Develop common genomic approaches and tools for cucurbits & Genomic assisted breeding for disease resistance
View the pdf version of this report
Watermelon Team members: Amnon Levi (USDA, ARS), Shaker Kousik (USDA, ARS), Kai-shu Ling (USDA, ARS), Cecilia McGregor (Univ. Georgia), Pat Wechter (USDA, ARS), Todd Wehner (North Carolina St. Univ.)
Overall objectives: Identifying quantitative trait loci (QTL) associated with resistance to major and emerging diseases, developing useful molecular markers and utilizing the genomic tools to incorporate resistance into watermelon cultivars.
Major diseases: Gummy stem blight, Fusarium wilt, Powdery mildew, Phytophthora fruit rot, Papaya ringspot virus (PRSV) and Cucumber green motile mosaic virus (CGMMV).
Work in progress and plans
1.2. Perform GBS analysis of PI collections, establish core populations of the four species, and provide community resource for genome wide association studies (GWAS)
GBS.
The GBS is complete and a manuscript describing the work has been submitted. For further information, see Genomics section
Core populations.
We are collecting and increasing Citrullus PI accessions, heirloom cultivars, and gene mutant type-lines. Seed increase of the 2000 PI accessions is being accomplished by seed companies, USDA scientists, and university researchers. Each is increasing 1 to 10 accessions per year using controlled pollination in greenhouse or field.
A core collection was developed, consisting of 420 PI accessions that had traits of interest to researchers. Of those, 250 germinated and were increased by self pollination. Seeds from self pollination and leaf tissue of those core accessions were sent to Michigan State University.
Gene type lines. Collection and seed increase of the watermelon gene type-lines will include all cultivars, breeding lines, and PI accessions in the gene mutant list at Cucurbit Genetics Cooperative. Examples include: PI 189225 (db, Ar-2-1), NC-517 (C), PI 482261 (Ctr), Bush Charleston Gray (dw-1), PI 595203 (zym-CH, zym-FL).
Selfing of PIs to form core population is in progress.
2.1, 2.2., 2.3 QTL map resistance, marker development and verification, introgress resistance
Fusarium wilt race 2 (Pat Wechter, Sandra Branham, and Amnon Levi, USDA, ARS, U.S. Vegetable Laboratory (USVL), Charleston, SC)
Genetic mapping of QTL associated with resistance to Fusarium wilt race 2- Two-hundred and twenty F2:3 families derived from the cross USVL-252FR x PI 244019-PRSV-R(S3) were constructed in collaboration with Dr. Nihat Guner and team at Sakata Seeds. The 220 families were evaluated for resistance to Fusarium wilt (FW) race 2 resistance (Figure 1) in two separate experiments at the U.S. Vegetable Laboratory. The distribution of FW race 2 resistance in the population indicates polygenic inheritance (Figure 2). Genotyping-by-sequencing (GBS) of the F2:3 population identified a major QTL on Chromosome 1 of USVL-252FR2 associated with resistance to FW race 2. KASP markers are being developed and will be validated for utility of incorporating the resistance into the genetic background of watermelon cultivars. We conducted a genetic mapping study to identify quantitative trait loci (QTLs) associated with resistance to Fon race 1 in segregating populations (F2:3 and recombinant inbred lines) of Citrullus amarus (citron melon) derived from the Fon race 1 resistant and susceptible parents, USVL246-FR2 and USVL114, respectively. A major QTL (qFon1-9) associated with resistance to Fon race 1 was identified on chromosome 9 of USVL246-FR2. This discovery provides an additional host-resistance source of resistance to Fon race 1 in watermelon and as it co-locates with the QTL for Fon race 2 resistance in the same population, may provide non-race specific resistance (Branham et al. 2017, 2019).
Figure 1. Two-hundred and twenty F2:3 families derived from the cross USVL-252FR x PI 244019-PRSV-R(S3) being evaluated for Fusarium wilt race 2 in a greenhouse at the U.S. Vegetable Laboratory (Summer, 2017).
