Summary of CucCAP Accomplishments
The CucCAP project provided the opportunity for 21 research groups from 11 universities and USDA laboratories to collectively engage our expertise in plant breeding, genetics, genomics, bioinformatics, plant pathology, extension and economics to leverage applied genomics to improve disease resistance in cucurbit crops. Our objectives were to:
- Develop genomic and bioinformatics breeding tools for cucurbit crops
- Perform genomic-assisted breeding to introgress disease resistance into cucurbit cultivars
- Perform economic impact analyses of cost of production and disease control and provide readily accessible information to facilitate disease control.
An overview of accomplishments include:
- Developed state-of-art centralized databases, genomic and bioinformatic tools for cucurbit crops
- Genotyped by sequencing (GBS) USDA PI collections for watermelon, melon, cucumber, and squash, and developed genomically-informed core populations
- As appropriate to the crop-disease combination (described below), identified sources of disease resistance, characterized inheritance, identified QTL, developed markers for marker-assisted selection (MAS), released resistant breeding lines
- Developed a centralized, web-based resource providing information for cucurbit disease control
- Provided training opportunities for 12 post-docs and 30 graduate students (fully or partially supported by CucCAP) as well as an additional 12 post-docs, 26 graduate students, and 88 undergraduates in the areas of bioinformatics, genomics, plant breeding, plant pathology, extension, and economics
- Published and presented:
- 104 refereed publications (125 includes additional related publications by the CucCAP team, e.g., analysis of fruit quality traits facilitated by CucCAP genomic resources, reports of new cucurbit diseases or cucurbit cultural practices)
- 144 extension publications, web-materials, or webinars
- 212 presentations at scientific conferences and universities
- 151 presentations in commodity-based venues
Activities, Results, Outputs by Objective
Objective 1: Genomic and bioinformatics breeding tools for cucurbit crops
A. Development of the Cucurbit Genomics Database
The CucCAP bioinformatics team developed the Tripal toolkit-based Cucurbit Genomics Database (CuGenDB) (http://cucurbitgenomics.org) (Zheng et al., 2019). This comprehensive, multifunctional genomic resource, which is continually being updated, contains all available genome and expressed sequence tag (EST) sequences, genetic maps, sequence annotation and transcriptome profiles for cucurbit species, and provides a central portal to store, mine, analyze, integrate and disseminate rapidly accumulating genomic and genetic data in cucurbits. The Cucurbit Genomics Database provides a critical resource for cucurbit genomics and breeding widely used by academic and industry researchers throughout the world.
In addition to key functions such as BLAST and genome browser capabilities, novel analytic capacities were developed, including ‘RNA-Seq’ modules for differential gene expression analyses facilitating comparisons among experiments, and a ‘Synteny Viewer’ to identify and visualize conserved syntenic regions within and across species. The synteny viewer will benefit our efforts to inquire whether QTL identified for resistance to a specific disease in one cucurbit crop can provide guidance for potentially relevant genomic regions in other crops. Several other genome databases have since incorporated ‘Synteny Viewer’, indicating the broad value of this tool.
B. Genetic characterization of the U.S. National Plant Germplasm System (NPGS) collections for cucumber, melon, watermelon, and squash.
Germplasm collections are a critical resource for plant breeders. They are frequently the first place to turn for new sources of valuable traits, including disease resistance. Molecular genetic characterization allows us to evaluate the extent and nature of variation that exists within our collections to help manage and preserve diversity and better facilitate breeding efforts. For each of the cucurbit crops there are approximately 1000-2000 PIs in the U.S. collections
The CucCAP project performed genotyping by sequencing (GBS) to genetically characterize the full USDA NPGS PI collections for cucumber, melon, watermelon (Citrullus sp.), and squash (Cucurbita pepo). For each species, 24,000-48,000 filtered SNPs were identified with an average density of 1 SNP per 5-15 kb, providing approximately 3000 markers per chromosome. The GBS data and identified SNPs are freely available.
The SNP data allow for numerous analyses providing information about the genetic structure of the PI collections. Principal component analysis (PCA), K-means, and phylogenetic analysis provideinsight into evolutionary and domestication trajectories and relationships among the accessions (Wang et al., 2018; Wu et al., 2019). Understanding of the genetic relationships among accessions also informs breeders about relationships among sources of disease resistance.
C. Development of core populations as a community resource for genetic mapping.
The genetic data from the PI collections for the four crops was used to identify a subset of ~300 accessions that capture >95% of the allelic diversity present within the collections. The selected accessions were supplemented with historical cultivars providing key horticultural and disease resistance traits. These functional panels provide a set of diverse lines, associated sequence data, SNP datasets and genetic maps for future genotypic and genome-wide association (GWAS) analysis of any traits of interest. The functional panels will be re-sequenced for high resolution mapping. Self-pollinations and seed multiplication are currently underway for each of the four crops. Re-sequencing has been initiated for cucumber.
