Performing Department
Agricultural & Environmental Res Station (AERS)
Non Technical Summary
In this proposal, we expand our deliverables to include nutraceutically enriched germplasm accessions for on-farm trials and strategies coupled with marker-assisted selection that result in resistant varieties. These activities, which will be supported by intensive education and outreach that would serve to flow existing varieties and those bred using MAS and other genomics-assisted approaches to pumpkin and squash farming communities in the U.S. Given the importance of pumpkin and squash in fundamental U.S. foods and the feasibility of Cucurbita spp. for high-throughput analysis and manipulation due to their smaller genome sizes, the public investment in pumpkin and squash genomic research is highly justified. Selected germplasm accessions with unique phytonutrient features and desirable fruit traits will be demonstrated for comparative performance and will be recommended to breeders and growers. All information and results of both commercial and academic interest will be shared in public database in dedicated web sites. Students will be trained on breeding, genomics, phytonutrient extraction and quantification, biomedical evaluation and also in extension. A website (http://www.wvpumpkinpark.com/) is already available integrating various activities of pumpkin cultivation, utilization and other outreach activities in the state of West Virginia. The website will be the primary outreach tool for the current project and will serve as a portal to all the deliverables produced in the project. It will be used to keep the public informed about the project and how their tax dollars are being spent. It will also be used to facilitate communication between the project participants and breeders, seed companies, farmers, extension personnel, agribusinesses, and others. Information about on-farm trials conducted will be included here. Demonstration plots of the advanced breeding lines will be established. Information of superior pumpkin and squash cultivars with favorable with enhanced nutraceutical traits and resistance to Fusarium wilt and CPW will be presented. During WVSU field days and at the special WV pumpkin festival, surveys will be conducted to elicit consumer preferences for fruit attributes (including shape, size, skin color and pattern, flesh color and texture, sweetness, flavor and aroma) and nutraceutical traits. On-line leaflets will be created for all appropriate new material that is created in the project. These will support the webinars, videos, and WV pumpkin festival. This resource will facilitate the selection of novel traits for the introgression of exotic germplasm into elite backgrounds. The proposal has possibilities for patents in the discovery of new pumpkin and squash marker panels and introgressed novel accessions for specialty markets with enhanced resistance and nutraceutical traits. We will develop simple horticultural procedures to maintain breeding lines, seed production, taste and flavor panels and cost-effective genomic protocols such as SNP genotyping for pumpkin and squash trait identification.
Animal Health Component
(N/A)
Research Effort Categories
Basic
40%
Applied
30%
Developmental
30%
Goals / Objectives
Among cultivated cucurbit crops, pumpkins and squashes (here onwards, pumpkin and squashes will be jointly referred as "pumpkins" throughout the current proposal) rank highly in levels of health-promoting compounds and demand for these types has increased in U.S. markets. Pumpkins contain very high amounts of lycopene and β-carotene. Because of its function in vitamin A biosynthesis, β-carotene will be of particular interest. In sub-Saharan Africa and the Americas, 17-30% children under age of five are deficient in vitamin A, which results in xerophthalmia (progressive blindness), increased infant morbidity and mortality, and depressed immunological responses. Breeding to increase β-carotene levels in crops has been suggested as a feasible approach for improving dietary vitamin A. Unfortunately, these types of pumpkin have become highly susceptible to cucurbit powdery mildew (CPM) and Fusarium Wilt (FW) when grown in West Virginia and elsewhere in the U.S., and produce consistently lower yields. We propose to use a multi-disciplinary approach to characterize pumpkins for nutraceutical and resistant traits in combination so that the derivative cultivars can not only be grown for greater profit but also have a greater potential to reduce cancer, type II diabetes and cardiovascular diseases. Further, we propose to transfer nutraceutical traits from germplasm accessions into the pumpkins that are locally adaptable using a genomics driven plant-breeding approach. The proposal will address