC population(s). Begin to make selections from the ABC population(s) for germplasm release. - Making selected lines available as a germplasm release will allow US rice breeders to have access to novel disease resistance genes that can be easily incorporated into their experimental breeding lines for cultivar development. Objective 3 Milestone 2 (FY2006) 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. - Lesion mimic and blast susceptible mutants will be crossed to the wild-type parents to identify loci that are involved in disease resistance. Two-hybrid library will be screened using Pi-ta and AVR-Pita as bait to identify more candidate genes. Milestone 3 (FY2007) Complete and publish characterization of interacting genes both in vivo and in vitro. - Loci identified from the crosses between the mutant and wild-type parents will be fine mapped using microsatellite markers and candidate genes identified from two- hybrid will be further analysis for their roles in disease resistance. Milestone 4 (FY2008) Complete and publish Pi-ta interacting genes - The role of candidate genes in disease resistance will be further verified by association studies and tightly linked microsatellite markers will be recommended for use by rice geneticists in marker-assisted selection. Objective 4 Milestone 2 (FY2006) Complete analysis of candidate genes. Mapping a few promising candidate genes. - Differentially expressed genes will be mapped using a recombinant inbred population that is under development. Milestone 3 (FY2007) Complete the sequence analysis of candidate genes in elite US germplasm for identification of DNA markers. - Mapped differentially expressed genes in resistance loci will be determined. Milestone 4 (FY2008) Complete DNA markers for marker-assisted selection. - Confirmed candidate genes in breeding parents will be sequenced for identifying 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 LongSerial 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.

Impacts
(N/A)

