Progress 05/16/06 to 09/30/10
Outputs
Progress Report 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. 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. Another critical step was completed towards the deregulation of the virus resistant genetically engineered papaya in Japan. The Ministry of Agriculture Forestry and Fisheries approved our environmental safety dossier. We obtained raw DNA sequences representing >12X � 19X coverage of the nuclear genome of Oriental fruitfly (Bactrocera dorsalis), a very important agricultural pest. This information will be used to apply molecular strategies for control of B. dorsalis and related pests. Raw DNA sequence of anthurium (Anthurium andraeanum) chloroplast was obtained for use in the development of novel floral colors. Deep genomic sequencing of the commercial genetically engineered SunUp papaya was initiated to expand the 3X draft sequence of the genome. In collaboration with Hawaiian pineapple industry we are conducting research to study flowering manipulation using genetic engineering. We obtained the Australian genetically engineered lines that are resistant to natural flowering and are currently propagating these plants under quarantine conditions. Improving synchronization of flowering is important to the Hawaii Coffee Industry because mechanical harvesters are recovering less than 50% of cherries as ripe fruits due to lack of flowering uniformity, and hand harvested operations also incur high labor cost due to multiple harvests of non-synchronized ripened cherries. We are evaluating a combination of plant growth regulators and foliar fertilizers to synchronize flowering in commercial fields in Kona, Hawaii. The etiology of newly emerging viral diseases in a commercial tomato farm on the Hamakua Coast of Hawaii was established. Potato virus Y and Tobacco Etch virus were predominantly detected in symptomatic plants. Integrated pest management strategies to control these viruses were implemented. Bacterial blight is one of most costly problems faced by the anthurium industry. Large scale screening of genetically engineered anthuriums against bacterial blight is ongoing. More than 60 new genetically engineered anthurium lines were tested this last year for resistance or tolerance to bacterial blight. In addition, 8 lines showing promise for increased tolerance have gone through more extensive testing. The efficacy of 9 disinfectants against the blight bacterium was determined to develop a short term management solution to combat bacterial blight. Three disinfectants showed promise for inhibiting the spread of the blight pathogen on cutting tools. Screening is being conducted on 55 lines of genetically engineered �Marion Seefurth� and �Midori� anthuriums for resistance to burrowing nematodes. After dracaena exports were suspended due to reniform nematode contamination, educational outreach was provided to the growers and a large scale steam sterilization method was tested in multiple systems for its effectiveness at eradicating plant parasitic nematodes in volcanic cinder media. A germplasm of economically important plant parasitic nematodes was established in vitro and in greenhouse cultures.
Impacts
(N/A)
Publications
- Paret, M. L., Cabos, R., Kratky, B. A., and Alvarez, A. M. 2010. Effect of plant essential oils on Ralstonia solanacearum race 4 and bacterial wilt of edible ginger. Plant Dis. 94:521-527.
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report 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. 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. Significant Activities that Support Special Target Populations The most critical step was completed in our efforts to deregulate the virus resistant genetically engineered (transgenic) papaya in Japan. In May 2009 the Food Safety Committee (FSC) of the Ministry of Health Labor and Welfare approved our petition on the food safety of the transgenic papaya. Following a public comment period, the FSC again confirmed the safety of the transgenic papaya in July 2009. The Ministry of Agriculture Forestry and Fisheries had previously approved our petition on the environmental safety of the transgenic papaya. We anticipate the transgenic papaya will be deregulated and approved for importation to Japan around February 2010. A 140 of the approximately 700 lines of �Marion Seefurth� and �Midori� transgenic anthurium were screened for resistance to bacterial blight. Fifteen promising lines each of Marion Seefurth and Midori were selected and are being further evaluated for bacterial blight tolerance and horticultural characteristics. Screening of additional lines is continuing. Last year, a project was initiated to use genetic engineering to develop low acid pineapple that is resistant to natural flowering that is triggered by environmental stresses. Callus production and plantlet regeneration were optimized and conditions for transforming pineapple are being optimized. Environmental data and plant tissue samples have been collected from commercial field sites on Maui and Oahu to elucidate precise conditions that induce natural flowering of pineapple. To quickly develop conditions for testing transgenic pineapple for resistance to natural flowering under field conditions, transgenic �Smooth Cayenne� will be imported into Hawaii under a collaborative project with Australia Queensland Department of Agriculture. We have completed the necessary Animal Plant Health Inspection Service and Hawaii Department of Agriculture Permits to import the transgenic pineapple. Gene flow and co-existence between transgenic and nontransgenic crops under commercial field conditions are being addressed with the virus resistant transgenic papaya. Seeds of fruit from commercial nontransgenic Kapoho fields growing adjacent to transgenic Rainbow papaya fields were sampled. Gene flow was detected in 0.9 percent of the seeds tested in the first two rows of the Rainbow fields which were a maximum of 22 feet from the transgenic field. Gene flow was not detected in rows sampled greater than 60 feet from the transgenic field. In another experiment, seeds of fruits were tested from a nontransgenic Kapoho tree that was surrounded by four transgenic Rainbow trees at a distance of five feet. The transgene was detected in 2.7 percent of the tested seeds.
