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?
Diverse germplasm resources (breeding lines, cultivars, landraces, wild species relatives, etc.) exist for enhancement of cultivated crop plants. Genes that may be valuable for crop improvement are often not well characterized, associated with undesirable traits, or difficult to effectively transfer to adapted cultivars or breeding lines due to low heritability and/or the presence of numerous genes, all of which are important to achieve the intensity of expression exhibited in the unadapted plant materials. The underlying genetic bases of key horticultural traits in major Solanaceous fruit crops ¿ tomato and pepper - will be elucidated, and will facilitate their deployment in improved germplasm. This project focuses on the genetic characterization and enhancement of attributes that influence quality, nutritive value and marketable yield of fresh and/or processed products. The specific objectives are:.
1)Development of tomato germplasm with enhanced quality attributes including fruit firmness and carotenoid pigments and characterization of the genetic control of these attributes. Fruit firmness and color contribute to tomato product quality and nutritive value. .
2)Identification of molecular markers linked to genes that influence resistance to tomato fruit rot caused by the anthracnose pathogen. Resistance-linked markers would facilitate development of resistant germplasm, thus reducing losses in yield and product quality. .
3)Development of new pepper germplasm for culinary and ornamental applications; characterization of the inheritance and genetic control of anthocyanin pigment accumulation in pepper fruit, flower and leaf tissue. The research will facilitate development of new pepper cultivars for diverse market applications.

Improvements in complex traits or traits with low heritability have lagged considerably and are often characterized by little or no progress or small incremental gains. This research is needed to determine what genes are most valuable, and efficiently transfer those genes in order to develop new germplasm containing those traits. New enhanced germplasm will reduce risks of production loss, lessen the need for chemical controls, address the needs of consumers who increasingly demand more nutritious products and improved product quality, and provide new value-added crops for growers faced with low profit margins. The research is relevant to public and private crop improvement programs that focus on cultivar development.

This research is directed towards the genetic improvement of horticultural crops which contributes to the Plant, Microbial and Insect Genetic Resources, Genomics, and Genetic Improvement National Program 301 (100 percent); Research Component II. Genomic Characterization and Genetic Improvement. Diverse genetic resources are utilized to improve commodity fresh and processing market quality, nutritive value, and to reduce the need for chemical control of disease and crop loss that occurs during production, processing, distribution, and storage.

2.List the milestones (indicators of progress) from your Project Plan.
Year 1 (FY 2004) Complete year 1 tomato fruit firmness evaluations of diallel population constructed to evaluate the contribution of firmness from different germplasm sources.

Complete molecular marker screening of a recombinant inbred line (RIL) population developed to identify quantitative trait loci (QTL) for anthracnose resistance.

Release pepper germplasm with improved ornamental/culinary attributes.

Identify GenBank sequences with homology to anthocyanin-related genes.

Year 2 (FY 2005) Complete year 2 tomato fruit firmness evaluations and determine heritability of firmness indices.

Develop populations for genetic studies of tissue-specific carotenoid accumulation in tomato.

Complete QTL analysis of tomato anthracnose resistance.

Identify structural and regulatory genes responsible for tissue-specific pigmentation in pepper.

Year 3 (FY 2006)

Conduct inheritance studies of tissue-specific carotenoid content in tomato.

Map genomic regions associated with tomato anthracnose resistance.

Identify structural and regulatory genes responsible for tissue-specific pigmentation in pepper.

Year 4 (FY 2007)

Release genetic stocks with unique firmness and carotenoid pigmentation.

Release pepper germplasm with novel ornamental or dual purpose ornamental/culinary attributes.

Develop pepper populations for inheritance studies of fruit and leaf anthocyanin pigmentation.

Develop selectable markers for tissue-specific anthocyanin accumulation in pepper.

Year 5 (FY 2008)

Characterize carotenoid-related gene expression in tissues of differentially pigmented tomato genotypes.

Evaluate sequence homology in carotenoid genes from contrasting genotypes.

Assess utility of QTLs for anthracnose resistance in marker assisted selection.

Conduct inheritance studies of tissue-specific anthocyanin accumulation in pepper.

Develop selectable markers for tissue-specific anthocyanin accumulation in pepper.

3.Milestones:
The milestones listed below were scheduled to be completed in Year 1 of the Project Plan. All milestones were completed.

Complete year 1 tomato fruit firmness evaluations of diallel population constructed to evaluate the contribution of firmness from different germplasm sources Complete molecular marker screening of recombinant inbred line (RIL) population developed to identify QTL for anthracnose resistance. Release pepper germplasm with improved ornamental/culinary attributes. Identify GenBank sequences with homology to anthocyanin-related genes.