View figure 1 on page 34 of the pdf
Figure 2. Distribution of F2:3 families derived from the cross USVL-252FR x PI 244019-PRSV-R(S3) for resistance to Fusarium wilt (FW) race 2 (Left). A major QTL associated with FW race 2 resistance on Chromosome 1 of USVL-252FR (right).
View figure 1 on page 35 of the pdf
Converting QTL to Kompetitive Allele Specific PCR KASP markers tightly linked to Fusarium wilt race 1 resistance- DNA of the resistant and susceptible parents (C. lanatus) and the F2 parental plants of the most resistant versus the most susceptible F2:3 families (Lambel et al. 2014) were used for a QTL-seq analysis. QTL-seq narrowed the Fon race 1 QTL interval on chromosome 1 of watermelon (Lambel et al. 2014) by 500 kb (Branham et al. 2018). SNPs from the interval were converted to KASP primers. The KASP primers were used in genetic mapping of the same population used for the initial mapping of QTL associated with FW race 1 resistance (Lambel et al. 2014). QTL mapping yielded several KASP markers tightly linked to race 1 resistance and narrowed the QTL interval further from 1.56 Mb to 315 kb (Figure 3). In collaboration with the HM.Clause team in Davis, California we conducted QTL-seq and developed KASP markers tightly linked to FW race 1 resistance (Figure 3; Branham et al. 2018). We have developed KASP markers for Fon races 1 and 2 QTL in C. amarus. The FW races 1 and 2 resistant lines USVL246-FR2 and USVL252-FR2 were crossed with Charleston Gray, Calhoun Gray and Sugar Baby to generate F1, F2, BC1 and BC2F2. The KASP markers will be used to incorporate resistance to FW races 1 and 2 into the genome background of watermelon cultivars.
Figure 3. KASP markers tightly linked to Fusarium wilt race 1 resistance (qFon1-1) on chromosome 1 of watermelon (Branham et al. 2018).
View figure 1 on page 35 of the pdf
What do you plan to do during the next reporting period to accomplish the goals?
Complete development and validation of KASP markers and incorporate FW races 1 and 2 resistance from USVL246-FR2 FR and USVL-252FR into genomic background of watermelon cultivars
(Wechter, Branham and Levi).
Papaya ringspot virus (PRSV) resistance
(Amnon Levi, Kai-shu Ling, and Sandra Branham, USDA, ARS, U.S. vegetable Laboratory (USVL), Charleston, SC)
Identifying QTL associated with PRSV resistance
Several F2 and BC1 populations derived from the cross USVL-252FR x PI 244019-PRSV-R(S3) were constructed in collaboration with Dr. Nihat Guner and team at Sakata Seeds. The genetic populations were evaluated for resistance to PRSV-resistance at the U.S. Vegetable Laboratory. The distribution of PRSV-resistance in the population confirmed inheritance by a single homozygous recessive gene in PI 244019 (Guner, 2004; Guner and Wehner, 2008). Genotyping-by-sequencing (GBS) of an F2 population identified a major QTL on Chromosome 3 of PI 244019 associated with PRSV-resistance (Figure 4). The major QTL interval comprises several ribosomal genes, among them the eukaryotic elongation factor eIF4E known to be associated with resistance to potyviruses in cucurbit crops (Ling et al. 2009). KASP markers are being developed and will be used for incorporating the resistance into the genomic background of watermelon cultivars.
What do you plan to do during the next reporting period to accomplish the goals?
Complete development of KASP markers tightly linked to PRSV-resistance in PI 244019-PRSV-R(S3) and use them to incorporate resistance into genome background of watermelon cultivars (Levi, Ling, Branham).
Figure 4. A major QTL associated with PRSV resistance identified on chromosome 3 of Citrullus amarus PI 482019 using GBS-SNP data analysis.
