Brief Summary of Minutes of Annual Meeting

2009 Outcomes " Determine specific genetic fingerprints of tested cultivars through the use of microsatellite markers and find loci useful for parentage analysis (Fulbright) " Refine the genetic linkage and genome sequence maps for map-based cloning of fungal vic and pathogenicity genes " Tom Kubisiak used the C. parasitica Genome sequence generated by the JGI Community Sequencing Program to identify 689 simple sequence repeats (SSRs) and designed 141 primer pairs. One hundred and thirty Four of the 141 primer pairs amplified discrete products, with 96 of the 134 showing polymorphism for the JA17 and X17.8 parents of the mapping cross. Allele data were generated for 96 progeny from the mapping cross for 32 polymorphic SSRs and a total of 30 of these markers were placed within the context of the published C. parasitica linkage map (Kubisiak and Milgroom, 2006, FG&B 43:453-463). The new linkage data were used by the JGI finishing group to connect a number of scaffolds in Version 1 of the genome assembly to generate Version 2 that was released July 10, 2009. Ï Complete assembly and community manual annotation of the C. parasitica genome sequence Nine-hundred and ninety-five of 11,251 transcripts have been manually curated by the C. parasitica annotation team to date. Version 2 of the genome assembly has been released by the JGI consisting of 26 scaffolds. Five of the new scaffolds contain two teleomers and are of a length consistent with a complete chromosome. Six additional scaffolds contain one teleomer and are in excess of 1 MB in length. The assembly release Version 2 of whole genome shotgun reads was constructed with the Arachne assembler and improved with finishing reads. This release contains 26 main genome scaffolds totaling 43.9j Mb. Five scaffolds are considered complete teleomere on one end. The remaining 15 scaffolds are smaller and do not contain teleomers. Roughly half of the genome is contained in four scaffolds all at least 5.1 Mbp in length. Annotation of Version 2 assembly was produced by the JGI Annotation Pipeline using a variety of homology-based and ab initio predictors. The Version 1 Gene Catalog and its manual curations also were mapped to the Version 2 assembly and were included in the filtering procedure that determined the initial Version 2 Gene Catalog. After filtering for EST support, completeness and homology support, a total of 11, 609 genes were structurally and functionally annotated. Ï Use the C. parasitica genome sequence to develop new microarray chip and proteomics platforms for analysis of global gene expression in the blight fungus when challenged by viral pathogens " Orchard established in WV with advanced, back-cross chestnut trees from VA for assessment of host resistance with hypovirulence in the Cryphonectria parasitica populationreplanted in 2009. 2010 Outcomes Ï Complete characterization of C. parasitica antiviral RNA silencing pathways. Two Dicer genes were identified in the C. parasitica genome, cloned and disrupted. Dicer DCL2 was shown to be required for antiviral RNA silencing while Dicer DCL1 was not required (Segers et al., PNAS 2007, 104:12902-12906). Four Argonaute genes were identified in the C. parasitica genome, cloned and disrupted. Only Argonaute AGL2 was required for antiviral RNA silencing (Sun et al., PNAS, IN PRESS). Three RNA dependent RNA polymerases and an orthologue of the QIP exonuclease involved in transgene silencing in Neurospora crassa have been identified in the C. parasitica genome and are currently being cloned and disrupted. The C. parasitica gene oah, encoding the enzyme Oxaloacetate acetylhyrdolase (OAH), a member of the PEP mutase (PEPM)/isocitrate lysase (ICL) superfamily, that catalyzes the hydrolysis of oxaloacetate to oxalic acid and acetate, was cloned, characterized and disrupted. Knockout of the oah gene reduced the ability to form cankers on chestnut trees, which suggest that the enzyme plays a key role in virulence. Completion of milestones not proposed: " A proposal to sequence the C. parasitica genome was approved by the Department of Energy Community Sequencing Program in June of 2006. The assembled 8.5 X C. parasitica genome sequence was released to the public on September 30, 2008. This is a tremendous resource for future studies on the chestnut blight fungus and its interaction with the chestnut tree. " Demonstration that hypovirus p29 suppresses RNA silencing in C. parasitica and in heterologous plant system. This is the first report of a mycovirus-encoded suppressor of RNA silencing. Segers, G.C., van Wezel, R., Zhang, X., Hong, Y. and Nuss, D.L. Hypovirus Papain-like protease p29 suppresses RNA silencing in the natural fungal host and in a heterologous plant system. Eukaryotic Cell 5:896-904, 2006. " Demonstrated that RNA silencing serves as an antiviral defense mechanism in C. parasitica (first example for any fungus) against hypoviruses and mycoreoviruses. Segers, G.C., Zhang, X., Deng, F., Sun, Q. and Nuss, D.L. Evidence that RNA silencing functions as an antiviral defense mechanism in fungi. PNAS USA 104:12902-12906, 2007. " First report of the cloning and sequence analysis of mycovirus-derived small RNAs (vsRNAs) generated by RNA silencing. The vsRNAs were shown to be produced, in a dicer dcl-2-dependent manner, from both positive and negative hypovirus RNA strands at a ratio of 3:2 and to be non-randomly distributed along the viral genome. C. parastiica was shown to respond to mycovirus infection with a 10-15 fold increase in dcl-2 transcript acuumulation while the expression of dcl-1 was modestly increased. The expression of dcl-2 was further increased (~35 fold) following infection by a CHV1-EP713 mutant that lacks the p29 suppressor of RNA silencing. A similar response in dicer gene expression following virus infection of plants or animals has not yet been reported. In this regard, it is anticipated that the evolutionary position of fungi relative to animals and plants will provide insights into additional novel mechanisms for the induction and suppression of RNA silencing pathways yet to be revealed in the other organisms. Zhang, X., Segers, G.C., Sun, Q., Deng, F. and Nuss, D.L. Characterization of hypovirus-derived small RNAs generated in the chestnut blight fungus by an inducible DCL-2-dependnet pathway. Journal of Virology 82:2613-2619, 2008. " Virus RNA recombination is an important component of virus evolution that contributes to the emergence of new viruses and the generation of internally deleted mutant RNAs, termed defective interfering (DI) RNAs, that are derived from, and dependent on, the parental viral genomic RNA. We provided the first experimental evidence that a host RNA silencing pathway is required for DI RNA production and virus vector RNA instability for a single-strand, positive sense RNA virus. Zhang, X. and Nuss, D.L. A host dicer is required for defective viral RNA production and recombinant virus vector instability for a positive sense RNA virus. Proc. Natl. Acad. Sci. USA. Early Edition, October 13, 2008.