the need for an interdisciplinary approach to integrate cutting edge technologies to impact pumpkin and squash genomics, biomedical investigations and breeding so that value added, high quality and resistant varieties are developed. Students will get opportunities to participate in different disciplines of plant science such as Genetics, Genomics, Biotechnology, Plant Breeding, and Horticulture. Apart from the known important compounds, we wish to explore all other compounds present in pumpkin and allied species using LC-MS Q-TOF (High performance Liquid Chromatography couples with Quadruple Time of Flight mass spectrometer with MS/MS capability). We plan to take advantage of association mapping approach to link the Cucurbita genome with the metabolome. Association mapping and QTL identification through this proposal will open up opportunities for genomic comparisons for various novel phytonutrient traits, fruit quality and resistant traits within and across the important cultivated species of the Cucurbita genus. The current proposal will address the need to integrate modern technologies that can create a set of common tools for marker assisted breeding through the following specific objectives; 1) Germplasm evaluation for yield, fruit quality and resistant traits and advancing accessions with favorable alleles and superior accessions, 2) Development of high-throughput SNP marker platforms for association mapping for location of QTLs/markers of importance 3) Assays for antioxidant properties and for biological activity in cell culture and 4) Integration of extension activities with proposed research.
Project Methods
Evaluation of fruit yield, quality and resistance traits: We propose to conduct phenotypic evaluations of approximately 400 (100 each) C. pepo, C. moschata, C. argyrosperma and C. maxima cultivars with three replications for a number of traits associated with field performance and fruit quality. Sixteen traits measured in the field, ten at harvest, and six after processing would be measured. Plants will be field inoculated and symptoms will be evaluated after four weeks. Metabolome Studies: Metabolomic profiles for flesh of ripe fruits of the association populations will be carried by the Metabolomics Center of University of Illinois. Compound identification will be performed using an LC-MS Q-TOF (High performance Liquid Chromatography couples with Quadruple Time of Flight mass spectrometer with MS/MS capability). This system accurately identifies compounds based on retention time, absorbance values, and molecular weight. Many compounds can be identified by comparing existing metabolomic databases such as MetAlign. Genotyping by Sequencing: Falling sequencing costs and new techniques for reduced genome representations now open the next generation sequencing technique to be utilized for genotyping hundreds of individuals, even when the genome is complex. The method of reducing the genome size or complexity is important for producing the most useful genotyping results; it must reliably sample the same regions across the individuals and preferably will sample DNA randomly dispersed across entire genome. A subset of 3 diverse individuals from each species will be prepared into a PstI RAD library and sequenced. The SNP identification will be performed with samtools/bcftools, a MAQ-style variant calling algorithm. Association mapping, Marker assisted Selection (MAS) and validation of QTLs in mapping populations and other early generation segregants: Once we identify candidate SNPs that are linked to various important traits, MAS and validation will be carried using Infinium platform (Illumina, San Diego, CA). iSelect HD Custom Genotyping BeadChips offer the ability to interrogate SNP genotypes. Assays for antioxidant properties and for biological activity in cell culture using pumpkin fruit extracts: Crude preparations will be made by homogenization of the mesocarp with 80% EtOH and evaporation on a rotary evaporator. The DPPH assay evaluates free radical scavenging. Cell culture: Vascular smooth muscle cell, cardiac muscle cell, adipose cell and established tumor cell lines will be used for the bioassays. Stock solutions of pumpkin extracts will be made by diluting the pumpkin extracts in DMEM. Determination of oxidative damage to proteins: Oxidation of amino acid residues on proteins results in formation of carbonyls that are introduced into protein side chains. These will be measured using the OxiSelect Protein Carbonyl Spectrophotometric Assay (Cosmo-Bio, Tokyo, Japan). Determination of oxidative lipid damage: Oxidative lipid damage will be determined using the AK-170 Lipid Peroxidation Assay to measure MDA in combination with 4-hydroxyalkenals (BIOMOL International, Plymouth Meeting, PA).