Cucurbit Coordinated Agricultural Project Annual Progress Report
CucCAP researchers and stakeholders met on October 27, 28 & 29, 2022 to present and discuss the grant’s accomplishments, ongoing research, plans and expectations.
- Yiqun Weng, USDA-ARS, University of Wisconsin Madison
- Rebecca Grumet, Michigan State University
- Kai-Shul Ling, USDA-ARS Charleston
- Anthony Keinath, Clemson University, SC
CucCAP Affiliated Postdocs and Graduate Students
- Feifan Chen – Post Doc at University of Wisconsin Madison (Weng)
- Ying-Chen Lin – Graduate Student, Michigan State Univ (Grumet)
- Junyi Tan – Graduate Student, University of Wisconsin Madison (Weng)
Objective 1. Develop genomic, bioinformatic, mapping approaches and tools for cucurbits
1.1 Genomic and bioinformatics
Provide seeds of core collection for re-sequencing, pan genome analysis.
1.2 Seed multiplication of cucumber core collection
PI line purification, seed increase.
From previous work, a cucumber core was developed which is composed of 399 accessions from diverse taxonomic groups, geographic origins, and market groups. A dozen important US historical varieties were also included. Most lines have undergone at least two generations of self pollination and are morphically uniform. Seeds of the 399 lines have been or are being distributed to five industry collaborators for seed increase by self pollination. It is expected that least 2,000 seeds will be returned in two years.
Objective 2. Map and develop markers for disease resistance
2.1 QTL mapping of resistances
- Downy Mildew:
2.1.1 Downy mildew (Yiqun Weng & Anthony Keinath)
In 2021, two F2:3 populations, WI7747 and WI7769, were developed for QTL mapping of DM resistance in two cucumber inbred lines, WI7631 (Chinese Long type), and WI7773 (an introgression line with DM resistance derived presumably from C. hystrix), respectively. Ninety-six F3 families of the two populations (two replications, eight plants per rep) were grown in open fields at Clemson, SC to exam inoculation responses to natural infection of the DM pathogen. Phenotypic data for general impression of DM symptoms were recorded at three time points. Data for yellowing, necrosis and sporulation were also collected from each plant at one time point. The frequency distribution of mean disease scores for the four parameter is shown in Figure 1.
Figure 1. Bar graphs for downy mildew disease scores among 96 families of the WI7769 F2:3 population. A. Disease scores for GI (general impression) from three time points in two replications. B. Distribution of mean disease scores for chlorosis (Chl), necrosis (Nec) and general impression (GI2).
Recombinant plants used for fine mapping of the dm4.1 (from WI7120), and dm5.3 (from PI 197088) major-effect QTL for DM resistance were also tested for DM inoculation responses. In 2022, these populations will continue to be tested in both field and growth chamber trials.
2.1.2 Phytophthora fruit rot (Rebecca Grumet)
- QTL mapping in PI 109483. QTL analysis on PFR resistance was conducted using segregating population from the cross between Gy14 and A4-3 (from PI 109483-DH). QTL-Seq identified two peaks on Ch5 and Chr6. Subsequent testing of F2 progeny homozygous for either the Gy14 or PI 109843 allele verified a role for qPFR5.1, which spanned a 4.5 Mb region overlapping with two DM resistance loci, dm5.2 and dm5.3. A4-3 also has been crossed with Poinsett 76 to verify the allele effect in a second genetic background. These plants are currently being grown in the greenhouse.
- GWAS for PFR resistance. Of 395 lines in the cucumber core population, we planted 267, 20, and 379 lines in the field in 2019, 2020, and 2021 field season, respectively. Young fruit (20-40 per line from multiple harvests per season) were harvested for PFR testing at 5-7 days post-pollination. At least two seasons of data are available for the majority (70%) of lines. There was good correlation for disease scores (r=0.775) between lines tested in 2019 and 2020, indicating reliability of phenotyping (Figure 2). Data have not been processed yet for fruit tested in 2021.
Figure 2. Correlation in phenotype scores between PI lines tested in 2019 and 2020
2.1.3 CGMMV (Kai-Shul Ling and Yiqun Weng)
We are screening 50 cucumber inbred lines for CGMMV resistance in a greenhouse in USDA-ARS Charleston, SC using a bioassay through mechanical inoculation. Symptom observation and testing for virus concentration will be conducted using a serological test (ELISA). In 2022, we will continue screening efforts on cucumber germplasm, first using the 400 core collections of cucumber and then with USDA cucumber germplasm collection.