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/


Progress 10/01/03 to 09/30/04

Outputs
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 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 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 better 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 resistance (R)-genes in the Kaybonnet lpa 1-1/Zhe733 population. Milestone 2 (24 months) - Continue molecular characterization of core subset. Continue identification of R-genes in the Kaybonnet lpa 1- 1/Zhe733 population. Conduct Targeted Local Lesions in Genomes (TILLING) for R-gene identification. Attempt to identify single nucleotide polymorphism (SNP) markers for AGPase/sucrose synthase. 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 of marker(s) for starch biosynthesis. Milestone 4 (48 months) - Publish TILLING for disease resistance. Publish marker(s) for R-gene and/or starch biosynthesis. 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 recombinant inbred line (RIL) population development. Milestone 2 (24 months) - Complete and publish confirmation of trisomic lines using bacterial artificial chromosome (BAC) clones. Publish molecular information on Oryza spp. Select cross for RIL population. Survey databases for markers associated with nucleotide binding site- leucine rich repeat (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 yeast two-hybrid (Y-2H) 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 enhanced 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. 3. Milestones: A. List the milestones that were scheduled to be addressed in FY 2004. How many milestones did you fully or substantially meet in FY2004, and indicate which ones were not fully or substantially met, briefly explain why not, and your plans to do so. This is a new project, started on 06/10/2004. Not enough time has elapsed to complete all the milestones listed for the first 12 months. As part of the milestones for objective 1, a Molecular Geneticist was employed, Steven Brooks reported for duty August 23, 2004. B. List the milestones that you expect to address over next 3 years. What do you expect to accomplish, year by year, over the next 3 years under each milestone? Objective 1 Milestone 1 (FY05) Conduct molecular characterization of core subset selected in companion DB NRRC project "Use of Diverse Germplasm for Genetic Improvement of Rice". Begin identification of R-genes in the Kaybonnet lpa 1-1/Zhe733 population. Milestone 2 (FY06) Continue molecular characterization of core subset. Continue identification of R-genes in the Kaybonnet lpa 1-1/Zhe733 population. Conduct TILLING for R-gene identification. Attempt to identify SNP markers for AGPase/sucrose synthase. Milestone 3 (FY07) Publish molecular characterization of core subset. Continue TILLING for disease resistance. Develop additional mapping population(s) to identify R-genes. Continue identification of marker(s) for starch biosynthesis. Objective 2 Milestone 1 (FY05) 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 advanced backcross (ABC) population development. An ABC population will be developed rather than a RIL population to decrease the variability in the progenies. Milestone 2 (FY06) Complete and publish confirmation of trisomic lines using BAC clones. Publish molecular information on Oryza spp. Select cross for ABC population. Survey databases for markers associated with NBS-LRR regions. Milestone 3 (FY07) Enter trisomic lines into the genetic stocks collection. Identify useful markers associated with the NBS-LRR regions to use in germplasm development. Objective 3 Milestone 1 (FY05) Complete and publish pathogenicity assays of lesion mimic plants. Complete construction of the yeast two-hybrid (Y-2H) library and construct verification. Milestone 2 (FY06) 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 (FY07) Complete and publish characterization of interacting genes both in vivo and in vitro. Objective 4 Milestone 1 (FY05) Complete DNA microarray analysis and sequence of differentially expressed genes from subtracted cDNA library. Complete verification of expression of differentially expressed genes. Milestone 2 (FY06) Complete analysis of candidate genes. Mapping a few promising candidate genes. Milestone 3 (FY07) Complete the sequence analysis of candidate genes in elite US germplasm for identification of DNA markers 4. What were the most significant accomplishments this past year? A. Single most significant accomplishment during FY 2004. Identification and use of lesion mimic mutant - Lesion mimic mutants are useful for studying the mechanism of disease resistance. At the Dale Bumpers National Rice Research Center, Stuttgart, AR a lesion mimic mutant of Katy rice induced by fast neutrons was identified, analyzed and released. Genetic analysis indicates that the lesion mimic phenotype is conditioned by a single recessive gene. This mutant should be useful to researchers studying programmed cell death and defense systems in higher plants. B. Other accomplishments Prediction of blast resistance genes in wild species - In order to identify novel blast resistance genes, it is important to identify known blast resistance genes in the Oryza species. At the Dale Bumpers National Rice Research Center, Stuttgart, AR, the blast resistance gene Pi-ta was predicted in seven additional accessions that included the species O. barthii, O. nivara, and O. rufipogon; and blast resistance gene Pi-b in five accessions represented by O. barthii, O. glumaepatula, and O. rufipogon. All these species except O. glumaepatula are ancestors of cultivated rice, which indicates this blast resistance gene is a very old and stable blast resistance gene. The fact that this gene is found in only a few Oryza species accessions suggests many novel blast resistance genes, including those not found in cultivated rice, may be found in the Oryza species. Induction and use of blast-susceptible mutants - Induction of blast-susceptible mutants of Katy will allow the isolation of novel blast resistance genes. At the Dale Bumpers National Rice Research Center, Stuttgart, AR, 42 blast-susceptible mutants of Katy were identified from a fast neutron mutagenized population. Mutants that exhibit different susceptibilities to blast were identified. These mutants should be useful to researchers studying the molecular mechanisms of disease resistance. Identification of expressed genes after sheath blight inoculation - Identification of a large number of expressed genes after inoculation with the sheath blight pathogen will lead to the development of molecular strategies to control the sheath blight disease. At the Dale Bumpers National Rice Research Center, Stuttgart, AR, 200 expressed rice and fungal genes were identified. Some of these genes appear to be involved in signal recognition and transduction at the interface of rice and R. solani, the sheath blight pathogen. These genes should be useful to researchers attempting to develop methods for controlling the sheath blight disease. Construction and use of SAGE libraries - Construction of two serial analysis gene expression (SAGE) libraries will allow the identification of molecular strategies to control the rice diseases. In cooperation with Ohio State University, at the Dale Bumpers National Rice Research Center, Stuttgart, AR, 10,000 SAGE clones were isolated and approximately 100 SAGE genes were sequenced and 700 tags were identified. This information should be useful to researchers attempting to identifying genes that can be used to control the disease sheath blight. C. Significant activities that support special target populations: Served on the advisory committee for the Capacity Building Grant entitled "Scientific Instrumentation and Curriculum Development for Teaching Plant Biotechnology" awarded to the University of Arkansas at Pine Bluff, an 1890 University. As part of the review meeting, a brief summary of DB NRRC research was presented. Made presentations on "Biotechnology in Rice Improvement" as part of the "Community Outreach Symposium on Agricultural Biotechnology Arkansas Biotechnology sponsored by the University of Arkansas at Pine Bluff, Department of Agriculture/Southern AgBiotechnology Consortium for Underserved Communities Project. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Nothing to report, project began 06/10/2004. 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? Marker-assisted selection is a tool that rice breeders can use to accelerate the speed and precision of their selection for new rice varieties using molecular markers. The molecular strategies to employ four molecular markers for tagging the durable and effective blast resistance gene Pi-ta were successfully transferred to US rice geneticists and breeders. The molecular methods to employ Pi-ta should be useful to rice geneticists and breeders worldwide for identifying and selecting blast resistance lines. Hosted an Open House on July 21, 2004, for over 75 industry leaders, in which latest research results were showcased. Participated in the annual University of Arkansas Rice Field Day through poster presentations on molecular marker research with rice germplasm and research to control the rice diseases, blast and sheath blight.

Impacts
(N/A)

Publications

  • Jia, Y., Wang, Z., Singh, P., Rutger, J.N., Martin, R., Pinson, S.R. 2004. Development and characterization of rice mutant populations for functional genomics of host-parasite interactions [abstract]. 15th International Plant Protection Congress Proceedings. p. 64.
  • Jia, Y., Wang, Z., Fjellstrom, R.G., Moldenhauer, K.A., Azam, M., Correll, J., Lee, F.N., Xia, Y., Rutger, J.N. 2004. Rice pi-ta gene is closely linked with resistance to the major pathotypes of the rice blast fungus in the U.S. Phytopathology. 94:296-301.
  • Jia, Y., Wang, Z., Singh, P., Redus, M., Fjellstrom, R.G., Johnson, V., Correll, J., Lee, F., Rutger, J.N. 2004. Rice Pi-ta gene confers resistance to the major pathotypes of the rice blast fungus in the U.S. [abstract]. Annual International Plant & Animal Genome Conference. Abstract P293, p. 145.
  • Jia, Y., Rutger, J.N., Wang, Z., Singh, P., Martin, R., Pinson, S.R. 2004. Development and characterization of rice mutant populations for functional genomics of host-parasite interactions. American Phytopathological Society Annual Meeting. Phytopathology. 94(6):47.
  • Jia, Y., Singh, P., Winston, E.M., Wamishe, Y., Correll, J., Valent, B. 2004. Molecular evolution of rice Pi-ta gene and fungal Magnaporthe grisea AVR-Pita gene. American Phytopathological Society Annual Meeting. Phytopathology. 94(6):47.
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