CURATION OF CUCURBIT GERMPLASM AT CORNELL UNIVERSITY

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0231436
• Project Start Date: Oct 1, 2012
• Project End Date: Sep 30, 2015
• Program Code: [(N/A)]- (N/A)

Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853

Performing Department
Plant Breeding

Non Technical Summary
Many of the cucumbers, melon, pumpkin, summer squash and winter squash grown in NY, nationally and globally depend on historical genetics from the vegetable breeding program at Cornell University for their successful agricultural production. As new needs in cucurbit crop production emerge locally or afar, these genetic stocks serve as a source of new traits to address emerging issues. Notably, these materials have served as critical sources of powdery mildew and virus resistance. Recently, we have rediscovered resistance to the new strain of downy mildew that is challenging cucumber production in NY and globally in these materials. To ensure our continuing ability to mount responses to these needs with genetic improvements of cucurbits, it is vital that we regenerate and characterize important seed stocks so that the traits are available and we know how to efficiently deploy them when needed. In addition to the use of these materials in the seed industry and the current Cornell breeding program, these materials are also released to growers for participatory plant breeding projects, which are an integral component of sustainable, local and organic aspects of agricultural production that are restoring the vitality of small farms, increasing food security and supporting local economies. Previously our group has used these materials as the source of traits for new cucurbit crop development in work supported by federal formula funds and earlier, similar projects have resulted in cultivar releases that are now available commercially.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	1421	1080	10%
202	1420	1080	20%
202	1421	1080	20%
202	1429	1080	20%
212	1420	1080	10%
212	1421	1080	10%
212	1429	1080	10%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants; 202 - Plant Genetic Resources; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1420 - Melons; 1429 - Cucurbits, other; 1421 - Cucumber;

Field Of Science
1080 - Genetics;

Keywords

cucurbit

germplasm

genotyping by sequencing

cucumber

melon

squash

plant breeding

disease resistance

Goals / Objectives
The overall objective of our proposal is to have viable, pure seed stocks of the cucurbit breeding materials that are well characterized and documented, represent authentic samples, and meet phytosanitary criteria as determined by NYSIP (New York Seed Improvement Program) inspection so they can be readily distributed. Our specific objectives are as follows. First, identify important seed accessions based on review of breeding notes and planting plans. This process will produce the most advanced seed lots and underutilized materials that may have promise for current needs and markets while generating preliminary information related to pedigree relationships. Second, reconstruct the relevant pedigrees, in order to better understand the genetics of the traits in question, the diversity and composition of the germplasm, efficacies of past strategies and reveal the breeding process itself. Third, regenerate the identified seed lots under the purview of NYSIP. Sharing seed internationally requires the seed crop to be inspected during production to satisfy import permit requirements. Fourth, test germination of the resulting seed lots and store them under the ideal conditions of our new seed storage facility for posterity. Fifth, the genomic characterization of cucumbers through genotyping by sequencing analysis. High resolution sequence analysis of breeding lines will complement the pedigree reconstruction and produce SNP markers that can be used to track these traits in current breeding efforts. This process should maintain a nimble and enduring resource for cucurbit genetic improvement.

Project Methods
All seed samples in the Cornell vegetable breeding program are given specific lot numbers and all records indicate the current and previous generation lot numbers which are cross-referenced on the seed packets themselves as internal controls. It is thus possible to trace lineages very accurately from 1942 to present. Lot numbers that appear in the literature, release articles, historical reports, and figure prominently in pedigrees will be targeted for preservation. Where multiple lot numbers exist of a given accession, the specific lot that is preserved will be determined by germination, the thoroughness of characterization and the frequency of distribution and utilization in breeding. Original sources of resistance will be key priorities along with most advanced breeding lines with traits of interest. Pedigrees will be reconstructed stepwise, using these seed lot linkages. Seed will be advanced by a greenhouse generation of a small number of plants, followed by a larger seed increase in 12 x 48 foot pollination cages of selected lines. In both circumstances we will be able to limit disease and insects. Seed crops will be inspected by NYSIP during the growing season and certified to be free of diseases of concern for these crops and consistent with phytosanitary certificate requirements. Germination tests will be performed on subsamples of seed from each seed regeneration lot. Plants will be observed for pertinent characteristics during their growth phase and assayed with target pathogens with appropriate controls in a manner that does not expose the seed production to the pathogen. Cucumbers regenerated though this process are especially appropriate for genomic characterizations by genotyping by sequencing. DNA will be extracted using Qiagen Plant DNeasy 96 well kits and submitted for genotyping by sequencing at the Institute for Genomic Diversity at Cornell University. The resulting SNP data will be aligned to the reference genome to identify regions under selection that were conserved during the breeding process that are derived from key sources of disease resistance and quality traits and reveal the genetic architecture of the breeding program.

