Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695
Performing Department
Horticultural Science
Non Technical Summary
While considerable research has been done linking consumption of fruits and vegetables to human health, tremendous amounts of variation in the levels of these compounds have been observed among different varieties of the same crop. Some of this variation is due to differences in where and how the crop is grown, to how it is handled and processed after harvest, and how it is prepared by the consumer; but a significantly large portion of this variation is due to differences in specific genes in the fruits and vegetables. There are 2 important consequences of this: First, medical researchers who utilize whole foods (fruits and vegetables) or extracts of whole foods in their nutritional or health studies, often don't know how different these foods can be. Replicating these studies can be problematic if the levels of suspected compounds in the plant vary by a factor of 10. Working with the isolated compounds is also problematic, as they don't always function the same way in our body when they are isolated as they do when we consume the whole fruit or vegetable. The second consequence is that the consumer may not be receiving all of the health benefits they expect when they purchase broccoli or blueberries from their local grocery or farmers market. The objectives of our research is to identify those specific genes in broccoli and blueberry that lead to this observed variation in health promoting compounds such as glucosinolates, lutein, and vitamin k in broccoli and anthocyanins in blueberry. We have created large populations of these plants and through the use of molecular markers, identified specific regions on broccoli and blueberry chromosomes that are consistently associated with differences in this compounds. Through this project, we will identify the specific genes present in those chromosomal segments that are responsible for variation and also we will determine how those genes function. This is accomplished by comparing known genes from model crops like Arabidopsis to suspected genes in broccoli and blueberry. We will also develop molecular markers that can be used by plant breeders to reduce the time it takes to develop new and improved broccoli and blueberry varieties and will be used by us to develop broccoli lines that are different from each other in only those genes that result in higher (or lower levels) of compounds associated with health. Medical researchers will then be able to use the whole food in feeding studies and be confident that the varieties are different only in one particular compound.
Animal Health Component
25%
Research Effort Categories
Basic
75%
Applied
25%
Developmental
(N/A)
Goals / Objectives
Understanding how plant secondary pathways: flavonoid/anthocyanin, isoprenoid and sulfur containing derivatives of methionine biosynthesis (glucosinolates and their byproducts) are regulated in broccoli and blueberry will provide the basis for developing vegetables and fruits that have enhanced or modified secondary metabolite profiles in respect to human health.The objectives of the project are to utilize the genetic and genomic resources we have generated to elucidate the genetic regulation of lutein, phylloquinone, and glucosinolates in broccoli; and the modifications of anthocyanins in blueberry. The specific objectives are: Identify specific genes corresponding to identified QTL associated with variation in lutein, phylloquinone, and glucosinolates in broccoli. Identify specific candidate genes corresponding to observed anthocyanin variation (acylation and glycosylation) in blueberry. Identify specific sequence variations in candidate genes and promoter regions. Determine if sequence variations affect transcription. Develop Co-dominant PCR markers for candidate genes. Develop near-isogenic broccoli lines (NIL) that can be used in nutritional studies and that can be used as donor sources for development of elite broccoli plant material.
Project Methods
Identify candidate genes in broccoli We will use anchored markers flanking the previously identified broccoli QTL associated with variation in glucosinolates, carotenoids, and vitamin K to identify specific candidate genes in the Brassica oleracea reference genome. For example, the major QTL we have identified associated with carotenoid variation has a 95% confidence interval on chromosome 2 between 1.5 and 4.0 Mbp. A search of this region of the reference genome has identified 3 putative genes that could function as transcription factors and a fourth gene that has >90% homology to Arabidopsis phytoene synthase (the first committed step in carotenoid accumulation). The same approach will be used for QTL associated with glucosinolates and vitamin K. Identify candidate genes in blueberry We will identify putative acyl- and glyco-transferases in the draft genomic sequence of blueberry utilizing known genes from these families in grape and Arabidopsis. Likely, these will represent large families of genes. Putative candidates such as Vitis vinifera UDP_glucose:flavonoid 3-O-glucosyltransferase, however, are specific to anthocyanin activity and should reduce the number of genes considerably. Sequence candidate genes By utilizing Brassica reference genome and our genomic scaffolding of blueberry, we will design staggered gene based primers to amply complete sequence of candidates from low and high producing lines including non-translated regions and promoters. We will identify changes in the coding regions that could alter function and differences in binding or regulatory motifs in the promoter region. TranscriptionWe will utilize real-time PCR to examine expression of candidate genes (low versus high lines) by isolating RNA at various times during head development (in broccoli) and berry development (in blueberry). Co-dominant markers. We will utilizing sequence information from to design PCR markers. The type of marker to be developed will be determined based by the observed polymorphism between high and low performing lines and could include SNPs, simple sequence repeats (SSR), sequence characterized amplified regions (SCAR) or other systems. Markers will be validated with populations that are segregating for the traits of interest. Develop NIL broccoli lines By using molecular markers, and a marker assisted backcrossing (MAB) strategy, we will conduct 3-4 cycles of MAB to develop NIL inbred lines that differ only in the genes represented by the molecular markers (on average, our map density is 1 marker every 1.5 cM and this represents approximately 600 kB in physical distance). We will provide validation of NILs in replicated trials by genotyping the lines and analyzing phytochemical profiles through UPLC and LC-MS.