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.
3. Progress Report:
This report summarizes progress made for this project, in place since 2010, to accommodate unit personnel changes and merger of NP 302 into NP 301 under which all objectives for this project fall. This project expired in May, 2013, and was replaced with project 5320-21000-015-00D, "Molecular Resources for the Improvement of Tropical Ornamental and Fruit Crops". Increased genome coverage and assembly of genetically engineered (GE), virus resistant SunUp papaya genome compared to the previous draft genome assembly was achieved. The improved SunUp papaya genome assembly will serve as a molecular and bioinformatic resource for papaya cultivar identification, characterization and selection for the development of improved disease resistance, production and fruit quality of papaya, an important economic and nutritional fruit crop in Hawaii and in other subtropical and tropical regions of the world. In a cooperative effort led by ARS researchers at Hilo, Hawaii, biosafety and other formal regulatory requirements were completed and import for human consumption of fresh (GE) Rainbow (line 55-1 derivatives) papaya fruit into Japan was granted by the Japanese government in December, 2011, followed by initial shipments by Hawaii growers/shippers. The import and marketing of GE Rainbow papaya in Japan will support the US Hawaiian Papaya industry by increasing US market share lost due to production decrease caused by the industry wide onslaught of the papaya ringspot virus in the 1990’s and represents one of the first fresh GE products from the US, accepted and marketed in Japan. Hot water drenches were evaluated and found to control reniform nematodes in potted dracaena. A PCR technique was designed to analyze the gut contents of predatory and omnivorous nematodes. An in-vitro rearing method was developed for Neoactinolaimus sp., a predatory nematode with potential as a biological control agent against plant-parasitic nematodes. In 2013, chemical pesticides were evaluated for their efficacy in controlling burrowing nematodes in anthurium fields. A survey was conducted of natural populations of insect-parasitic nematodes that exist in Hawaiian soils. A new species of Heterorhabditis, an entomopathogenic nematode used in biological control of insect pests, was discovered. Research to provide information on genome sizes, chloroplast DNA sequence and biochemical composition of floral pigments in Anthurium cultivars and accessioned species were initiated to obtain baseline information lacking in this group of plants. Newly available baseline molecular and biochemical information will serve as a reference platform for further development of biochemical resources and molecular tools for cultivar and species characterization, selection and production of new cultivars using traditional and modern genetic engineering approaches to support the ornamental and flower industry. In 2013, further refinement of methods for detection, determination and comparison of floral pigments and related compounds across commercial Anthurium cultivars and species were achieved in collaboration with researchers at the University of Hawaii at Hilo, College of Pharmacy.