2008 Annual Report 1a.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. 1b.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. 3.Progress Report 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) 4.Accomplishments 1. Stacking resistance genes to the rice blast fungus: Pyramiding resistance genes with overlapping resistance to a wide range of races of blast is essential for developing disease-resistant cultivars. Scientists at USDA-ARS Dale Bumpers National Rice Research Center in Stuttgart, AR, analyzed the reaction of two major blast resistance genes Pi-ta and Pi-k to 11 races of the rice blast fungus using a recombinant inbred line population derived from a cross of a US cultivar Katy and a breeding line RU9101001. Results demonstrated that Pi-ta confers resistance to races IA-45, IB-1, IB-45, IB-49, IC-17, IG-1, ID-1, IE-1, and IH-1; and Pi-k confers resistance to IA-45, IB-45, IG-1, and IH-1. These results suggest that it is useful to stack both Pi-ta and Pi-k into advanced breeding lines to provide long-lasting resistance to the rice blast fungus. (NP 301, Component 2C) 2. Identification of a new component in plant disease resistance: The Pi-ta resistance gene is predicted to interact with the pathogen avirulence gene AVR-Pita in triggering resistance response to blast disease. However, it is unknown if another rice gene is also required in this interaction. Scientists at the USDA-ARS Dale Bumpers National Rice Research Center in Stuttgart, AR, created rice genetic stocks using fast neutron irradiation, and mapped a nuclear gene Ptr(t) required for Pi-ta mediated signaling recognition and transduction. Ptr(t) was mapped at the Pi-ta region and was found to co-segregate with Pi-ta along with a 9 megabase genomic region of the chromosome 12. Identification of rice mutants and mapped Ptr(t) gene are important resources to clone Ptr(t). These results advance our understanding of molecular mechanisms of Pi-ta-mediated signal recognition and transduction that lead to complete resistance. (NP 301 Component 2C; NP 303 Component 3C) 3. Molecular markers for a virulent race of the rice blast fungus in the US: A virulent race of the rice blast fungus has been recovered from commercial rice fields and has been observed to cause significant yield losses in the southern US. Scientists at the USDA-ARS Dale Bumpers National Rice Research Center in Stuttgart, AR, identified and mapped two major resistance genes named as Pi41(t) and Pi42(t) to this race using a recombinant inbred line population derived from a cross of a genetic stock Kaybonnet lpa1-1 with a newly introduced resistant germplasm, Zhe733, from China. Results demonstrated that both Pi41(t) and Pi42(t) confer resistance to this race as well as to the other 10 common races of the rice blast fungus found in the US. DNA markers linked to these two resistance genes can be used in marker-assisted breeding to develop improved cultivars. Recombinant inbred lines containing either Pi41(t) or Pi42(t) can be used for fine mapping and cloning to better understand the mode of action of these resistance genes. (NP 301, Component 2C) 4. A new host of the rice blast fungus: Epidemics of rice blast disease in the southern US are erratic, thus understanding the primary source of overwintering inoculum of the pathogen is important. Scientists at the Dale Bumpers National Rice Research Center in Stuttgart, AR, determined that Carolina foxtail, a winter annual grass species, can be a host for rice blast fungus under favorable conditions. Results showed that symptoms of blast on this weed produce atypical blast lesions, but several other common weeds, rice flatsedge, yellow mutsedge, northern jointvetch and palmleaf morning glory, were not hosts for the rice blast fungus. This demonstrates that the blast fungus is capable of using a common weed as a means to overwinter, providing inoculum to infect rice in subsequent cropping seasons. This information is useful for developing effective disease management strategies. (NP 301, Component 2C; NP 303, Component 3C) 5. Expression profiles of rice in response to the sheath blight pathogen: Large-scale expression analysis of rice genes that respond to infection by the sheath blight pathogen allows the identification of resistance genes that are induced. Scientists at the USDA-ARS Dale Bumpers National Rice Research Center in Stuttgart, AR, identified genes regulating the response to sheath blight using DNA microarray and sequence analysis of 20,000 tags using a robust long serial analysis of gene expression (SAGE). This is the first identification of important pathways of rice in response to the infection of Rhizoctonia solani. This accomplishment lays a foundation for mapping and tagging critical genes that can be used for preventing sheath blight disease. (NP 301, Component 2C; NP 303, Component 3C) 6. Improved phenotyping method for studying resistance to sheath blight: Current methods of evaluation require relatively large amounts of seed, and thus screening is postponed until the later stages of breeding. 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 U.S. rice production. Scientists at the Dale Bumpers National Rice Research Center, in Stuttgart, AR, developed an improved method, one that is a modified unpublished method used in Bangladesh, and extended this technology for evaluating US germplasm. This method will fill a critical need for fast and reliable data for evaluating for sheath blight resistance because it saves time, money, and space, and facilitates screening of large numbers of accessions and breeding lines. These evaluation methods can also be modified to evaluate other diseases on other crops caused by blast and sheath blight pathogens. (NP 301, Component 2C; NP 303, Component 3C) 7. Identification of major regulatory genes: The US farmers prefer to plant rice earlier to save water; however, rice is susceptible to cold, and early planting would require cold-tolerant rice cultivars. Genetic mechanisms and molecular profiles of cold tolerance are unclear. Scientists at the Dale Bumpers National Rice Research Center, in Stuttgart, AR, were involved in designing DNA microarray chips to identify genes regulating the