View figure 1 on page 36 of the pdf
Powdery mildew of watermelon
(Shaker Kousik, Patrick Wechter, Sandra Barnham, Amnon Levi; USDA, ARS, U.S. vegetable Laboratory (USVL), Charleston, SC)
Inheritance of powdery mildew resistance, identification of QTL and RNAseq
USVL608-PMR (S6), a red fleshed watermelon line with high levels of resistance to PM was used as the female parent (P1) and crossed with USVL677-PMS which is highly susceptible (P2). The parents, F1, backcrosses to both parents (BC1, BC2) and a large F2 population were inoculated with a local isolate of PM and assessed for disease severity on a 0-10 scale of increasing disease severity. All susceptible parent (USVL677-PMS) plants were rated >7 [mean disease severity (DS) = 94%], whereas most resistant parent (USVL608-PMR) plants were rated as 1 (DS=2.5%). Majority of the BC1 plants were rated ≤2 and considered as resistant. Of the 466 F2 plants, 221 were rated ≤2 (DS=3.1%). Of the 76 BC2 plants, 23 were rated ≤2 (DS=2.9%). Chi-square analyses of the observed segregation of phenotypes for the F2 plants indicated that two genes control PM resistance with a good fit for a 7:9 resistance to susceptibility ratio. The proposed model for this ratio is two genes with one recessive for high resistance and one dominant for high resistance. This is supported by a backcrossing segregation ratio of 1:3. We have observed some highly and moderately resistant plants in the F2 indicating the cumulative effect of the two genes. QTL-seq analysis on the most resistant and most susceptible DNA bulks from the F2 populations identified a major QTL in chromosome 2.
We have also completed RNA-seq analysis of the parents during PM infection. Plants of the resistant line USVL608-PMR and the susceptible line USVL677-PMS were with inoculated with 105 conidia-ml of P.xanthii. Symptom development was observed every day. In addition, leaf samples were collected for microscopy and for RNA extraction. RNA-seq profiling was done on leaf samples collected at 0, 1, 4, and 9 days post inoculation (DAI). Powdery mildew symptoms were visible on USVL677-PMS 4 DAI whereas leaves of USVL608-PMR were clean. We have completed RNA-seq on all these samples. Data analysis is in progress. A quick analysis of the differentially expressed genes (DEG) indicated several resistance genes in chromosome 2.
We also completed inheritance studies on the egusi type watermelon (C. mucosospermus) line USVL531-MDR. This line was found to be resistant to 11 PM isolates from across the U.S.A. and was released by USDA ARS in 2018. This line was used as the female parent (P1) and crossed with USVL677-PMS which is highly susceptible (P2). The parents, F1, backcrosses to both parents (BC1, BC2) and a large F2 population were inoculated with a local isolate of PM and assessed for disease severity on a 0-10 scale of increasing disease severity. The susceptible parent (USVL677-PMS) had mean disease severity of 8.14 on the 0-10 scale, whereas it was 1.17 for the resistant parent. Segregation patterns point to single gene inheritance, but also indicated another gene is inherited maternally. Chi-square analyses of observed segregation of phenotypes for the F2 populations fit models for these gene models and were further supported by segregation patterns in the backcross populations. QTL-seq analysis on the extremes from the F2 populations and RNA-seq analysis of the parents during PM infection are being conducted to identify the chromosomal regions involved in resistance. USVL531-MDR will serve as a useful source to incorporate PM resistance into commercial cultivars. We have developed several red fleshed resistant lines (at F7) using USVL531-MDR as the source of resistance.
Advancing Powdery mildew resistant inbred lines
Fruit from F2 plants from a cross of USVL531-MDR and USVL677-PMS with powdery mildew resistance, uniform red flesh and decent brix (>7) were collected and have been advanced till F6 and further advancement to F7 is in progress. We are currently evaluating 10 red fleshed F7 lines that were homozygous for resistance. We completed assessment of fruit quality from F5 and F6 progenies that were homozygous for resistance to PM and had red flesh and brix >7 in 2018.