2.2 Marker development and verification
2.2.1 Downy mildew (Yiqun Weng & Anthony Keinath)
For QTL mapping of DM resistances in WI7773 and WI7631, leaf samples from 96 F2 plants in each of the WI7747 and WI7769 population were collected. DNA extraction is underway. In 2022, we plan to do genotyping-by-sequencing of the two populations for QTL analysis.
Our second objective is to conduct fine mapping of the major-effect DM QTL,dm4.1, and dm5.3, and introgress them into different genetic backgrounds through marker-assisted QTL pyramiding. For fine mapping, we have developed near isogenic lines (NILs) for dm4.1 and dm5.3 in two backgrounds: the Chinese Long inbred line 9930 and the US pickling cucumber line Gy14 that also carries dm1 (CsSGR). Phenotyping and genotyping of recombinants among NIL-derived F2 and BC plants revealed multiple sub-QTL at the dm4.1 locus in both WI7120 and PI 197088 Recombinants defining each sub-QTL are being identified. For dm5.3, extensive genotyping and phenotyping were conducted among segregating populations, which allowed to delimit the dm5.3 locus into ~650 kb region on chromosome 5. In 2022, we will further narrow down the candidate gene regions for dm4.1.1, and dm4.1.2B, and dm5.3.
2.2.2 Phytophthora fruit rot (Rebecca Grumet)
- To refine map location of qPFR5.1, F2 progeny (n=768) were screened with KASP markers flanking the QTL to identify recombinant individuals within the qPFR5.1 region, which were then self-pollinated to produce F4 lines. Nine recombinant lines were planted in the field and 30-100 fruit were phenotyped for each family. KASP SNP genotyping narrowed the pPFR5.1 QTL to a 1-2Mb region that falls between dm5.2 and dm5.5 (Figure 5)
- Age-related PFR resistance. QTL-seq analysis among F2 progeny and DH lines derived from Gy14 (ARR-) X Poinsett 76 (ARR+) identified a single strong QTL for ARR on chromosome 3. KASP markers flanking the QTL were used to genotype 768 F2 seedlings. Selected recombinant and non-recombinant individuals were self-pollinated to produce F4 lines. Phenotyping was performed in a replicated trial of 22 recombinant and 14 non-recombinant lines (5 plants/line; RCBD) in the greenhouses. The non-recombinant lines verified the effect of the QTL. Genotyping by KASP markers is being used to refine the QTL (Figure 6).
Figure 3. Testing F4 lines recombinant in the QTL PFR5.1 region for young fruit resistance
Figure 4. Verification of the QTL effect for age-related resistance.
Obj. 3. QTL introgression into breeding or advanced lines, and release to breeders
3.1 Downy mildew (Yiqun Weng & Anthony Keinath)
During fine mapping of dm4.1 and dm5.3, plants carrying different combinations of dm4.1, dm5.2 and dm5.3 resistance alleles were identified and backcrossed with Gy14 aiming to develop Gy14 carrying all permutations of the three QTL. We have developed homozygous dm4.1+dm5.2 QTL in Gy14 backgrounds (Gy14Q2). In the 2021 Summer field season, the Gy14Q2 inbred line was provided to the extension team for evaluation of its DM resistance and horticultural performance. In 2022, we plan to advance these plants to BC2F2 to identify those that are homozygous at all three loci.
3.2 QTL pyramiding of DM and PFR resistances (Yiqun Weng, Rebecca Grumet and Anthony Keinath)
We have developed cucumber plants that carry DM resistance QTL dm4.1, dm5.2 (both from WI7120), and dm5.3 from PI 197088. Marker-assisted selection was practiced using four markers at the three loci (two for dm5.3) to select plants that were homologous at all three loci (Gy14Q3). A plant carrying homozygous qPFR5.1 QTL for PFR resistance was crossed with Gy14Q3. The resulting F1 plant carrying all four QTL (dm4.1, dm5.2, dm5.3, and qPFR5.1) was further backcrossed with Gy14 to advance to BC1, which were subjected to marker-assisted selection. Since dm5.2-qPFR5.1-dm5.3 were located in a 5 Mbp block in repulsive phase on cucumber Chromosome 5, ideal recombinants combined with expected alleles at three loci were not identified so far. In 2022, we will continue to do marker-assisted selection with more plants to identify desired allele combinations. The selected BC1 plants will be backcrossed again with Gy14 to advance to BC2.