Impacts
(N/A)
Publications
- Gonsalves, D., Ferreira, S., Suzuki, J., Tripathi, S. 2008. Papaya. In: Kole, C., Hall, T.C., editors. Compendium of Transgenic Crop Plants. Oxford UK:Blackwell Publishing. p. 131-162.
- Tripathi, S., Suzuki, J., Ferreira, S., Gonsalves, D. 2008. Papaya Ringspot Virus: Characteristics, Pathogenicity, Sequence Variability and Control. Molecular Plant Pathology. 9:269-280.
- Ming, R., Hou, S., Feng, Y., Yu, Q., Dionne-Laporte, A., Saw, J., Senin, P. , Wang, W., Ly, B.V., Lewis, K.L., Salzberg, S.L., Feng, L., Jones, M.R., Skelton, R.L., Murray, J.E., Chen, C., Qian, W., Shen, J., Du, P., Eustice, M., Tong, E., Wang, X., Lyons, E., Paull, R.E., Michael, T.P., Wall, K., Rice, D., Albert, H.H., Wang, M., Zhu, Y., Schatz, M., Nagarajan, N., Agbayani, R., Guan, P., Blas, A., Wai, C., Ackerman, C.M., Ren, Y., Liu, C. , Wang, J., Wang, J., Na, J., Shakirov, E.V., Haas, B., Thimmapuram, J., Nelson, D., Tang, H., Bowers, J.E., Gschwend, A.R., Delcher, A.L., Singh, R., Suzuki, J.Y., Tripathi, S., Neupane, K., Wei, H., Irikura, B., Paidi, M., Jiang, N., Zhang, W., Presting, G., Windsor, A., Perez, R., Torres, M. J., Feltus, F., Porter, B., Li, Y., Burroughs, M., Luo, M., Liu, L., Mount, S.M., Christopher, D.A., Moore, P.H., Sugimura, T., Jiang, J., Schuler, M. A., Mitchell-Olds, T., Shippen, D., Depamphilis, C.W., Palmer, J.D., Freeling, M.R., Paterson, A.H., Gonsalves, D., Wang, L., Alam, M. 2008. The draft genome of the transgenic tropical fruit tree papaya (Carica papaya Linnaeus). Nature. 452:991-996.
- Suzuki, J.Y., Tripathi, S., Fermin, G., Hou, S., Saw, J., Ackerman, C., Schatz, M., Pitz, K., Yepes, M., Fitch, M., Manshardt, R., Slightom, J.L., Ferreira, S., Salzberg, S.L., Alam, M., Ming, R., Moore, P., Gonsalves, D. 2008. Characterization of insertion sites in Rainbow papaya, the first commercialized transgenic tree-fruit crop. Tropical Plant Biology. 1:293- 309.
