Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) The goal is to facilitate the adoption of molecular tools as an aid to the improvement of important floral crops. The first objective will be to identify the floral crops most likely to benefit from these tools in the near term. Secondly, we will broaden available information regarding repetitive sequences (SSR's) and retrotransposons. Final objective will be to test these sequences in selected floral crops to develop protocols for fingerprinting and/or markers for simply inherited traits. Approach (from AD-416) We will obtain input from the floral industry to identify those crops that currently have significant breeding efforts in the United States and are most likely to benefit from this information in the relatively near term. We will conduct exhaustive surveys of the target crops for SSR's and retrotransposons. We will utilize the high throughput sequencing capabilities of the GRL located at NCSU for screening the genomes of target crops for SSR's. We are already using the facility for sequencing and have all techniques functioning in the lab. We have already identified African retrotransposon sequences in poinsettia, chrysanthemum, petunia and violet. We will use these techniques to identify and characterize the spectrum of retrotransposons, as well as flanking sequences, in the target crops. The final phase will be to establish the suitability of these sequences as markers for desirable traits in floral crops and/or in generating cultivar fingerprints. Significant Activities that Support Special Target Populations This report serves to document research conducted under a specific cooperative agreement between ARS and North Carolina State University. It was recently transferred to Fort Pierce from North Carolina and now falls under the parent CRIS 6618-22000-034-00D, Domestic, Exotic and Emerging Diseases of Citrus, Vegetables and Ornamentals (DEED). Amplified Fragment Length Polymorphism (AFLP) and Microsatellite Analysis (SSR) were both shown to be highly effective methods for cultivar indentification in New Guinea impatiens, and AFLP was additionally useful for identification of breeder and series. Retrotransposons were detected in multiple floral crops. To date, 442 kilobases of the New Guinea impatiens genome have been sequenced, 14 microsatellite markers for New Guinea impatiens have been developed and 58 retrotransposons have been sequenced in 8 crops. Cooperator�s performance was monitored by multiple phone and email discussions of program goals and accomplishments. Additionally, Cooperator and ADODR met during the American Phytopathological Society meeting in July 2007 to further discuss project. Accomplishments There has traditionally been a lack of molecular methods for development and protection of important floral crops. Retrotransposons were identified in African Violet, Begonia, Chrysanthemum, Poinsettia, Geranium, New Guinea Impatiens, Petunia and Rose. The presence and diversity of retrotransposons in these crops indicated that retrotransposons are ubiquitous in floral crops and a good source of polymorphism in these crops. This method may be useful for cultivar protection, patent validation and enhancement of breeding programs. Addresses National Program Component 2 (Biology, ecology, epidemiology, and spread of plant pathogens and their relationships with hosts and vectors).
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Progress 03/01/02 to 02/28/07
Outputs
Progress Report Objectives (from AD-416) The goal is to facilitate the adoption of molecular tools as an aid to the improvement of important floral crops. The first objective will be to identify the floral crops most likely to benefit from these tools in the near term. Secondly, we will broaden available information regarding repetitive sequences (SSR's) and retrotransposons. Final objective will be to test these sequences in selected floral crops to develop protocols for fingerprinting and/or markers for simply inherited traits. Approach (from AD-416) We will obtain input from the floral industry to identify those crops that currently have significant breeding efforts in the United States and are most likely to benefit from this information in the relatively near term. We will conduct exhaustive surveys of the target crops for SSR's and retrotransposons. We will utilize the high throughput sequencing capabilities of the GRL located at NCSU for screening the genomes of target crops for SSR's. We are already using the facility for sequencing and have all techniques functioning in the lab. We have already identified African retrotransposon sequences in poinsettia, chrysanthemum, petunia and violet. We will use these techniques to identify and characterize the spectrum of retrotransposons, as well as flanking sequences, in the target crops. The final phase will be to establish the suitability of these sequences as markers for desirable traits in floral crops and/or in generating cultivar fingerprints. Significant Activities that Support Special Target Populations Bad Clob Data for Accn/FY/Question 405677 2008 30
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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 project is aligned with NP301, Plant Genetic Resources, Genomics, and Genetic Improvement. The floral crop industry lags behind other commodities in their use of molecular tools to better identify individual varieties of specific floral species. The objective of this research is to first identify the floral crops most likely to benefit from molecular tools; to then determine the ability of molecular markers, such as repetitive sequences (SSRs) and retrotransposons to fingerprint specific varieties; and the final objective is to develop protocols for fingerprinting simply inherited traits in the plants. 2. List by year the currently approved milestones (indicators of research progress) The milestones for this project are: FY2003-Determine the molecular markers most likely to work on
the target crops FY2004-Characterize the molecular marker sequences; test markers for variability and usefulness in New Guinea impatiens, geraniums, and petunia. FY2005-Use markers to differentiate specific varieties of New Guinea impatiens, geraniums, poinsettia, and petunia. FY2006- Write papers on the investigations of SSRs for fingerprinting of Poinsettia and New Guinea impatiens together with a comparative analysis of previous work using molecular marker polymorphisms; Complete the characterization of retrotransposons in poinsettia and New Guinea Impatiens. FY2007- Test SSR and retrotransposon sequences in selected floral crops to develop protocols for fingerprinting and/or markers for simply inherited traits. 4a List the single most significant research accomplishment during FY 2006. NP301, Component 2, Genomic Characterization and Genetic Improvement. Identification of Fluorescent-AFLP and SSR Markers for Differentiation and Analysis of New Guinea Impatiens. Fluorescent