responses to cold that are associated with cold tolerance at the seedling stage in rice. This accomplishment has laid the foundation for the development of rice cultivars with enhanced resistance to cold, along with new tools for further dissection of the interaction between biotic stress response pathways. (NP 301, Component 2C; NP 303, Component 3C) 8. Identification of resistance to rice grain smuts: False smut and kernel smut represent important emerging and chronic diseases of rice (respectively), and no sources of resistance were known in U.S. rice cultivars. At the Dale Bumpers National Rice Research Center in Stuttgart, AR, in cooperation with the University of Arkansas, resistance to kernel smut has been identified and confirmed. Furthermore, a potential source of resistance to false smut was identified, and will be confirmed in future season(s). These newly identified sources of smut resistance will be used to identify the corresponding disease resistance genes. (NP 301, Component 2C; NP 303, Component 3C). 9. Identification of cultural management practices that mitigate the severity of false smut and kernel smut of rice: The severity of rice grain smuts is highly influenced by environmental factors, including cultural crop management. A thorough understanding of the genotype x management interactions is necessary in order to clearly evaluate disease resistance in rice germplasm. Simultaneously, management practices that reduce disease incidence on susceptible cultivars can be identified. At the Dale Bumpers National Rice Research Center in Stuttgart, AR, in cooperation with the University of Arkansas, the effects of crop rotation, tillage, fertility, and irrigation on smut severity were evaluated. Specific management schemes were identified to (1) eliminate false smut incidence, (2) minimize kernel smut severity to very low levels, and (3) maximize disease pressure for resistance evaluation. (NP 301, Component 2c; NP 303, Component 3A) 10. Cloning of a new allele for pericarp color in rice: The Rc locus controls pigmentation of the rice bran layer. Selection for white pericarp (rc) occurred during domestication of the crop, and the allele is now ubiquitous among white-bran cultivars worldwide. Researchers at the Dale Bumpers National Rice Research Center in Stuttgart, AR, in cooperation with the University of Arkansas, recently identified a reversion mutant allele at the Rc locus. The Rc-g allele has immediate value for specialty red rice production as it fits a niche rice market and possesses none of the non-desirable traits associated with Rc in exotic germplasm. (NP301 Component 2C) 11. Adequate molecular diversity identified in a collection of rice and its wild relatives for association mapping of phenotypic traits: Use of marker-trait association mapping techniques enables DNA markers to be associated with agronomically important traits, like pest resistance, without the time-consuming development of mapping populations. To apply marker-trait association mapping strategies the appropriate molecular diversity must be present in the collection of rice accessions. At the Dale Bumpers National Rice Research Center in Stuttgart, AR, the molecular diversity of three different groups of rice germplasm accessions representing international sources, U.S. rice varieties, and rice wild relatives, was evaluated using 176 SSR markers spread out over the entire rice genome. This statistical evaluation divided the accessions into three different clusters, representing the three original groups of rice accessions, and revealed that the molecular variation within and between the three groups was more than adequate for marker-trait association mapping techniques to ascertain possible novel genes for agronomically important traits like pest resistance. Use of association mapping techniques will substantially reduce the work and time necessary to identify DNA markers linked to agronomically desirable traits so that rice breeders can incorporate desirable genes into U.S. rice varieties more efficiently. (NP301, Component 2C) 12. DNA markers used to ascertain the background of a collection of rice wild species accessions: Rice wild species (Oryza spp.) are a source of new genes for rice improvement. It is important to know how closely related the numerous Oryza spp. accessions are so that new genes being incorporated into U.S. elite rice varieties that are currently being developed come from different backgrounds. At the Dale Bumpers National Rice Research Center in Stuttgart, AR, a collection of Oryza spp. accessions was genotyped with 123 SSR markers, and the data analyzed with cluster and structure analyses. These analyses showed the Oryza spp. accessions represented eight different backgrounds. Accessions with genes for sheath blight and blast resistance will be chosen from different backgrounds in an attempt to incorporate a diverse group of pest resistance genes. (NP301, Component 2C) 13. Purification of the southern U.S. rice cultivar, LaGrue: Molecular efforts aimed at developing mapping populations to identify molecular markers associated with phenotypic traits and DNA sequencing of parents used to develop a population require purified accessions. At the Dale Bumpers National Rice Research Center in Stuttgart, AR, a single LaGrue plant was selected from LaGrue foundation seed, based on genotyping with 120 SSR markers and the plant’s phenotype. This purified LaGrue accession is being resequenced and will subsequently be aligned with the known Nipponbare rice sequence, and is being used to develop several mapping populations. Having this purified LaGrue line available for a variety of molecular genetic studies will promote a better understanding of U.S. rice cultivars at the molecular level. Subsequently, these findings will be employed to benefit the U.S. rice industry by improving rice yield, pest resistance, and other agronomic characters using 'next generation' molecular genetic tools. (NP301, Component 2C) 6.Technology Transfer Number of Non-Peer Reviewed Presentations and Proceedings 17 Review Publications 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. 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.