2007 Annual Report 1a.Objectives (from AD-416) To develop new knowledge about the genetics, genomics, and transgenics of selected tropical crops by completely sequencing and characterizing the non-recombinant region of the papaya sex chromosome, producing a draft genomic sequence of the entire papaya genome, characterizing a set of papaya's flower organ and disease resistance genes, developing an improved ability to regulate gene expression, and producing and evaluating new transgenic approaches for resistance to papaya ringspot virus disease. Results will include the development of a papaya genomics database, a high-density genetic map combining AFLPs and microsatellites with markers flanking major genes controlling fruit size and disease reaction. Markers developed for genetic and physical mapping and marker assisted selection in papaya will be shared with researchers worldwide. The genetic maps generated from different mapping populations will be linked together by a common set of microsatellite markers. Results of the proposed research will significantly advance the development of genomic tools and knowledge for papaya improvement. The genetic resources generated from this project will enhance the capacity for positional cloning of important novel traits from this little-studied tropical fruit crop. 1b.Approach (from AD-416) (1) Fingerprint and end-sequence approximately 40,000 clones from our existing bacteria artificial chromosome (BAC) library for anchoring the whole genome shotgun (WGS) sequence data that will be produced from two WGS libraries of the papaya genome, (2) mine the papaya BAC end and genomic sequences to develop 4,000 microsatellite markers (simple sequence repeats or SSRs) for constructing a high density genetic map of the papaya genome of at least 1,000 SSRs for combining with our amplified fragment length polymorphism (AFLP) map, (3) assemble and annotate the papaya genome sequences, (4) select a core set of evenly distributed SSRs to map major genes controlling fruit size and disease reactions, (5) develop a transient gene silencing system for functional genomic analysis in papaya, (6) characterize novel papaya disease resistance genes with the functional genomic tool, (7) determine the relationship between transgene copy number and gene silencing, (8) characterize the activity of SCYLV P0 and other viral suppressors of post-transcriptional gene silencing (PTGS) in Nicotiana benthamiana as a model system for application to sugarcane, (9) identify papaya genes with tissue-specific expression patterns for developing tissue-specific promoters, (10) use segmented and synthetic gene technology to develop and subsequently characterize transgenic papaya with resistance to wide range of papaya ringspot virus (PRSV) strains, (11) measure the extent, if any, of gene flow from commercial transgenic papaya to adjacent nontransgenic papaya fields, (12) develop and commercialize a transgenic Kapoho with segmented coat protein genes for the Hawaiian papaya industry, (13) develop data that are necessary to have the Rainbow transgenic papaya deregulated in Japan, and (14) develop, transfer, and commercialize transgenic papaya for developing countries with focus on Bangladesh. 4.Accomplishments Characterization of a carotenoid biosynthetic gene of papaya fruit opens the way for improving fruit quality. Depending on the cultivar, papaya fruit may be the yellow color of carotenoids or it may be the red color of lycopenes. Since carotenoids are the source of human vitamin A and lycopenes are antioxidants that help prevent cancers, there are nutritional and health implications tied to the color of papaya fruit flesh. ARS scientists at the U.S. Pacific Basin Agricultural Research Center, in collaboration with researchers from the Hawaii Agricultural Research Center, the University of Hawaii, and the University of Illinois isolated and characterized a key gene regulating the carotenoid biosynthetic pathway. This work has potential to improve the nutritional quality of papaya and will be used for understanding the genetic linkage between fruit color and fruit flesh firmness, a post-harvest characteristic important for shipping and handling. NP 302 Component II, Biological Processes that Improve Crop Productivity and Quality, Problem Area c) Developing High-Value Products. A papaya bacterial artificial chromosome (BAC) library of papaya is developed as a valuable genomic tool and surprisingly reveals a closer relationship to distantly related poplar than to closely related arabidopsis. BAC libraries have become important in crop genomics for constructing physical maps, mapping genes of agricultural importance, performing comparative genomics between crop species, and analyzing genome structure. ARS scientists from Hilo, HI collaborated with scientists from the University of Hawaii, the Hawaii Agriculture Research Center, the University of Hawaii, and analyzed the DNA sequences of more than 50,000 papaya BAC ends to reveal a plant genome containing all of the major repeat classes of DNA, a protein coding content very similar to that of Arabidopsis, a large number of useful microsatellites, and a surprising amount of co-linearity with the genome of poplar, the model tree for DNA sequence analysis. The papaya BAC end sequences will provide a valuable resource for future physical mapping and accelerate the construction of a new generation of genetic maps to assist in crop improvement. NP 301 Component II, Genomic