2012 Annual Report
1a.Objectives (from AD-416):
Objective 1: Develop the genomics of papaya for producing new knowledge about the regulation of transgenic disease resistance. [NP 301, C4, PS 4B]
Objective 2: Develop methods for improved manipulation and expression of transgenes in key tropical/subtropical ornamental and fruit crop species. [NP 301, C4, PS 4A]
Objective 3: Evaluate biotechnology risk and develop methods for practical adoption of selected transgenic crops. [NP 301, C4, PS 4C]
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.
Papaya is an important nutritional fruit crop in Hawaii and in other subtropical and tropical regions of the world. Gene annotation on an improved assembly of the geneticially engineered (GE) SunUp papaya genome is continuing with the aim of applying bioinformatic information towards papaya cultivar characterization, selection and development for improved disease resistance, production and fruit quality.
Anthurium is one of Hawaii’s most valuable commercial flowers. A collection of accessioned material for the development of molecular and biochemical tools for cultivar characterization and identification was initiated.
In collaboration with researchers at the University of Hawaii at Hilo, College of Pharmacy, development and refinement of biochemical methodologies to profile floral pigments and related compounds in commercial Anthurium are continuing for the purpose of cultivar and species characterization, selection and improvement of ornamental traits.
An in-vitro rearing method was developed for Neoactinolaimus sp., a predatory nematode with potential as a biological control agent. A cinder box trial was initiated to evaluate chemical nematicides against burrowing nematode in anthurium. Twenty-three ginger cultivars were collected and propagated in bag culture and tissue culture for future screening for bacterial and nematode resistance/tolerance.
New genome sizes reported for Anthurium. Little is known of the genome organization of the important horticultural crop Anthurium, information that is vital for understanding inheritance, origins of genes and traits and for developing modern molecular tools for cultivar improvement. To gain fundamental knowledge in the area of genome organization in Anthurium, genome size estimates for 78 Anthurium accessions representing 34 species and nine cultivars were obtained and reported by ARS researchers in Hilo, Hawaii. The new genome sizes include first estimates for 33 Anthurium species, the smallest (A. obtusum) and largest (A. roseospadix) Anthurium genome sizes reported to date and the first genome sizes for commercial cultivars. The new knowledge of Anthurium genome sizes will support advancement of molecular approaches for improvement and development of new cultivars for the Anthurium industry.
Import and consumption of genetically-engineered (GE), PRSV-resistant Rainbow papaya in Japan was approved. A major portion of U.S. market share of papayas in Japan was lost due to devastation to the Hawaiian papaya industry in the 1990’s caused by the papaya ringspot virus (PRSV). The first shipments of Rainbow papaya to Japan were made in December, 2011 following final approval of the application by the Japanese government. The GE, Rainbow import application package was prepared and submitted through a cooperation between ARS researchers in Hilo, Hawaii, and researchers or representatives from various Universities and Industry. This is the first fresh genetically engineered fruit to be commercialized in Japan.
Novel flavones identified from Anthurium. Anthocyanins are flavonoid compounds that are known to comprise the colorful pigments that are the commercially attractive feature of Anthurium flowers, but little is known of the factors governing its synthesis. Through a collaboration between University and ARS researchers in Hilo, Hawaii, modern analytical techniques were applied to identify flavonoid-related compounds produced in Anthurium. The molecular structures of new, previously undescribed flavone C glycosides, a type of flavonoid compound were determined from a Hawaiian cultivar of Anthurium andraeanum. Discovery of the novel flavone C glycosides, provides new information on the biosynthesis of flavonoids and anthocyanin in Anthurium, information that will advance research for improvement of floral traits and advance research in flavonoids, compounds important to human health and nutrition.
Bliss, B.J., Suzuki, J.Y. 2012. Genome size in Anthurium evaluated in the context of karyotypes and phenotypes. AoB Plants. doi:10.1093/aobpla/pls006.
Cabos, R.Y.M., A.H. Hara, M.M. Tsang. 2012. Hot water drench treatment for control of reniform nematodes in potted dracaena. Nematropica. 41:72:79.
Clark, B.R., Suzuki, J.Y., Bliss, B.J., Borris, R.P. 2012. Flavone C-glycosides from Anthurium andraeanum. Natural Product Communications. 7(6):747-748.
Fermin, G., Keith, R.C., Suzuki, J.Y., Ferreira, S.A., Gaskill, D.A., Pitz, K.Y., Manshardt, R.M., Gonsalves, D., Tripathi, S. 2011. Allergenicity assessment of the papaya ringspot virus coat protein expressed in transgenic Rainbow papaya. Journal of Agricultural and Food Chemistry. 59(18):10006-10012. DOI: 10.1021/jf201194r.
Gonsalves, D., Gonsalves, C., Carr, J., Tripathi, S., Matsumoto, T., Suzuki, J., Ferreira, S., Pitz, K. 2012. Assaying for pollen drift from transgenic Rainbow to nontransgenic Kapoho papaya under commercial and experimental field conditions in Hawaii. Tropical Plant Biol. 5(2):153-160. DOI:10.1007/s12042-011-9090-5.
Melzer, M.J., J.S. Sugano, D. Cabanas, K.K. Dey, B. Kandouh, D. Mauro, I. Rushanaedy, S. Srivastava, S. Watanabe, W.B. Borth, S. Tripathi, T. Matsumoto, L. Keith, D. Gonsalves, J.S. Hu. 2012. First report of Pepper mottle virus infecting tomato in Hawaii. Plant Dis. 96(6):917.