Identifying and developing multiple disease resistant lines from accessions
Majority of the watermelon plant introductions (PI) considered as resistant or tolerant display varying levels of disease resistance. Hence it is important to screen and select for several generations to develop highly resistant lines from these PI. We have developed 36 lines with high levels of resistance to powdery mildew from various PI. Of these 13 are also resistant to Phytophthora fruit rot and can be considered as multiple disease resistant (MDR). These lines were evaluated for resistance to powdery mildew and Phytophthora fruit rot in the field and displayed high levels of resistance compared to susceptible lines including Mickey Lee and USVL677-PMS. These lines will serve as useful sources of resistance for future studies.
In 2018 we released four red fleshed lines with high levels of resistance to powdery mildew. We have completed making crosses with these powdery mildew resistant lines (USVL608-PMR, USVL313-PMR, USVL585-PMR and USVL225-PMR) with USVL677-PMS and ‘Dixie Lee’ to develop populations for conducting inheritance studies in 2019 and developing resistant inbred lines with high fruit quality. A paper documenting the release of these four PM resistant watermelon lines was published in HortScience in 2018.
Phytophthora fruit rot of watermelon
(Shaker Kousik; USDA, ARS, U.S. Vegetable Laboratory, Charleston, SC)
Inheritance of resistance, identification of QTL and RNAseq
The U.S. Vegetable Laboratory (USDA, ARS) in Charleston has developed several germplasm lines with high levels of resistance to Phytophthora fruit rot. In these studies we used the germplasm line USVL531-MDR which was resistant to 20 different P. capsici isolates from across the U.S.A. Studies to determine inheritance of resistance to Phytophthora fruit rot using the same population described for powdery mildew (USVL531-MDR X USVL677-PMS) were conducted as USVL531 is resistant to both these diseases. However, based on this study it was difficult to assess the number of genes controlling resistance and hence we are in the process of developing a recombinant inbred line (RIL) population and are currently at the F8 stage.
We completed growing out the F3 families in summer-fall of 2018 (total 40 families, about 600 plants) and screened them for Phytophthora fruit rot. The data is being analyzed. We have extracted DNA from parents and F2 plants for GBS. However, since resistance to powdery mildew is a dominant trait we will pool the DNA from 20 most susceptible lines and 20 most resistant lines and send it out for sequencing. We will perform QTLseq analysis on the resulting data.
We are currently phenotyping the populations from USVL003-MDR x USVL677-PMS for resistance to powdery mildew and Phytophthora fruit rot.
Fruit rind samples were collected from individual fruit after 12h, 24h, 48h, 72h, and 96h after inoculation and immersed in liquid nitrogen to quench all the metabolomics processes. Rind samples were then processed for extraction of RNA and sent to Duke University Genomic center for RNA sequencing. Sequencing has been completed and we are currently analyzing the RNA-seq data. We completed experiments to determine the transcriptomic profile during P. capsici infection of resistant and susceptible genotypes. Our studies with melatonin have also shown that it can suppress the growth of Phytophthora capsici in culture plates. Our research has also indicated that 1000mM melatonin solution is capable of reducing development of Phytophthora fruit rot on cucumbers.
Advancing resistant inbred lines
Advanced germplasm lines of USVL531-MDR, USVL0020-PFR, Charleston, Gray and Sugar Baby were grown in the field and fruit were harvested when mature. Fruit of each of these lines was inoculated with 104 zoospores/ml and maintained in a humid chamber (26 ± 1 °C >95%RH). We have identified three red fleshed (plants) with tolerance to Phytophthora fruit rot and high level of resistance to Powdery mildew (at the F5 stage). These will be screened for resistance to both the diseases and advanced further to F6
Project metrics (time line) for research on Phytophthora fruit rot and powdery mildew of watermelon
- Develop germplasm lines with resistance to Phytophthora fruit rot and powdery mildew for watermelon: Completed.