amplified fragment length polymorphism (F-AFLP) and microsatellites (SSRs) were used to evaluate New Guinea impatiens (Impatiens hawkeri W. Bull) cultivars. Ninety-five quality-selected polymorphic fragments from ten F-AFLP+3 primer combinations were used to evaluate 100 cultivars representing a variety of colors, forms, and breeding programs. Jaccard similarities and unweighted pair-group method of the arithmetic average (UPGMA) clustering formed a dendrogram with three cultivar groups, to a large extent clustering the cultivars by breeder with a high cophenetic correlation coefficient. A small insert genomic library was created and 442kb of New Guinea impatiens sequence was screened for repetitive motifs, resulting in 14 microsatellite markers. A subset of 46 cultivars representing five commercial breeding companies and 11 cultivar series was selected for microsatellite analysis. Seven loci were polymorphic, with two to six alleles per locus. Although both methods were equally
effective in distinguishing the cultivars from one another, the topologies of the dendrograms for the two methods were different. The topology of the AFLP dendrogram reflected possible relationships based on cultivar series and breeding company, while the SSR dendrogram did not. The objectives of this research were to develop and validate both F-AFLP and SSR methodologies for New Guinea impatiens, identify markers that can be reliably used for fingerprinting, and create a database for future cultivar comparisons. 4d Progress report. This report serves to document research conducted under a specific cooperative agreement between ARS and the Department of Plant Pathology, North Carolina State University. Additional details of research can be found in the report for the parent CRIS 22000-015-00D. 5. Describe the major accomplishments to date and their predicted or actual impact. NP301, Component 2, Genomic Characterization and Genetic Improvement. In this research, we have developed
molecular markers that distinguish between closely related varieties of impatiens. This will enable the industry to tag individual varieties as the plants are move throughout the world. 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 research benefits the floral industry directly, and provides them with specific molecular markers they can use.
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Progress 10/01/04 to 09/30/05
Outputs
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? What does it matter? The floral crop industry lags behind other commodities in their use of molecular tools to better identify individual varieties of specific floral species. The objective of this research is to first identify the floral crops most likely to benefit from molecular tools; to then determine the ability of molecular markers, such as repetitive sequences (SSRs) and retrotransposons to fingerprint specific varieties; and the final objective is to develop protocols for fingerprinting simply inherited traits in the plants. This research is relevant to National Program 303, Plant Diseases. 2. List the milestones (indicators of progress) from your Project Plan. The milestones for this project are: FY2002-Work with floral companies to determine the crops most likely to benefit; Survey the target crops for
molecular markers. FY2003-Determine the molecular markers most likely to work on the target crops FY2004-Characterize the molecular marker sequences; test markers for variability and usefulness in New Guinea impatiens, geraniums, and petunia. FY2005-Use markers to differentiate specific varieties of New Guinea impatiens, geraniums, poinsettia, and petunia. FY2006- Write papers on the investigations of SSRs for fingerprinting of Poinsettia and New Guinea impatiens together with a comparative analysis of previous work using molecular marker polymorphisms; Complete the characterization of retrotransposons in poinsettia and New Guinea Impatiens. 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? In FY2006, scientific publications will be written, which describe the use of molecular markers for identification of specific varieties of several floral
crops. The markers developed will be the first ones used specifically in the floral industry for variety fingerprinting. 4a What was the single most significant accomplishment this past year? Fingerprinting of varieties of New Guniea impatiens. The ability to rapidly determine differences in varieties of various floral crops will allow the industry to better track where and when varieties are moved through commerce. Molecular markers were developed and were able to distinguish between 46 varieties of impatiens. 4d Progress report. This report serves to document research conducted under a specific cooperative agreement between ARS and the Department of Plant Pathology, North Carolina State University. Additional details of research can be found in the report for the parent CRIS 22000-015-00D. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. In this research, we have developed molecular markers that distinguish between
closely related varieties of impatiens. This will enable the industry to tag individual varieties as the plants are move throughout the world. 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 research benefits the floral industry directly, and provides them with specific molecular markers they can use.
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Progress 10/01/03 to 09/30/04
Outputs
4. What were the most significant accomplishments this past year? This report serves to document research conducted under a reimburseable agreement between ARS and North Carolina State University. Additional details of research can be found in the report for the parent CRIS #6645- 22000-014-00D, "Control of Fungal Pathogens of Samll Grains". The Poinsettia genome was searched by direct sequencing of genomic libraries and by sequencing PCR products amplified using conserved primer sequences. Approximately 700,000 nucleotides of poinsettia genome sequence generated in this lab were searched for repetitive sequences. Motifs of at least six, five, four and four repeats were selected for analysis of polymorphisms for di-, tri-, tetra- and pentamers, respectively. A sextanucleotide repeat was also identified. More complex, compound repeats were also identified. A preliminary evaluation of primers designed from 18 SSR's was conducted to determine their suitability to
detect polymorphisms. They revealed one to four alleles. Twelve were selected for evaluation of 42 cultivars. SSR's may provide a suitable alternative to AFLP's for defining cultivar families but did not provide the resolution to consistently distinguish between some cultivars. This work is in preparation for publication. We are extending this to include New Guinea Impatiens and have identified a similar number of SSR's from approximately 250,000 nucleotides. The retrotransposon analysis of floral crops includes poinsettia, geranium, chrysanthemum, petunia and rose as well as a detailed analysis of diversity within poinsettia. This research is nearing completion.
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