Recipient Organization
WEST VIRGINIA STATE UNIVERSITY
PO BOX 1000
INSTITUTE,WV 25112
Performing Department
Agricultural & Environmental Res Station (AERS)
Non Technical Summary
The present work focuses mainly define the genomic features and to increase understanding interrelationships of its cultivar-groups so that we can use genetic resources more efficiently in breeding programs. 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. 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 highthroughput 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. The U.S. pumpkin and squash growers are seeking to diversify the varieties and rotate them every season, while protecting this vulnerable crop from diseases and pests.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
Pumpkin and squash species include a variety of high value crops (summer and winter squashes) that play an important role both in local diets and as export crops in the U.S. Squash and pumpkins production worldwide exceeds 20 million tons from more than 1.5 million hectares. The fruits, seeds or other parts of squash and pumpkins (pumpkins) possess compounds with antioxidative, anti-inflammatory, hypoglycemic and anti-hyperglycemic actions and hence are active as anticancer and antidiabetic agents. Pumpkins for processing (pie filling, puree, and baby food) and culinary seed production are important in the U.S. According to the most recent classification the genus Cucurbita comprises 20 species (2n = 2x = 40). All summer squashes are essentially Cucurbita pepo, but winter squash can be C. pepo, C. moschata, C. argyrosperma, or C. maxima. Domesticated forms of subspecies pepo include pumpkins, zucchini, orange and miniature ball. Subspecies ovifera includes acorn, delicata, and sweet dumpling types of winter squash and yellow crookneck of summer squash. The present work focuses mainly define the genomic features and to increase understanding interrelationships of its cultivar-groups so that we can use genetic resources more efficiently in breeding programs. 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. Students will be trained on breeding, genomics, phytonutrient extraction and quantification, biomedical evaluation and also in extension.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. We have already generated 20,456 SNPs segregating in our pumpkin collection which consists of 389 summer squashes (Cucurbita pepo). We also generated partial reference genome assembly for jack-o'-lantern, a pumpkin type of C. pepo. Reference genome assembly is fundamental for use in mapping GBS reads so that SNPs with chromosomal information can be launched in association mapping, comparative genomics, molecular breeding. Hence, for the first time, we aim to complete the assemble high?quality reference genome of jack-o'-lantern using advanced genomic technologies including large-scale next generation sequencing and high-throughput chromosome conformation capture (Hi-C) data. Proposed reference genome with chromosome-scale assembly along with functional annotation and analysis will be an invaluable resource for molecular research in jack-o'-lantern.Specific objectives of the proposal are:Germplasm evaluation for nutraceutical traits and resistant traits and advancing accessions with favorable alleles and superior accessionsDevelop of high-throughput SNP genotyping by sequencing for association mapping for location of QTLs/markers of importance
Project Methods
Objective 1: Germplasm evaluation for yield, fruit quality and resistant traits and advancing the accessions with favorable alleles and superior accessionsWe propose to conduct phenotypic evaluations of approximately 300 (100 each) C. pepo, C. moschata, and C. maxima cultivars with replication 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. Accessions would be grown in replicated plots at the Vegetable Research Farm using standard horticultural techniques. Cultivars would be direct seeded then thinned to 60 cm within rows and three meters between rows. Plots would receive optimum fertilization (450 lb/A 12-12-12 banded in the row) and major insect pests would be controlled with herbicides (fungal and bacterial diseases will be left uncontrolled). Plants will be field inoculated and symptoms will be evaluated after four weeks. Fruit parameters will be evaluated at harvest and separate subsamples will be saved for processing and storage evaluation. The processing would take place at our Food Science and Technology Pilot Plant. Fruit pieces would be placed on a conveyor belt steamer to cook and will be frozen and stored for subsequent analysis. Total solids, soluble solids, and viscosity will be measured on processed samples. A subset of fruit from each accession will be cured and placed in storage at 10C and evaluated at six, 8 and 12 weeks for fruit decay and physiological decline in storage (shrivelling, pithiness). Data from field, laboratory and storage will be complied and combined with genotypic data for association mapping.β-carotene analyses will be performed by using fruit flesh color using the procedure of Cuevas et al. (2009). Mesocarp and endocarp color will be categorized into RED (RED), Yellow (YELL), and white (WH) using the Royal Horticulture Society mini-color chart. QβC content of each variant will be determined by reverse phase high performance liquid chromatography (HPLC). After QβC content is determined for different carotenoids gene-variants, β-carotene can be estimated using the correlation of mean mesocarp color ratings and amount of QβC (e.g. YELL=15.62 µg g-1 β-carotene). Such identified natural variants for several further used for evaluation in participatory selection approach for potential use as novel accessions.Objective 2: Develop of a whole genome sequence and mapping SNPs generated by genotyping by sequencing for association mapping for location of QTLs/markers of importanceGenomic DNA will be isolated from leaf tissues of jack-o'-lantern using DNeasy Plant Mini Kit according to the manufacturer's protocol (Qiagen). Next generation sequencing DNA libraries will be prepared using NEBNext Ultra II DNA Library Prep Kit according to the manufacturer's protocol (NEB). Multiple insert libraries will be prepared for short read sequencing with insert size of 170 bp, 500bp, 800 bp, 5000 bp, 10000 bp and 20000 bp. Genome size of the jack-o'-lantern will be estimated based on the kmer distribution. The short read sequeicng with different insert size will be produced using Illumina sequencing method. The short read sequencing data will be preprocessed and assembled de novo using SOAP Denovo, ABySS, MaSuRCA ((Luo et al. 2012; Simpson et al. 2009; Zimin et al. 2013) and gap closing was executed. These scaffolds were repeat masked using repeat masker (http://www.repeatmasker.org/) and annotated using MAKER tool ((Holt and Yandell 2011). We will be using the innovative advanced genomic technologies including Next Generation Sequencing with multiple insert libraries, Single-molecule real time sequencing, optical mapping to generate high quality reference scale de novo assembly for jack-o'-lantern for the first time. Additionally, high-throughput chromosome conformation capture (Hi-C) data will be used to identify chromatin interactions within the nucleus. Hi-C will be used to reconstruct haplotypes at the chromosomal level by placing the unanchored contigs to the right location. This innovative strategy, will generate the high-quality reference genome for jack-o'-lantern with functionally annotated and validated genomics data.Association mapping for location of QTLs/markers of importance GWAS analysis tools will be implemented in the database by building upon open source R packages of both EMMAX (Kang et al. 2010) and GenABEL (Aulchenko et al. 2007). Population structure and kinship matrix will be used as covariates in mixed linear models. Manhattan plots for associated SNPs will be visualized by use of GenomeBrowse v1 (Golden Helix, Inc.). The SNP P-values obtained by GWAS will undergo sequential Bonferroni correction (Holm 1979) and false discovery rate determination.