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Progress 10/01/07 to 09/30/08
Outputs
Progress Report 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. 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. Significant Activities that Support Special Target Populations A draft genome sequence of transgenic papaya 'SunUp' was published in the journal Nature. Major efforts were made to experimentally develop information for a revised petition to deregulate the transgenic papaya in Japan. In December 2007 a second revised petition was sent to the Food Safety Committee (FSC) of the Ministry of Health Labor and Welfare (MHLW). The FSC analyzed this petition and wanted several more questions answered. The main questions were 1) to determine the compositional properties of the transgenic papaya at a) when it is picked at color break, b) when it is picked at color break and allowed to ripen at room temperature and then analyzed, and c) when it is allowed to fully ripen on the tree and then analyzed, and 2) to quantitatively determine the amount of GUS protein in transgenic papaya from different generations. The information was experimentally obtained and this third revised petition was sent to our Japan counter parts for translation to Japanese on August 2008. We expect the petition to be sent to the FSC in September 2008 and are confident that the FSC will approve the petition. A protocol to screen transgenic anthurium for bacterial blight under screenhouse conditions was established. So far, 86 of the 700 transgenic lines have gone through the entire screening protocol. Of the 86 screened, 16 lines showed tolerance to bacterial blight. The rest of the 700 lines will be systematically screened. Those showing tolerance or resistance will be screened again and selected lines will be evaluated for their horticultural characteristics. Pollen for transgenic Rainbow papaya was used to pollinate nontransgenic Kapoho papaya flowers that were at different stages of development under field conditions. This was done to assess the potential of gene flow from Rainbow papaya to Kapoho at different flower development changes. Research was started on developing transgenic pineapple for resistance to precocious natural flowering. Three fresh fruit cultivars have been put into tissue culture and transformation of these cultivars will start within the next year. This relates to NP 301, Component 2
Impacts
(N/A)
Publications
- Ling, K., Zhu, H., Gonsalves, D. 2008. Resistance to Grapevine Leafroll Associated Virus-2 is Conferred by Post-Transcriptional Gene Silencing in Transgenic Nicotiana benthamiana. Transgenic Research. 17:733-740.
- Meng, B., Gonsalves, D. 2007. Grapevine rupestris stem pitting-associated virus: A decade of research and future perspectives. Plant Viruses. 1:52- 62.
- Gonsalves, D., Suzuki, J.Y., Tripathi, S., Ferreira, S. 2008. Papaya ringspot virus (Potyviridae). Encyclopedia of Virology, 5 vols, 3rd Edition, vol. 4, p. 1-8. Edited by B. Mahy & M. VanRegenmortel. Oxford:Elsevier.
- Suzuki, J.Y., Tripathi, S., Gonsalves, D. 2007. Virus Resistant Transgenic Papaya: Commercial Development and Regulatory and Environmental Issues. In: Punja, Z.K., DeBoer, S., Sanfacon, editors. Biotechnology and Plant Disease Management. Wallingford, United Kingdon: CAB International. p. 436- 461.
- Meng, B., Gonsalves, D. 2008. Grapevine rupestris stem pitting-associated virus. In: Rao, G.P., Myrta, A., Ling, K.-S., editors. Characterization, Diagnosis & Management of Plant Viruses. Texas: Studium Press LLC. p. 201- 222.
- Ling, K., Zhu, H., Gonsalves, D. 2008. Grapevine Leafroll Associated Viruses. Molecular Diagnosis of Plant Viruses. Horticultural Crops/Studium Press, Houston, Texas. pp 181-199.
- Chin, M., Rojas, Y., Moret, J., Fermin, G., Tennant, P., Gonsalves, D. 2007. Varying genetic diversity of papaya ringspot virus isolates from two time-separated outbreaks in Jamaica and Venezuela. Archives of Virology 152, 2101-2106.
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report 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. 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. 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. Technology Transfer Number of U.S. Patents granted: 5 Number of Non-Peer Reviewed Presentations and Proceedings: 17
Impacts
(N/A)
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.
- 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.
- 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.
- 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.
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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? This research supports NP 302, Plant Biological and Molecular Processes, primarily components II, Biological Processes that Improve Crop Productivity and Quality and III, Plant Biotechnology Risk Assessment. The research also contributes to NP 301, Plant, Microbial, and Insect Genetic Resources, Genomics and Genetic Improvement, primarily component II: Genomic Characterization and Genetic Improvement. Defined genetic improvements of crops can be facilitated by elucidating the genetic bases underlying crop agronomic/horticultural traits. Milestones that were set with the complete genome sequencing of arabidopsis, as the model dicot plant, and rice, as the model monocot plant, have rapidly advanced our understanding of numerous genetic systems. Nevertheless, it is now clear that model plant
genomics are not sufficient to explain the agronomic/horticultural traits of crops. There is a need for detailed comparative and functional genomics on a wide range of crop plants. Detailed knowledge of the genomics of the tropical fruit crop papaya is needed to develop new knowledge about the evolution and function of genes controlling important developmental states such as sex determination and flowering and physiological processes such as reaction to pathogens and expression of disease resistance. Papaya, family Caricaceae, and arabidopsis, family Brassicaceae, are both in the order Brassicales so that comparative genomic studies between these two species can reveal the level of DNA sequence conservation or divergence among homologous gene families. There is a need to develop papaya genome sequence data to evaluate evolutionary gene divergence. More knowledge of the regulation of expression of transgenes is needed to increase our efficiency in engineering disease resistance and
other valuable traits in tropical crops. Evaluating the potential risks of transgenic crops is an important prerequisite towards the commercialization of transgenic products. More knowledge is needed in directly assessing these products under a variety of conditions and during different stages leading up to commercialization. The planned research on tropical plant genomes will lead to better utilization of tropical crop genetic resources and increase the value of these resources by characterizing and facilitating the manipulation of genes important for increasing productivity, value, and safety of selected tropical crop species. Papaya genome analyses will identify genes conferring useful agronomic traits such as sex determination, flower development, and disease resistance for assisting breeders in the improvement of germplasm. Knowledge developed about regulation of gene expression will improve the efficiency and provide new approaches for producing disease resistant germplasm.