While the resequencing data will ultimately provide higher resolution mapping, the GBS data allowed for preliminary GWAS results. In cucumber, regions associated with disease resistance were identified for anthracnose, downy mildew, gummy stem blight, and root knot nematodes (Meloidogyne incognita race 3) (Wang et al., 2018). In watermelon, QTL were identified for resistances to bacterial fruit blotch, powdery mildew race 2W, and the watermelon strain of Papaya ringspot virus (Wu et al., 2019).
Objective 2: Genomic-assisted breeding for disease resistance in cucurbit crops
CucCAP breeding efforts focused on the priority diseases identified by each industry through consultation with growers, shippers and processors as represented by commodity organizations and with seed companies with significant cucurbit breeding programs. The importance of these problems was underscored by research investments cucurbit stakeholder groups have made to combat these diseases.
A. Identify QTL for resistance, develop markers to facilitate marker assisted selection (MAS).
For each crop and multiple diseases, progress has been made to identify QTL and develop markers. Molecular markers were developed for 12 crop-disease combinations; QTL for an 13 additional crop-disease combinations were mapped (Table 1). Several are already being used by the seed industry. Specific examples are provided by crop below in the following sections.
B. Introgress disease resistance into cucurbit crops.
In concert with QTL identification and marker development, the four CucCAP crop teams (watermelon, melon, cucumber, squash) are developing and releasing breeding lines with resistances to the priority diseases. Outputs include – identification of new sources of resistance; release of breeding materials; analysis of inheritance of resistance; and incorporation of resistance into commercial breeding material.
|Alternaria||Melon||chr 10, 12||Wechter, Levi||Daley et al. 2017|
|Angular leaf spot||Cucumber||chr 5||STAYGREEN||Weng||Wang et al. 2018|
|Anthracnose||Cucumber||chr 5, 7||STAYGREEN||Weng, Wehner, Fei||Pan et al. 2018
Wang et al. 2018; Wang et al., 2019
|Bacterial fruit blotch||Watermelon||multiple||Levi, Wechter, Wehner, Fei||Branham et al., 2019b; Wu et al 2019|
|CYSDV||Melon||chr 3,5||STS||Wintermantel, McCreight|
|Downy mildew||Cucumber Watermelon||chr 1,4,5 In progress||SSR, STAYGREEN||Weng, Wehner Wechter||Wang et al. 2018, 2019|
|R1. chr 9 (C. amarus) R2. chr 1, 9 (C. amarus) R1. chr1 (C. lanatus) R1. chr 2,7,11||KASP KASP KASP KASP||Levi, Wechter, McGregor
|Meru and McGregor 2016; Branham et al. 2019a Branham et al. 2017, 2020
Fall et al. 2018; Branham et al. 2018b Branham et al. 2018a
|Gummy stem blight||Watermelon Cucumber||multiple chr 2,5,7||In progress||McGregor Wehner, Fei||Wang et al.,2018|
|Phytophthora fruit rot||Cucumber
Young fruit chr5 In progress
|Powdery mildew||Squash Cucumber Melon Watermelon||chr. 10
|Mazourek, Weng, Wehner
Wechter, Kousik, McCreight Kousik, Wechter, Levi
|Holdsworth et al. 2016 Wang et al. 2018
Wu et al.,2019
|PRSV||Squash Watermelon||chr 16
|KASP||Mazourek, Wessel-Beaver Ling, Levi, Wechter||Branham et al., 2019b|
|Target leaf spot||Cucumber||chr 6||SNP/SSR||Weng|
|ZYMV||Watermelon Squash||chr 3
|Branham et al., 2019b|
Examples of breeding accomplishments:
- Watermelon. Multiple sources of resistance, QTL and markers have been developed for Fusarium wilt races 1 and 2 (Branham et al., 2017, 2018b, 2019a,b; Fall et al., 2018; Meru and McGregor, 2016). QTL and molecular markers also have been developed for Papaya ringspot virus, Zucchini yellow mosaic virus (Branham et al., 2019b) and gummy stem blight, and QTL have been identified for bacterial fruit blotch (Branham et al., 2019a) and powdery mildew. Breeding lines with foliar resistance to multiple powdery mildew isolates were released (Kousik et al., 2018a); lines with resistance to powdery mildew and Phytophthora fruit rot are being used to develop inbreds with red flesh and increased brix. Breeding is in progress to develop gummy stem blight resistant lines with good fruit quality. A source of resistance to Cucumber green mottle mosaic virus was identified and a resistant breeding line (Ling and Levi, 2019) has been approved for release.
- Melon. A major QTL and marker has been developed for Fusarium race 2 in melon (Branham et al., 2018a). Additional QTL have been identified for Alternaria (Daley et al., 2017) and powdery mildew. Breeding is underway to transfer resistance to powdery mildew and Fusarium wilt into commercial melon types. Inheritance studies have been performed for resistance to Cucurbit yellow stunting disorder virus (McCreight et al., 2017); QTL identification is in progress. Six lines have been identified with high resistance to Cucumber mosaic virus. Virus resistances are being transferred into western shipper type melons. Multiplex PCR-based systems were developed for identification and quantification of four common viruses infecting melon.