Progress 10/01/12 to 09/30/15

Outputs
Target Audience:Our target audiences include those interested in accessing important cucurbit germplasm. Seed companies, farmer breeders and growers were reached through reports, field days, workshops and presentations at conference. Changes/Problems:Given the volume of seed packets that were discovered that needed to be curated, less progress was made on the initial focus of reconstructing cucumber pedigrees with genome information and more effort was dedicated to the time sensitive matter of getting important germplasm into ideal storage for its preservation. What opportunities for training and professional development has the project provided?Three graduate students became familiar with the seed storage system and retrieved germplasm for research and breeding projects. Two further released cultivars based on germplasm were trialed and evaluated from this collection. They gave conference presentations on the results of their work. How have the results been disseminated to communities of interest?The results have been shared at field days andthrough conference presentations as listed. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1: We have searched through over 100,000 seed packets from past vegetablebreeding work at Cornell that represent efforts since 1942. Seed that were critical to pedigrees or represented endpoints of breeding lines that were shared with others were identified and removed for better storage. Seed were frozen to exterminate seed insect pests and packets were organized by breeding project and moved into appropriate storage for long term viability.Of this approximately 30 accessions of Chinese cabbage, 20 accessions of beans, 2,036accessions of cucumber germplasm, 160 accessions of squash bred for virus resistance, 3,590 accessions of melons bred for virus resistance, 80 accessions of melons bred for resistance to gummy stem blight, 490 accessions of squash bred for powdery mildew resistance, 50 accessions of pumpkins bred for powdery mildew resistance. Objective 2: We have reconstructed the pedigrees of Market more and Poinsett cucumbers, powdery mildew resistant pumpkins and virus resistance in squash and melon. Prominent milestones accessions along each pedigree have been archived and highlighted in the seed stocks listed under objective 1 above. Stable lines were regenerated but a significant number of these are still in the queue for regeneration given our discovery of approximately ten times more seed packets than we expected and some breeding programs for additional goals that were discovered during the process. Objective 3: We have regenerated over 200 seed lots from the most important breeding outputs. We have filled over 1,000 seed requests from seed companies and growers seeking to access these materials. Several have been rediscovered and are being offered commercially. Notablybacterial wilt resistance in cucumbers has been well received. One heirloom accession, 'Seminole' melon has been found to still have contemporary downy mildew resistance and is now available commercially. Objective 4. We have developed genotyping by sequencing pipelines for all these crops. We have optimized genotyping by sequencing using an ApeKI digest and 96plex libraries that are being sequenced on the Illumina NextSeq. We are in the process of mapping several resistance loci and are contributing germplasm to the recently funded Cucurbit Coordinated Agricultural Project (CuCAP).We have pioneered an approach of using breeding lines and cultivars with introgressions that conferdisease resistanceto quickly generate molecular markers for plant breeding programs using the accessions we discovered. We have used this approach to map powdery mildew resistance in Cucurbita and are processing the data on an array of traits in other species.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Holdsworth WL, Summers CF, Glos M, Smart CD, Mazourek M. 2014. Development of downy mildew-resistant cucumbers for late-season production in the Northeast. HortScience. 49:10-17.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Gardner J., Hoffmann MP, and Mazourek M. 2015. Striped cucumber beetle (Coleoptera: Chrysomelidae) aggregation in response to cultivar and flowering. Environmental Entomology, 44:309-316.
• Type: Journal Articles Status: Submitted Year Published: 2016 Citation: Brozowski L, Leckie BM, Gardner J, Hoffmann M, Mazourek M. Submitted. Subspecies delineates striped cucumber beetle (Acalymma vittatum) preference in Cucurbita pepo. Hort Research.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Wyatt LE, Strickler SR, Mueller LA, Mazourek M. 2015. An acorn squash (Cucurbita pepo ssp. ovifera) fruit and seed transcriptome as a resource for the study of fruit traits in Cucurbita. Hort. Research. 2:1-7.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Holdsworth W, Mazourek M. Developing Downy Mildew-Resistant for the Eastern U.S. Oomycete Molecular Genetics Network. Asilomar, CA; March 14-17, 2015
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Holdsworth W, Mazourek M. Genotyping by Sequencing Yields Dense Marker Datasets Effective for Genetic Characterization of Squash. Cucurbitaceae. Bay Harbor, MI. October 12-16, 2014.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Holdsworth W, Summers C, Glos M, Smart C, Mazourek, M. Breeding for downy mildew resistance in cucumber. Cucurbitaceae. Bay Harbor, MI. October 12-16, 2014.