Increased genetic resistance to pests and diseases will allow U. S. farmers to increase crop productivity, lower their production costs, and help technology transfer programs to foreign countries. Knowledge developed on the environmental effects of transgenic plants will support regulatory approval and public acceptance of the genetically enhanced lines. 2. List by year the currently approved milestones (indicators of research progress) Year 1 (FY 2006): Numbered items correspond to milestones from Project Plan. 1. Isolate BAC DNA from hermaphrodite BAC library. 2. Fingerprint the entire BA library using the 5-enzyme, 4-dye high- throughput technique with 70% success. 23. Characterize ca. 500 new clones from SSH library. 28. Analyze Ac-Ds transgenic sugarcane lines. 29. Determine effect of P0 on siRNAs in N. benthamani. 34. Conduct agrobacterium infiltrations to develop PTGS as a genomic tool for papaya. 39. Transform sugarcane with SCYLV P0, SRMV HcPro, TCV P38, PVX P25. 44. Analyze
tissue-specific gene expression by CDNA-AFLP. 49. Test segmented gene transgenic papaya for resistance to PRSV strains outside of Hawaii. 50. Transform papaya with synthetic genes. 57. Establish methodology for detecting transgene seeds of very young fruit. 58. Measure transgene flow to adjacent commercial transgenic and nontransgenic orchards. 65. Obtain all required information or data for deregulation in Japan. 66. Deregulate papaya in Japan. 67. Select & start characterization of transgenic Kapoho for deregulation. 72. Continue sequencing of coat protein gene of PRSV isolates from Bangladesh strains. 73. Construct segmented coat protein transgene against Bangladesh strains. 74. Test segmented gene papaya for resistance to Bangladesh strains. 75. Submit application for importing and confined testing of selected papaya in Bangladesh. 76. Transform papaya with synthetic genes. Year 2 (FY 2007): Numbered items correspond to milestones in Project Plan. 3. Edit fingerprint data using
GenoProfiler. 4. Assemble contigs with FPC program. 7. End-sequence BAC clones from entire papaya hermaphrodite BAC library with estimated 70% success rate. 9. Isolate plasmid DNA from 4 separate papaya cDNA libraries with 72,000 clones in total. 12. Design Overgo primers from conserved arabidopsis and Brassica sequences & papaya EST sequences. 16. Design SSR primers based on the BAC-end sequence. 20. Generate map and associate each linkage group from genetic maps with a chromosome. 24. Conduct time course & local vs. systemic comparison of CPBI genes. 30. Continue determine effect of P0 on siRNAs in N. benthamani. 35. Clone PRVHA & make infectious constructs. 40. Continue to transform sugarcane w/ SCYLV P0, SRMV HcPro, TCV P38, PVXP25. 45. Continue to analyze tissue-specific gene expression by cDNA-AFLP. 51. Test R0 synthetic gene plants for resistance to PRSV. 52. Analyze expression of coat protein segments in relation to resistance to PRSV strains. 53. Select promising lines for
deregulation. 59. Use small fruit methodology to measure transgene flow to trees over time. 60. Test for presence of transgenes in nontransgenic papaya orchards throughout Puna. 68. Continue characterization and deregulation of transgenic papaya. 77. Analyze R0 plants containing synthetic genes for resistance. 78. Transform papaya withy segmented transgenes developed for Bangladesh. 79. Establish and evaluate confined field trials of transgenic papaya in Bangladesh. Year 3 (2008): Numbered items correspond to milestones in Project Plan. 5. Optimize the physical map by manually merging contigs and adding singletons to contigs. 8. Clean the raw sequencing data with Phred and generate the database for it. 10. Sequence 72,000 cDNA clones with estimated 80% success rate. 13. Label and pool overgo probes hybridize papaya BAC filters. 17. Test SSR primers for polymorphism using parental plants of the mapping populations. 18. Test map populations using the SSR primers with polymorphism. 21.