- Cucumber. A major QTL and underlying gene conferring resistance to downy mildew (DM) and several other diseases (angular leaf spot, anthracnose) was identified (Pan et al., 2018; Wang et al., 2018, 2019). Sources of resistance to the new DM strains were tested in multiple years and locations (VandenLangenberg et al., 2016) and QTL identified (Wang et al., 2016). Marker-assisted backcrossing to introgress resistance into pickling cucumber is underway. Advanced inbreds of pickling and slicing types selected for yield, earliness, quality and downy mildew resistance are being prepared for release. A breeding line with young fruit resistance to Phytophthora (P. capsici) fruit rot was released (Grumet and Colle, 2017) and QTL have been identified for young fruit and age-related resistance. QTL and markers for resistance to powdery mildew (Wang et al., 2018b) and target leaf spot have been identified.
- Squash and Pumpkin. A QTL was identified and marker developed for powdery mildew resistance in squash (Holdsworth et al., 2016). New sources of resistance to Phytophthora crown rot in C. maxima and C. moschata (Mantooth et al., 2017; Kousik et al., 2018c) have been identified. A mapping population has been developed for resistance to Phytophthora in zucchini (C. pepo) and QTL identification is in progress. A QTL has been found for resistance to PRSV-W in squash and QTL identification is in underway for ZYMV. Genetic analysis of resistance to PRSV-W and ZYMV from ‘Nigerian Local’ and ‘Menina’ indicate potentially complementary sources (Seda-Martinez et al., 2019); resistance is being introgressed into advanced breeding lines of tropical pumpkin (C. moschata).
Objective 3. Analysis of cost of production and readily accessible information for cucurbit disease diagnosis and control.
- A. Economic analysis. Macro and micro economic variables (interest rates, input costs, production windows, existing crop budgets) were collected and 13 representative farms developed for different crops and production regions: watermelon – 3 CA, 3 FL, 1 TX; melon – 3 CA, 1 TX; cucumber – 1 PA; pickles – 1 NC). Production costs and risks analyses were developed using a risk-based simulation model indicating probabilities of profit or loss depending on crop, region, and farm size.
- B. Cucurbit disease diagnostic and control information. The CucCAP extension team developed the CucCAP website (https://cuccap.org/) to provide a centralized source of information ‘one stop shopping’ for cucurbit disease diagnosis and control. Cuccap.org, which has had more than >63,000 page views, provides diagnostic resources and disease control recommendations with links to plant disease clinics, production guides with disease control recommendations, disease alerts, links to forecasting tools, and links to state, regional, and national watermelon, melon, cucumber and squash commodity websites. A set of 16 cucurbit disease fact sheets with pathogen biology, diagnostic guides, and symptom and pathogen pictures at the macro and micro level, were developed for the website addressing detection and control of anthracnose, downy and powdery mildew, Fusarium wilt, Phytophthora capsici, gummy stem blight, Papaya ringspot virus and Zucchini yellow mosaic virus, among other diseases in cucurbits. Thirteen of the fact sheets were translated into Spanish and are accessible through https://cuccap.org/espanol/. The CucCAP website is continually updated with disease outbreaks, Cooperative Extension news posts, current factsheets, events calendar, and production manuals. The ‘CucCAP Chronicle’ newsletter is distributed monthly. The extension team also provides consulting, conference calls, diagnostics, disease management recommendations, field days, demonstration plots, workshops, agent in-service training, and publishes extension articles, bulletins, disease management reports, and cultivar evaluations.
The CucCAP proposal described how information would be translated into products and delivered.
How information will be translated into products and delivered to end-users (from the original CucCAP proposal)
- (a) Sources of disease resistance will be made available to the cucurbit breeding community through publications and germplasm releases.
- (b) Information regarding pathogen populations, distribution and spread, virulence, control measures and germplasm under development, will made available through web postings, extension venues and publications.
- (c) Genomics databases, tools, and bioinformatics platforms to facilitate genomics-assisted breeding will be made available through workshops, publications, and cucurbitgenomics.org.
- (d) Markers and sequence information for disease resistance traits will be made available to the breeding community through publications and web-based databases.
- (e) Improved germplasm and breeding lines for use in cultivar development will be made available to seed companies through publications and germplasm releases.
- (f) Economic analyses of farm-level impacts will be distributed via extension venues and publications.
As described above, each of these outcomes has been accomplished.
CucCAP provided the opportunity for extensive collaboration among breeders, geneticists, genomicists, bioinformaticians, plant pathologists, extension specialists, and economists to develop genomic and breeding tools to incorporate disease resistance in cucurbit crops and provide relevant disease control information. Collectively the CucCAP team:
- developed a centralized website providing genomic tools and databases; provided GBS and SNP data for full USDA PI collections of watermelon, melon, cucumber and squash (Cucurbita pepo) and developed genomically-informed core populations for each crop;
- identified sources of disease resistance, characterized inheritance, identified QTL, developed markers for screening, released resistant breeding lines, and have worked to introgress resistance into commercially valuable materials;
- developed informational materials and provided centralized access to diagnostic resources, disease control recommendations, disease alerts and forecasting tools for cucurbit crops