2009-2010 Publications Aguilar, F.X., M.M. Cernusca, and M.A. Gold. 2009. A preliminary assessment of consumer preferences for chestnuts (Castanea spp.) using conjoint analysis. HortTechnology 19: 216-223.

Anagnostakis, Sandra. 2010. Identification of chestnut trees. The Connecticut Agricultural Experiment Station. 29 Sept. 2010. .

Barakat, A., J. Carlson, M. Staton, T. Kubisiak, C. Smith, S. DiLoreto, K. Baier, M. Atkins, B. Blackmon, S. Ficklin, F. Hebard, P. Sisco, W. Powell, S. Anagnostakis, D. Nielsen, A. Abbott, N. Wheeler, and R. Sederoff. 2010. Analyses of the transcriptome of the Fagaceae species. Proceedings of Plant and Animal Genome XVIII Meeting, San Diego, California, January 9-13, 2010, W234.

Barakat, A., D.S. DiLoreto, Y. Zhang, C. Smith, K. Baier, W.A. Powell, N. Wheeler, R. Sederoff, J. E. Carlson. 2009. Comparison of the transcriptomes of American chestnut (Castanea dentata) and Chinese chestnut (Castanea mollissima) in response to the chestnut blight infection. BMC Plant Biology. 9:51 (pp. 1-11).

Carlson, J.E., A.G. Abbott, S. Anagnostakis, K. Baier, A. Barakat, J.B James, N. Islam-Faridi, S. Ficklin, F. Hebard, T. Kubisiak, C. Maynard, S.M. Merkle, W. Miller, C.J. Nairn, W. Powell, S.C. Schuster, L.P. Tomsho, T.K. Wagner and C.D. Nelson. 2010. Forest Health Initiative for American chestnut restoration. Proceedings of Plant and Animal Genome XVIII Meeting, San Diego, California, January 9-13, 2010, P523.

Chen, Chen, Q. Sun, B. Narayanan, D.L. Nuss and O. Herzberg. 2010. Structure of oxaalacetate acetylhydrolase, a virulence factor of the chestnut blight fungus. J. Biol. Chem. 285:26685-26696.