- Develop populations for phenotyping resistance to Phytophthora fruit rot and powdery mildew of watermelon: Completed
- Sequence and map Phytophthora fruit rot and powdery mildew QTL in watermelon: In progress.
- Introgress Phytophthora and powdery mildew resistance into cultivated type watermelon: In progress
- Participation in outreach to stakeholder groups per year via industry events and field days. Completed
Watermelon gummy stem blight resistance
(Luis Rivera and Todd C. Wehner; NC State Univ.; Cecilia McGregor, University of Georgia, Athens, GA)
Inheritance of resistance, identification of QTL
Wehner
Objective: a) Evaluate a RIL population of watermelon (Citrullus lanatus × C. amarus) for resistance to gummy stem blight and fruit quality traits and b) Map GSB resistant genes through genome-wide association studies (GWAS).
Phenotyping: A watermelon GSB population was developed by intercrossing the most resistant accessions of Citrullus four times (I4), followed by crossing with elite cultivars of watermelon (I4F1), followed by intercrossing without selection, while maintaining wild and elite types in the populations (I4F1I4), followed by self-pollinations of plants at random (I4F1I4S1). The 300 RILs and 20 controls (10 PIs and 10 commercial cultivars) were evaluated for resistance to gummy stem blight in greenhouses at North Carolina State University in Raleigh, North Carolina (Figure 1 and 2), and in the field at the Horticultural Crops Research Station at Clinton, North Carolina (Figure 3). We inoculated plants with Stagonosporopsis cucurbitacearum at a concentration of 5×105 spores/ml (Figure 4). To evaluate disease severity, we adopted an ordinal disease assessment scale (Gusmini et al. 2002). Plants were rated four times, in an experiment with, 2 locations, and 10 replications (at greenhouse and field). We also evaluated fruit quality in the gummy stem blight field trial. We also collected data of fruit shape, rind pattern and toughness, seed size and color, flesh color and intensity and hollow heart. We will identify RILs with high yield of excellent fruit quality.
Figure 3. Field test for resistance to gummy stem blight
View figure 3 on page 39 of the pdf
Additionally, genomic DNA of gummy stem blight isolates collected from field outbreaks was extracted, and a PCR-based marker test for distinguishing the three morphologically identical, but genetically distinct species causing gummy stem blight was performed (Figure 4). We used three sets of primers, including Db05 that produces a 216 to 224-bp fragment in all three species, Db06 that produces a 283- to 289-bp in S. citrulli and a 268-bp and slightly fainter fragment in S. cucurbitacearum, and Db01 that produces a 256- to 364-bp fragment in S. citrulli (Brewer et al. 2015). Two of the isolates were S. cucurbitacearum (syn. Didymella bryoniae) and one isolate was S. caricae.
Genotyping: The 300 RILs were planted in spring 2018, at greenhouses of NC State, to sample leaf tissue for DNA extraction. The DNA was sent for SNP discovery through genotyping by sequencing (GBS) method at Cornell University. We expect to get several thousand of SNPs for association analysis (GWAS). Resistance to GSB and fruit quality are being evaluated in 3 years (2017, 2018, 2019), 2 locations (field, greenhouse), and 10 replications on 300 lines (I4F1I4) at the S4, along with 20 controls (10 PIs and 10 commercial cultivars).
Association analysis: The phenotypic and genotypic data is being analyzed using R packages: GWASTools, GWASdata, SNPassoc, snpMatrix, GenABEL and pbatR. The result of the analysis will
allow us to locate and identify SNP markers associated with GSB resistance.
Figure 4. Gummy stem blight spore mass production and identification through PCR and electrophoresis
View figure 3 on page 40 of the pdf
Inheritance of resistance, identification of QTL
McGregor
Population Development:
Three populations are currently being used for this research: WPop GSB1 (PI 482276 x Crimson Sweet), WPop GSB2 (PI 189225 x Sugar Baby) and the ZxD population (ZXRM x PI 244019). The later population was previously developed.