Characterization and Genetic Improvement, Problem Area c) Genetic Analyses and Mapping of Important Traits. Characterizing the effect of SCYLV P0 on siRNAs advances our understanding of RNA silencing in plants and viral suppressors of RNA silencing. Dicot-infecting polerovirus P0 proteins suppresses local gene silencing but do not affect systemic gene silencing. The sugarcane yellow leaf virus P0 protein (SCYLV P0) suppresses local gene silencing, but in addition, and in contrast to the dicot P0 proteins, it suppresses systemic silencing and induces cell death in Nicotiana benthamiana ARS researcher at Hilo, HI carried out deletion analysis and revealed that elimination of 15 amino acids at the C terminus of SCYLV P0 abolishes both the suppression of systemic silencing and the induction of cell death, leaving the activity of the truncated protein similar to the dicot P0s. Further analysis of SCYLV P0 to identify interacting host proteins will allow a deeper understanding of plant anti-viral defense mechanisms and viral countermeasures. NP 301 Component II, Genomic Characterization and Genetic Improvement, Problem Area c) Genetic Analyses and Mapping of Important Traits. Towards a Virus Induced Gene Silencing (VIGS) system for papaya: a functional genomics tool. With the completion of the draft papaya genome sequence, systems for studying the functions of the newly sequenced genes will be of great importance. VIGS is one tool for functional genomics that has proved very useful in numerous other plant systems. To construct a VIGS vector for papaya, scientists in Hilo, HI cloned the entire genome of Papaya ringspot virus (PRSV) and made 2 types of infectious clones. Efforts are underway to test the infectivity of these clones. Future work includes modifying these clones to facilitate insertion of papaya genes for functional studies. NP 301 Component II, Genomic Characterization and Genetic Improvement, Problem Area c) Genetic Analyses and Mapping of Important Traits. Testing transgenic papaya lines for resistance to PRSV from India, Bangladesh, and Mexico. Strains of PRSV from several locations in India, Bangladesh and Mexico were tested against the commercialized SunUp and Rainbow papaya and a number of transgenic papaya lines with segmented coat protein genes. Tests were done at Cornell University through an SCA with Dr. Marc Fuchs because it is risky to import strains of PRSV into Hawaii. All transgenic papaya were overcome by India PRSV isolates. PRSV isolates from Bangladesh gave mixed results, as some overcame resistance of all transgenic lines but one strain in particular showed delayed infection and mild symptoms. SunUp and some selected segmented gene lines showed resistance to PRSV from Mexico. These results suggest that the present segmented gene lines will not work in India, may work in parts of Bangladesh, and should work in Mexico. With the latter, the transgenic papaya lines could serve as germplasm to create new papaya varieties for controlling PRSV in Mexico. A patent was obtained for the multiple virus resistance using segmented gene approach. NP 301 ComponentII, Genomic Characterization and Genetic Improvement, Problem Area c) Genetic Analyses and Mapping of Important Traits. Testing of RO papaya lines with synthetic genes for resistance to PRSV. Several R0 lines with synthetic genes were tested against PRSV from Hawaii in collaboration with Steve Ferreira of University of Hawaii. One line looked promising and was transplanted to the field for seed production. Seeds will be produced and the seedlings tested for resistance against PRSV from Hawaii. R0 synthetic transgenic lines produced for the Bangladesh project are being propagated and will be grown in the field for seeds and seedlings will be subsequently tested for resistance. A patent was obtained for the synthetic gene approach. NP 301 ComponentII, Genomic Characterization and Genetic Improvement, Problem Area c) Genetic Analyses and Mapping of Important Traits. Testing of papaya lines containing PRSV coat protein genes and genes that govern fruit softening. A number of R0 lines with PRSV resistance and fruit softening genes were tested for resistance to PRSV in collaborative work with Steve Ferreira of University of Hawaii. Seeds were obtained from resistant lines, and selected lines will be planted in the field in an effort to identify potential virus resistant lines that may show delay in fruit softening. Delay in fruit softening is an important characteristic for improving the shipping and shelf life of papaya. A patent was obtained for delayed softening of papaya. NP 301 ComponentII, Genomic Characterization and Genetic Improvement, Problem Area c) Genetic Analyses and Mapping of Important Traits. Selection of segmented gene lines for deregulation in the U.S. Two transgenic ‘Kapoho’ lines with segmented genes were selected for deregulation in collaborative work with Steve Ferreira of University of Hawaii. The two lines are in the R3 stage and show resistance and good fruit and horticultural characteristics. These lines will be a valuable addition to Hawaii as as trangsnenic Kapoho has not been developed. These transgenic lines may help to guard against new strains of PRSV that may be inadvertently introduced into Hawaii. NP 301 ComponentII, Genomic Characterization and Genetic Improvement, Problem Area c) Genetic Analyses and Mapping of Important Traits. Bangladesh transgenic papaya project. This work had been supported by USAID