Map the major gene and/or QTLs controlling fruit size on the high- density SSR linkage map. 25. Functional analysis of selected CPBIs by directed silencing. 31. Identify functional domains of SCYLV P0 by deletion/point mutation analysis. 36. Produce and test mutations in PRV Hc-Pro gene. 41. Analyze effects of SCYLVP0, SRMV HcPro, TCV P38, PVX P25 on gene expression. 46. Clone, sequence and verify differential expression of cDNA-AFLP tissue specific genes. 54. Continue analysis of PRSV synthetic gene plants in successive generations. 61. Develop recommendations for cost effective coexistence of transgenic and nontransgenic papaya in Puna. 62. Apply knowledge gained to work in developing countries. 69. Continue characterization and deregulation of transgenic Kaphho. 80. Send synthetic gene plants to Bangladesh. 81. Continue analysis of synthetic gene plants. 82. Continue confined field trials in Bangladesh. 83. Analyze R0 plants with segmented gene. Year 4 (2009): Numbered items
correspond to milestones in Project Plan. 6. Anchor BAC-ends and possibly ESTs on the physical map. 11. Clean the raw EST sequencing data with Phred and generate the database for it. 14. Score Overgo membranes and import into database. 15. Assemble and annotate the whole genome shotgun sequence. 19. Assemble a papaya high-density genetic map using 1,000 SSR markers. 22. Scale down the target region of fruit size genes from the SSR linkage map. 26. Continue functional analysis of selected CPBIs by directed silencing. 32. Continue to identify functional domains of SCYLV P0 by deletion/point mutation analysis. 37. Analyzed VIGS of GUS transgenic lines. 42. Analyze effects on endogenous miRNAs & target mRNAs. 47. Isolate selected tissue-specific genes from BAC library. 55. Continue analysis of synthetic gene plants. 63. Continue applying knowledge for coexistence of transgenic and nontransgenic papaya domestically and to developing countries. 70. Deregulate transgenic Kapoho in Japan. 85.
Establish confined field trials of synthetic gene papaya in Bangladesh. 86. Continue characterization of synthetic gene papaya in Hawaii. 87. Establish large scale field trials in Bangladesh with selected lines. 88. Continue deregulation efforts of transgenic papaya in Bangladesh. Year 5 (2010): Numbered items correspond to milestones in Project Plan. 27. Conduct functional analysis of selected CPBI homologs in Arabidopsis knock-outs. 33. Determine subcellular localization & P0 domains required for SCYLV P0 suppression of PTGS. 38. Evaluate VIGS of candidate disease resistance genes. 43. Continue to analyze effects on endogenous miRNAs & target mRNAs. 48. Isolate & transient test selected tissue-specific promoters from papaya. 56. Select and start the deregulation of synthetic gene plants. 64. Continue applying knowledge for coexistence of transgenic and nontransgenic papaya domestically and to developing countries. 71. Transgenic Kapoho released commercially. 89. Continue to
characterize promising lines of transgenic papaya for Bangladesh. 90. Deregulate earliest selected transgenic papaya in Bangladesh. 4a List the single most significant research accomplishment during FY 2006. This project started May 16, 2006. Please refer to report for terminated project 5320-21000-010-00D (accession 407217) which this project replaces. 4b List other significant research accomplishment(s), if any. This project started May 16, 2006. Please refer to report for terminated project 5320-21000-010-00D (accession 407217) which this project replaces. 4c List significant activities that support special target populations. This project started May 16, 2006. Please refer to report for terminated project 5320-21000-010-00D (accession 407217) which this project replaces. 4d Progress report. This project started May 16, 2006. Please refer to report for terminated project 5320-21000-010-00D (accession 407217) which this project replaces. 5. Describe the major accomplishments to
date and their predicted or actual impact. This project started May 16, 2006. Please refer to report for terminated project 5320-21000-010-00D (accession 407217) which this project replaces. 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? This project started May 16, 2006. Please refer to report for terminated project 5320-21000-010-00D (accession 407217) which this project replaces. 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). This project started May 16, 2006. Please refer to report for terminated project 5320-21000-010-00D (accession 407217) which this project replaces.
Impacts
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Publications
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