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Our target audiences include those interested in accessing important cucurbit germplasm. Seed companies, farmer breeders and growers were reached through reports, field days, workshops and presentations at conference. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Six undergraduate students participated in variety trials and were able to learn replicated trial design and maintenance and phenotyping of horticultural characteristics and leaned to identify pests like striped cucumber beetles and diseases like powdery mildew. A graduate student collected data, performed statistical analysis reconstructed pedigrees and wrote a draft of a manuscript to release and describe the breeding lines. How have the results been disseminated to communities of interest? Two field days were held in 2014. The Vegetable Breeding Institute field days showcased these breeding lines to plant breeders for an array of regional and global seed companies. The 3rd Annual Student Organic Seed Symposium and Seed School was hosted at Cornell. Attendees toured research plots on the organic farm. A manuscript describing the pumpkin breeding lines is in preparation. What do you plan to do during the next reporting period to accomplish the goals? A number of seed stocks with Cucumber mosaic virus resistance and potyvirus resistance have been identified in our seed collection and will be increased and screened for virus resistance.

Impacts
What was accomplished under these goals? A replicated trial was conducted of 15 powdery mildew resistant pumpkin breeding lines and representative controls. The data that was collected included growth habit, fruit characteristics such as earliness, fruit number, size, shape, and yield and powdery mildew resistance characteristics including the affected area on adaxial and abaxial leaf surfaces and petiole. Data was analyzed in JMP. Their pedigrees were reconstructed and made into a figure for publication. A draft manuscript was prepared that will be submitted in 2015. Seed was regenerated from 22 melon breeding lines that were previously bred in the Cornell vegetable breeding program for performance in organically managed production systems. These seed lots were screened for Squash mosaic virus and gummy stem blight through seedling screens and affected seed lots were eliminated or healthy individual seedlings were propagated if possible. An observation trial was performed with the melon breeding lines to determine which were candidates for a replicated trial for release. Plots with powdery mildew or severe striped cucumber beetle damage were eliminated.

Publications

Progress 10/01/12 to 09/30/13

Outputs
Target Audience: Our target audiences include those interested in accessing important cucurbit germplasm. Seed companies, farmer breeders and growers were reached through reports, field days, workshops and presentations at conferences. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? We host several undergraduate students annually that gain experience working on applied plant research, seed production and variety trial design, maintenance and analysis. Two graduate students worked with genotyping by sequence data and established pipelines for their analysis and mentored an undergraduate in comparing genomic and transcriptomic datasets. How have the results been disseminated to communities of interest? Results have been disseminated though field days and reports to the seed industry and seed samples distributed to growers and seed companies. Select materials have been utilized in breeding and trialing efforts related to a USDA-OREI project (Grant number:2012-51300-20006). What do you plan to do during the next reporting period to accomplish the goals? We will continue to regenerate cucumber, squash and melon lines with phytosanitary inspections. Pumpkins and select cucumbers will be trialed and manuscripts will be prepared for publication in horticultural journals.

Impacts
What was accomplished under these goals? We have thoroughly canvassed the seed inventory for cucubit breeding germplasm at Cornell and identified all the significant cucumber genetic stocks have been identified and prioritized for regeneration. Several have been regenerated by self-pollination of single plants and a subset of those have been further bulk increased in pollination cages, inspected by NYSIP for distribution, and preserved in seed storage. The sixteen best pumpkin lines were regenerated by single plant self pollinations, characterized for powdery mildew resistance, habit and fruit and peduncle traits in preparation for bulk regeneration and replicated trials. All seed maintained by the cucurbit breeding program, consisting of several thousand seed lots, was frozen to kill insect pests and placed into appropriate storage conditions. Melon and squash lines with virus resistance were identified and prioritized for regeneration. We have optimized a genotyping by sequencing protocol that yields abundant SNP markers in cucumber and squash (C. pepo). Plate based DNA extraction protocols have been established along with library preparation for sequencing. We have established bioinformatics pipelines to manage the resulting data including SNP identification without a reference genome and genetic mapping.

Publications