Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695
Performing Department
Horticultural Science
Non Technical Summary
Diet has a significant impact on human health. Some estimates project that a third of all cancer deaths in the United States could be avoided through the appropriate modification of diet. The consumption of fruits and vegetables is related to reduced rates of tumor initiation and progress, occurrence of cardiovascular disease, urinary tract infections, rheumatoid arthritis, diabetes, macular degeneration, gastric ulcers, and other disorders. A number of health promoting compounds (phytochemicals) in fruits and vegetables such as glucosinolates, alpha and beta-carotene, lutein, lycopene, alpha-tocopherol, ascorbate, folic acid, phytosterols such as beta-sitosterol and stigmasterol; triterpene esters; phenolic molecules such as anthocyanins, flavonols, proanthocyanidins, ellagitannins, and phenolic acids; and phytonutrients such as calcium and selenium have been suggested to act in a protective manner through a variety of different mechanisms. Efforts to encourage the public to consume greater quantities of fruits and vegetables have to date shown limited success. In 1991, the non-profit organization "Produce for Better Health" and the National Cancer Institute initiated the "5 a Day for Better Health" program to promote healthy eating through increased fruit and vegetable consumption. However, results from surveys conducted from 1992 to 2002 suggested that average daily fruit intake among persons over 2 years of age remained the same (1.6 servings) and that average daily vegetable consumption declined from 3.4 to 3.2 servings during the same period. In 2005, approximately 33% of the US adult population surveyed consumed fruit two or more times per day, and only 27% ate vegetables three or more times per day. Additionally, more than 60% of North Carolina adults are overweight or obese, significantly above the national average. More than 77% of North Carolina adults do not eat the recommended five fruits or vegetables per day. Physical inactivity and unhealthy eating combined are the second leading preventable cause of death in North Carolina and the nation at large, and both increase the risk of heart disease, certain types of cancer, diabetes, high blood pressure, stroke and obesity. In addition to encouraging the public to consume greater quantities of fruits and vegetables through programs dedicated to increasing awareness, a second approach to combating chronic disease would be to enhance the concentration of health promoting compounds in fruits and vegetables through conventional plant breeding techniques. Unfortunately, a number of obstacles exist. These health promoting compounds are difficult to measure precise evaluations require expensive instrumentation and specialized technical expertise. The current analyses are generally too time-consuming and cost prohibitive for most breeding programs. The development of molecular markers associated with genes' that impact the variability of health promoting compounds in fruit and vegetables crops would speed the development of enhanced fruits and vegetables and result in economic benefit to growers and health benefits to consumers.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The research will focus on the genetics and stability of health-promoting phytochemicals and phytonutrients (e.g. glucosinolates, beta carotene, lutein, lycopene, tocopherols, ascorbate, folic acid, phytosterols such as beta sitosterol and stigmasterol; triterpene esters; phenolic molecules such as anthocyanins, flavonols, proanthocyanidins, ellagitannins, phenolic acids, and selenium) in fruits and vegetables of economic importance to North Carolina consumers and producers. Multi-year, comprehensive surveys of the variability of these compounds in primary and secondary gene pools (elite germplasm and non-cultivated relatives) will be conducted and high throughput assays for phytochemical analysis will be developed. Four breeding programs in the Department of Horticulture at North Carolina State University (blueberry, strawberry, tomato, and watermelon) and a fifth program at the USDA facility in Charleston, SC (cruciferous vegetables) will collaborate and provide germplasm for these surveys. Priority will be given to blueberry, tomato and cruciferous vegetables, as appropriate populations for these surveys have already been established. Analysis will be conducted using HPLC and LC-MS in the Brown lab during 2009-2011 and will represent the most extensive survey of phytochemicals to date in blueberry. Molecular markers will be developed that are associated with variation in the plants secondary metabolomic profile, particularly with those compounds in the glucosinolate, flavonoid, and carotenoid biochemical pathways. Priority will be given to developing single-nucleotide polymorphic (SNP) markers within candidate genes of interest. The project will collaborate with existing breeding programs to identify and/or assemble appropriate populations for the genetic mapping of both structural and regulatory factors associated with variation. Linking a plant's secondary metabolomic profile to molecular markers, either through the use of evenly-spaced, genome wide marker scans or by utilizing select candidate genes with putative functions, will allow for a closer examination of how these plants accumulate phytochemicals and will also provide molecular markers that will ultimately lead to fruits and vegetables with enhanced phytochemical profiles.
Project Methods
Over 600 genotypes of 3-year-old blueberry are currently located at Piedmont Research Station, Salisbury N.C. This population includes elite germplasm, hybrids, progenies and open pollinated progenies. A broad representative sample of diploid, tetraploid and hexaploid species will be evaluated over 3 years. Blueberry fruit will be harvested from each of 600 plants at the Piedmont Research Station, Salisbury, NC, at a uniform statge. Sub-samples will be labeled and processed for extraction based on the chemical properties and polarity of the desired phytochemical class. More polar compounds such as the phenolic acids ascorbic acid will be extracted using water as a main inorganic solvent. Less polar compounds such as the anthocyanins and proanthocyanidins will be extracted for quantitative analysis using a mixture of organic and inorganic solvents followed by high-speed centrifugation to separate extracts containing target compounds from pellets. Carotenoids and fat-soluble vitamins, the least polar compounds will be extracted using non polar solvents such as ethanol and saponified. Assaying samples for phytochemical content will require utilizing multiple platforms. The confirmation of compound identity will be performed using HP Liquid Chromatography coupled with Quadrupole Time of Flight mass spectrometer with MS/MS capability. The accuracy of this procedure allows exact mass measurements of small molecules such as quercetin, and of much larger molecules such as proanthocyanidins. The estimation of compound concentration will be performed using Ultra Performance Liquid Chromatography that allows for faster and more cost efficient quantification of compounds. Analysis will be conducted during 2009-2011 and will represent the most extensive survey of phytochemicals to date in blueberry. Surveys of carotenoid and flavonoid variability will also be conducted using 32 diverse tomato genotypes. These 32 genotypes represent cultivated and wild. Surveys will be conducted at the Research stations at Mills River and Salisbury, NC, in 2010 and 2011. Phytochemical analysis of tomatoes will be as described previously for blueberries. Candidate gene sequences will be identified through a variety of techniques. Where draft genomic sequences are available (Blueberry), the sequences will represent the best candidates based on either homology or predicated function, or conserved domains of interest. Where draft genomic sequences are not available, candidate genes will be identified utilizing degenerate primers based on sequences of related or model crops. The identified candidate gene sequences will be used to design primers (PRIMER3). A panel of accessions will be used to identify and validate SNPs. SNP assays will be developed through a number of vendors (ABI Biosystems and Illumina.). The populations described previously will be genotyped with the SNP assays and several software programs will be utilized to determine the statistical significance of detected associations between markers and traits of interest (SAS, NTsys, STRUCTURE, and TASSEL).