Costa, R., F. Tavares, H. Machado, S. Serrazina, L.T. Dinis, J. Gomez-Laranjo, M.S. Pais, and T. Kubisiak, T. 2009. Understanding disease resistance to Phytophthora cinnamomi in Castanea sp. Proceedings of the Genomics of Forest and Ecosystem Health in the Fagaceae (Beech Family), Nov 10-13, 2009, Research Triangle Park, NC, USA. p.32.

Donis Gonzalez. I.R., M. Mandujano, C. Medina-Mora and D.W. Fulbright. 2009. Presence of mycotoxins after 90 days of storage in fresh chestnuts. 4th International Chestnut Symposium, Beijing, China. Acta Horticulturae 844: 69-74.

Donis Gonzalez, I.R., E.T. Reyser, D. Guyer and D.W. Fulbright. 2010. Efficacy of postharvest treatments for reduction of molds and decay in fresh Michigan chestnuts. First European Chestnut Conference. Acta Horticulturae 866: 563-570.

Donis Gonzalez, I.R., E.T. Reyser, D. Guyer and D.W. Fulbright. 2010. Shell mold and kernel decay of fresh chestnuts in Michigan. First European Chestnut Conference. Acta Horticulturae 866: 353-358.

Fulbright, D.W. 2006. Chestnut culture in Italy. Annual Rept. Northern Nut Growers Association. 1-41 (published in 2010).

Fulbright. D.W., M. Mandujan and S. Stadt. 2010. Chestnut production in Michigan. First European Chestnut Conference. Acta Horticulturae 866: 531-537. Gold, M.A., M.M. Cernusca and L.D. Godsey. 2009. Agroforestry Product Markets And Marketing. Chapter 11. In: Garrett, H.E. (ed). North American Agroforestry: An Integrated Science and Practice. 2nd Edition. Agronomy Society of America, Madison, WI.

Gold, M.A. and H.E. Garrett. 2009. Agroforestry Nomenclature, Concepts and Practices. Chapter 3. In: Garrett, H.E. (ed). North American Agroforestry: An Integrated Science and Practice. 2nd Edition. Agronomy Society of America, Madison, WI.

Gold, M.A. and M.H. Hall (eds.). 2009. Agroforestry Comes of Age: Putting Science into Practice. 11th North American Agroforestry Conference Proceedings. Columbia, MO. May 31-June 3, 2009. 517 p.

Guyer, D. J. Xing, M. Mandjuano and D.W. Fulbright. 2010. Influence of selected factors on efficiency and effectiveness of a peeling machine for chestnut. First European Chestnut Conference. Acta Horticulturae 866: 595-603.

Hunt, K.L., M.A. Gold and M.R. Warmund. 2008. Status of Chinese Chestnut (Castanea mollissima) Cultivar Performance in Missouri. 4th International Chestnut Congress Sept. 25-28, 2008, Beijing, China.

Islam-Faridi, N., C.D. Nelson, P.H. Sisco, T.L. Kubisiak, F.V. Hebard, R.L. Paris and R.L. Phillips. 2009. Cytogenetic analysis of American chestnut (Castanea dentata) using fluorescent in situ hybridization. Acta Hort. (ISHS) 844:207-210.

Liang, H, P.A. Kumar, V. Nain, W.A. Powell, J.E. Carlson. Selection and Screening Strategies. Chapter 4. In: C. Kole, C.H. Michler, A.G. Abbott and T,C. Hall (eds) Transgenic Crop Plants: Volume 1: Principles and Development. Springer-Verlag  Berlin, Heidelberg, New York, Tokyo, 2010, pp 85-144.

Lin, Y-C., J-M. Frigerio, C. Cabane, C. Bodenes, A. Daruvar, P. Rouze, M. Staton, A. Barakat, T. Kubisiak, A. Kremer, C. Plomion and Y. Van de Peer. 2009. In silico screening of conserved orthologous sequences (COS) from Fagaceae ESTs resources. Proceedings of the Genomics of Forest and Ecosystem Health in the Fagaceae (Beech Family), Nov 10-13, 2009, Research Triangle Park, NC, USA. Poster No.17, p.54.

Medina-Mora, C.M. and D.W. Fulbright. 2010. Evaluation of simple sequence repeats (SSR) for genetic analysis of chestnut trees in Michigan orchards. First European Chestnut Conference. Acta Horticulturae 866: 127-133.

Milgroom, M.G., K. Sotirovski, M. Risteski and M.T. Brewer. 2009. Heterokaryons and recombinants of Cryphonectria parasitica in two clonal populations in southeastern Europe. Fungal Genet. Biol.46: 849-854.