B. List the milestones (from the list in question #2) that you expect to address over the next 3 years (FY2005, 2006, and 2007). What do you expect to accomplish, year, by year, over the next 3 years under each milestone?

Year 2 (FY 2005) Complete year 2 tomato fruit firmness evaluations and determine heritability of firmness indices. Second year evaluations were fortuitously completed in year 1. Genetic analysis will be completed and as part of a contingency on this objective, an assessment made on the feasibility of pursuing development of selectable markers for firmness. Feasibility is contingent on adequate heritability and availability of skilled technical help.

Develop populations for genetic studies of tissue-specific carotenoid accumulation in tomato. Development of several populations will be completed to investigate the genetic control of tissue-specific carotenoid accumulation in tomato fruit.

Complete QTL analysis of tomato anthracnose resistance. A statistical analysis of the marker data collected from screening of the RIL population described in year 1 will be conducted to identify QTL linked to genes for anthracnose resistance in tomato.

Identify structural and regulatory genes responsible for tissue-specific pigmentation in pepper. Building upon the work from Year 1, probes for various regulatory and structural genes will be used to begin characterization of the genetic control of anthocyanin accumulation in different pepper tissues.

Year 3 (FY 2006)

Conduct inheritance studies of tissue-specific carotenoid content in tomato. Available populations developed from prior years of the project will be used to characterize the genetic control of tissue-specific carotenoid accumulation in different fruit tissues to account for varying pigmentation in these tissues.

Map genomic regions associated with tomato anthracnose resistance. QTL identified in Year 2 will be mapped to the tomato molecular marker linkage map. The results will facilitate development of selectable markers for anthracnose resistance that may be used in development of disease resistant cultivars.

Identify structural and regulatory genes responsible for tissue-specific pigmentation in pepper. Building upon the research in Year 2, we expect to have a well characterized model to explain tissue-specific anthocyanin accumulation in different pepper tissues.

Year 4 (FY 2007)

Release genetic stocks with unique firmness and carotenoid pigmentation. Germplasm lines with defined firmness and/or carotenoid attributes will be made available for distribution to public and private researchers conducting crop improvement.

Release pepper germplasm with novel ornamental or dual-purpose ornamental/culinary attributes. New ornamental or dual-purpose ornamental/culinary pepper germplasm with novel fruit, foliar, and plant habit will be released in cooperation with a CRADA partner.

Develop pepper populations for inheritance studies of fruit and leaf anthocyanin pigmentation. Adequate populations will be available to complete inheritance studies of fruit and leaf anthocyanin pigmentation. This aspect of the project may be accelerated to facilitate related Year 3 objectives.

Develop selectable markers for tissue-specific anthocyanin accumulation in pepper. Results of gene expression studies and classical inheritance studies will be utilized to develop selectable markers for tissue-specific anthocyanin pigmentation in pepper. The results will facilitate development of new germplasm.

4.What were the most significant accomplishments this past year?
Considerable untapped genetic diversity exists in wild relatives of tomato to enhance the nutritive value of cultivated forms of the crop. Utilizing an accession of the wild tomato relative L. cheesmanii, genes that influence fruit carotenoid content were transferred to the cultivated tomato to enhance fruit nutritional content. Two cherry tomato breeding lines designated 02L1058 and 02L1059 that produce fruit with high levels of the carotenoid beta-carotene were released by ARS for use in the development of specialty hybrids. Beta-carotene can be synthesized to vitamin A and is an essential nutrient. Analysis of chemical constituents and taste panel evaluations indicated that fruit quality of these lines was equal or superior to conventional cultivars containing little beta-carotene. These breeding lines are available for research purposes, including development and commercialization of new cultivars.

B. Other significant accomplishment(s), if any.

New pepper germplasm is needed to enrich the commercial pepper genepool which lacks diverse new cultivars to meet market demands for novel ornamental, culinary, and dual-purpose cultivars. Utilizing the rich genetic diversity available in pepper landraces and related species, new pepper germplasm has been developed in a collaborative project between scientists in the Vegetable Laboratory, Floral and Nursery Products Research Unit, and a private partner. This germplasm has unique gene combinations for leaf characteristics, plant habit, and fruit shape size, color and pungency for use in ornamental and dual-purpose ornamental/culinary applications. A new cultivar has been developed in collaboration with the CRADA partner. The CRADA partner has requested confidentiality on the cultivars attributes pending application for PVP. This cultivar has been submitted for a national award in the All America Selection trials. A previously developed dual-purpose pepper cultivar "Tangerine Dream" is now available for sale at the retail level. Ornamental peppers have the highest per unit value of any pepper product and have become a profitable crop for greenhouse production and an innovative way for small farmers to produce a high value alternative crop.