with the ultimate objective of developing transgenic papaya to help the rural people of the country. An application for the introduction and confined field testing of selected transgenic lines in Bangladesh was prepared by our lab and sent to BARI (Bangladesh Agricultural Research Institute) for submission to the proper governmental agencies. Unfortunately, the application was not processed through all of the steps, and the grant ended at the end of June 2007. We will not continue work on this project unless new funding is made available and unless the authorities provide a reasonable outlook for acceptance of an application for introducing and confined field testing of transgenic in their country. NP 301 ComponentII, Genomic Characterization and Genetic Improvement, Problem Area c) Genetic Analyses and Mapping of Important Traits. Deregulation efforts of transgenic SunUp papaya in Japan. The revised application package for submission to the Ministry of Health Labor and Welfare for deregulation of the SunUp papaya in Japan is complete except for the characterization of the gentamycin gene fragment insert that was detected by southern blot of SunUp DNA. However, we have failed to detect the insert and its papaya border sequences by a directed PCR approach, by analysis of a BAC library that was produced by Ray Ming and Paul Moore’s group, and by analysis of a shotgun sequence library produced by the group of Maqs Alam have given negative results. As an added approach, a fosmid library was developed of SunUp DNA and this library is being screened for the gentamycin gene fragment insert. This last part of the Japan deregulation package has been very difficult to complete. NP 301 ComponentII, Genomic Characterization and Genetic Improvement, Problem Area c) Genetic Analyses and Mapping of Important Traits. 6.Technology Transfer Number of U.S. patents granted 5 Number of non-peer reviewed presentations and proceedings 17 Review Publications Gonsalves, D., Vegas, A., Prasartsee, V., Drew, R., Suzuki, J., Tripathi, S. 2006. Developing Papaya to Control Papaya Ringspot Virus By Transgenic Resistance, Intergeneric Hybridization, and Tolerance Breeding. Plant Breeding Reviews. 26:35-781. Klas, F.E., Fuchs, M., Gonsalves, D. 2006. Comparative spatial spread over time of Zucchini Yellow Mosaic Virus (ZYMV) and Watermelon Mosaic Virus in fields of transgenic squash expressing the coat protein genes of ZYMP and WMV, and in fields of nontransgenic squash. Transgenic Research. 15:527-541. Lai, C., Yu, Q., Hou, S., Skelton, R., Jones, M., Lewis, K., Murray, J., Eustice, M., Guan, P., Agbayani, R., Moore, P.H., Ming, R., Presting, G. 2006. Analysis of papaya BAC end sequences reveals first insights into the organization of a fruit tree genome. Molecular Genetics and Genomics. 276:1-12. Ling, K., Zhu, H., Petrovic, N., Gonsalves, D. 2007. Sensitive Detection of Grapevine Leafroll Virus-2 with Elisa Using an Antiserum Against the Recombinant Coat Protein. Journal of Phytopathology. (2007) 155:65-69. Mccafferty, H., Zhu, Y.J., Moore, P.H. 2006. Improved Carica papaya tolerance to the carmine spider mite by expression of Aanduca sexta chitinase transgene. Transgenic Research. 15:337-247. Ming, R., P.H. Moore 2007 Genomics of sex chromosomes. Current Opinion in Plant Biology. 10:123-130. Ming, R., Wang, J., Moore, P.H., Paterson, A. 2007. Sex chromosomes in flowering plants. American Journal of Botany. 94(2): 141-150. Sakuanrungsirikul, S., Sarindu, N., Prasartsee, V., Chaikiatiyos, S., Siriyan, R., Sriwatanakul, M., Lekananon, P., Kitprasert, C., Boonsong, P., Kosiyachinda, P., Fermin, G., Gonsalves, D. 2005. Update on the development of virus-resistant papaya: Virus-resistant transgenic papaya for people in rural communities of Thailand. Food and Nutrition Bulletin. 26(4):422-426. Skelton, R.L., Yu, Q., Scrinivasan, R., Manshardt, R., Moore, P.H., Ming, R. 2006. Tissue differential expression of lycopene beta-cyclase gene in papaya. Cell Research. p.731-739. Ming, R., Wu, K., Moore, P.H., Patterson, A.H. 2006. Sugarcane genomics and breeding. In: Lamkey and Lee (eds). Plant Breeding: The Arnel R. Hallauer Symposium. Blackwell Publishing. Chap 20. pp. 283-292. Yu, Q., Hou, S., Hobza, R., Feltus, F.A., Wang, X., Jing, W., Blas, A., Lemke,C., Saw, J.H., Moore, P.H., Alam, M., Jiang, J., Paterson, A.H., Vyskot, B., Ming, R. 2007. Chromosomal location and gene paucity of the male-specific region on papaya Y chromosome. Mol Genetics Genomics 278:177-185. Gonsalves, C., Lee, D.R., Gonsalves, D. 2007. The adoption of genetically modified papaya in Hawaii and its implications for developing countries. Journal of Developmental Studies 43:177-191. Klas, F., Fuchs, M., Gonsalves, D. 2007. Geostatistical analysis of spatial virus spread overtime provides new insights into the environmental safety of commercial virus-resistant squash. Information Systems for Biotechnology News Report May 2007, 2-8. Zhou, F., Wang, M., Albert, H.H., Moore, P.H., Zhu, Y.J. 2006. Efficient transient expression of human gm-csf protein in nicotiana benthamiana using potato virus x vector. Applied Microbiology and Biotechnology 72:756:762. Zhu, Y.J., Agbayani, R., Moore, P.H. 2007. Ectopic expression of the Dahlia merckii defensin peptide DmAMP1 improves resistance against Phytophthora palmivora by reducing pathogen vigor. Planta 226:87-97. Gonsalves, D. 2006. Transgenic Papaya: Development, Release, Impact, and Challenges. Advances in Virus Research. 67:317-354.