Nelson, C.D., J.E. Carlson, S.A. Merkle, W. Powell, C.J. Nairn, C. Maynard, T.L. Kubisiak, F.V. Hebard, S. Anagnostakis and A. Abbott. 2009. Biotechnological tools for accelerating species restoration. Proceedings of the Genomics of Forest and Ecosystem Health in the Fagaceae (Beech Family), Nov 10-13, 2009, Research Triangle Park, NC, USA. p.39.

Salamon, J.A., R. Acuña and A. L. Dawe. 2010. Phosphorylation of phosducin-like protein BDM-1 by protein kinase 2 (CK2) is required for virulence and Gb subunit stability in the fungal plant pathogen Cryphonectria parasitica. Mol. Microbiol. 76: 848-860.

Sederoff, R., N. Wheeler, J. Carlson, A. Barakat, W. Powell, K. Baier, T. Kubisiak, P. Sisco, F. Hebard, S. Anagnostakis, J. Tomkins, A. Abbott, M. Staton, C. Smith, D. Nielsen and S. Ficklin. 2010. A genome platform for chestnut. Proceedings of Plant and Animal Genome XVIII Meeting, San Diego, California, January 9-13, 2010, W253.

Sederoff, R., N. Wheeler, J. Carlson, A. Barakat, W. Powell, K. Baier, T. Kubisiak, P. Sisco, F. Hebard, S. Anagnostakis, J. Tomkins, A. Abbott, M. Staton, C. Smith, D. Nielson and S. Ficklin. 2009. The chestnut genome project: looking ahead, opportunities, and challenges. Proceedings of the Genomics of Forest and Ecosystem Health in the Fagaceae (Beech Family), Nov 10-13, 2009, Research Triangle Park, NC, USA. p.7.

Sisco, P.H., R.R. Sederoff, J.P. Tomkins, J.E. Carlson, T.L. Kubisiak, M.E. Staton, F.V. Hebard, S.L. Anagnostakis, W.A. Powell and C.P. Smith. 2009. The United States National Science Foundation project on developing tools for the study of the Fagaceae: Castanea, Quercus, and Fagus. Acta Hort. (ISHS) 844:267-274.

Spinger, J.C., A.L.D. Baines and A.M. Jarosz. 2009. Hypovirus mediated recovery of blight infected American chestnut trees in Michigan. Phytopathology 99: S123.

Springer, J.C., M.T. Chansler, A.L.D. Baines and A.M. Jarosz. 2010. Vegetative incompatibility diversity in Michigan populations of the chestnut blight fungus, Cryphonectria parasitica. 95th Meeting of the Ecological Society of America, Pittsburgh, PA.

Staton, M., D. Henry, M. Monsanto, S. Ficklin, C-H. Cheng, G., Fang, B. Blackmon, A.Barakat, J. Tomkins, F. Hebard, T. Kubisiak, J. Carlson, R. Sederoff, R. and A. Abbott. 2009. Comparative genome analysis of Chinese chestnut to model plant species and Fagaceae family species. Proceedings of the Genomics of Forest and Ecosystem Health in the Fagaceae (Beech Family), Nov 10-13, 2009, Research Triangle Park, NC, USA. p.20.

Sun, Qihong, G.H. Choi and D.L. Nuss. 2009. A single argonaute gene is required for induction of RNA silencing antiviral defense and promotes viral RNA recombination. Proc. Nat. Acad. Sci. 106:17927-17932.

Valdivia, C., L. Zabek, J. Arbuckle, M. Gold and C. Flora. 2009. Human and Institutional Dimensions of Agroforestry. Chapter 13. In: Garrett, H.E. (ed). North American Agroforestry: An Integrated Science and Practice. 2nd Edition. Agronomy Society of America, Madison, WI.

Wheeler, N., D. Nielson, C. Smith, J. Tompkins, A. Abbott, M. Atkins, B. Blackmon, M. Staton, S. Ficklin, F. Hebard, P. Sisco, J. Carlson, A. Barakat, W. Powell, K. Baier, S. Anagnostakis, T. Kubsiak, C. Burdine and R. Sederoff. 2009. Annotated EST-based SSR and SNP genetic maps for chestnut. Proceedings of the Genomics of Forest and Ecosystem Health in the Fagaceae (Beech Family), Nov 10-13, 2009, Research Triangle Park, NC, USA. p.11.