Genes from diverse wild species are known to influence fruit firmness but have not been adequately evaluated to broaden the narrow genetic base for tomato firmness. A uniform measure of fruit firmness is also lacking. Utilizing breeding lines developed from crosses with the wild tomato species Lycopersicon hirsutum, a collaborative project between scientists from the Vegetable Laboratory, Produce Quality and Safety Laboratory, and Ohio State University, evaluated fruit firmness in lines of varying genetic background using a number of firmness measures. Analysis of tomato fruit firmness trials indicated that different measurement parameters account for varying aspects of fruit firmness. Firmness derived from firm-fruited parents can generally be transferred to superior breeding lines and there is little deviation from the predicted performance among firm donor parents. The analysis also indicated little influence of planting location on firmness. Our results demonstrate that genes for fruit firmness can be predictably transferred to commercially acceptable material and that selection of lines with superior firmness may be economized in one production location.

Model systems have long been available for studying biochemical and genetic aspects of anthocyanin pigmentation in plants. Despite these systems, our knowledge concerning regulation of the biosynthetic pathway and the action of regulatory genes that modify gene expression is limited. In order to evaluate gene expression, primers were developed that amplified three key genes in the anthocyanin pathway. We demonstrated differential expression of these genes in response to varying environmental conditions, thus accounting for genotype by environment interactions previously observed wherein varying levels of pigmentation were observed under extreme temperature conditions. The results will provide fundamental information to understand how regulatory genes control tissue specific structural gene expression and provide a basis for developing selectable markers for tissue-specific pigmentation.

C. Significant activities that support special target populations.

Collaborator with Alcorn State University, Alcorn State, MS, an 1890's institution, on a successful USDA, CSREES 1890 Institution Capacity Building Grant funded 09/2003. Project No. 2003-03965, Utilization of Genomics for Molecular Breeding of High Quality Disease Resistant Peppers.

D. Progress Report opportunity to submit additional programmatic information to your Area Office and NPS (optional for all in-house ("D") projects and the projects listed in Appendix A; mandatory for all other subordinate projects).

None.

5.Describe the major accomplishments over the life of the project, including their predicted or actual impact.
The start date for this CRIS was 07/24/2003. As a result, the major accomplishments for the five-year term are limited to those documented above in Question 4.

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?
Value-added cherry tomato breeding lines with enhanced nutritive value have been released to the public. Please see Question 4.A. for additional information. (Stommel, J.R. Notice of release of cherry tomato germplasm breeding lines 02L1058 and 02L1059. 2003. U.S. Dept. of Agriculture, Agricultural Research Service, Washington, DC. Germplasm release notice).

A previously developed dual-purpose ornamental/culinary pepper cultivar named Tangerine Dream developed under a Cooperative Research and Development Agreement is now available for sale to the general public via the CRADA partner. Tangerine Dream is not protected and is available for use by other scientists and the seed industry in research and the development and commercialization of new cultivars.

Plant Variety Protection is being applied for with a CRADA partner to protect a new ornamental pepper cultivar that the industry partner will license from ARS. Licensing will be completed subsequent to issuance of PVP. The new cultivar will be available for the spring 2005 production season. The CRADA partner has requested confidentiality on the cultivars attributes.

A new CRADA to conduct research on the genetic control of anthocyanin accumulation and develop ornamental and dual purpose ornamental/culinary pepper germplasm was developed between the Vegetable Laboratory, Floral and Nursery Products Research Unit, and a private partner. This CRADA succeeds a CRADA whose terms were completed in 2003.

Two additional private companies have approached us regarding our research on anthocyanins and have agreed to join with the partner described above to develop a joint CRADA to conduct research on the genetic control of anthocyanin accumulation and development of novel crops. When ratified by ARS, this larger CRADA will replace the new agreement noted above.

7.List your most important publications in the popular press and presentations to organizations and articles written about your work.
Bliss, R.M., Elstein, D. 2004. Scientists get under eggplants skin. Agricultural Research Magazine. 52(1):16-18.

Review Publications
Stommel, J.R., Griesbach, R.J. 2004. Capsicum annuum l. 'Tangerine Dream'. Hortscience. 39(2):448-449.