• WPop GSB1: PI 482276 x Crimson Sweet population of 225 F2:3 lines. Complete
o Backcross population: WPop GSB 1BC: BCF2 for PI 482276 x Crimson Sweet (recurrent) for trait introgression and marker validation. Complete
• WPop GSB2: PI 189225 x Sugar Baby population of 140 F2:3 lines. Complete
o Backcross population: WPop GSB 2BC: BC for PI 189225 x Sugar Baby (recurrent) for trait introgression and marker validation. In progress
Phenotyping:
• WPop GSB1: PI 482276 x Crimson Sweet population of 178 F2:3 lines (15 plants x 178 lines = 2670 plants) was phenotyped in a growth chamber using C. citrilli isolate 12178A (GA). Disease symptoms for each seedling were scored on a 0 – 5 scale and BLUPs calculated. Parents and F1 and 4 other control genotypes were also included. Complete
• WPop GSB2: PI 189225 x Sugar Baby. This population is currently being phenotyped. In progress
• ZxD population (ZXRM x PI PI 244019). This population is currently being phenotyped. In progress
Genotyping and QTL mapping:
• WPop GSB1: PI 482276 x Crimson Sweet population of 178 F2 plants were genotyped by GBS. The reads were aligned (Fei lab) to the C. amarus PI 296341 reference genome. A genetic map consisting of 1,237 high quality markers were created. Three QTL for GSB resistance was identified: qClGSB1.1 (R2 = 17%), qClGSB1.2 (R2 = 13%), qCLGSB8.1 (R2 = 10%).
Goals for 2018-2019
- Develop KASP markers to span QTL regions identified in WPop GSB1 (PI 482276 x Crimson Sweet population.). Validate makers in BCF2 and start introgression.
- Complete phenotyping for WPop GSB2, and use the data to create bulks for QTL-seq. Samples will be sent for sequencing (Georgia Genomics Facility), and ΔSNP index will be calculated to identify significant regions.
- Phenotype ZxD population and map QTL using existing SNP map.
Cucumber green mottle mosaic virus
(Kai-shu Ling and Amnon Levi USDA, ARS, U.S. Vegetable Laboratory, Charleston, SC)
- We have completed the initial screening of USDA watermelon germplasm (~1,400 accessions). In the repeated test, several selected lines showed promising level of tolerance to CGMMV (without visible symptom). However none of them was immune to CGMMV, the virus titer were detectable in the tolerant plants using ELISA tests.
- We made single plant selection of the promising lines and are developing segregating populations through crossing. S2 seeds have been generated from one of the most promising Citrullus colocynthis line.
- Seeds from seven PI lines with potential for resistance (tolerance) to CGMMV have been sent to the collaborator to generate plant tissue for support the re-sequencing efforts under the CucCAP project.
- We submitted a release notice ‘Virus-resistant desert watermelon (Citrullus colocynthis) germplasm line ‘USVL18-157VR’ useful for enhancing CGMMV-resistance in watermelon cultivars. The release notice is currently in the process of review and approval by USDA, ARS, National Program Leaders (NPL).
Table 1. Selected lines with potential tolerance to CGMMV were selected for re-sequencing
Test Item Number |
Taxon |
Seed |
| 138 | Citrullus colocynthis | 30 |
| 145 | Citrullus colocynthis | 30 |
| 151 | Citrullus colocynthis | 30 |
| 157 | Citrullus colocynthis | 30 |
| 565 | Citrullus lanatus | 30 |
| 570 | Citrullus lanatus | 30 |
| 714 | Citrullus lanatus | 30 |
What do you plan to do during the next reporting period to accomplish the goals?
We are advancing through single plant selection of the most promising Citrullus colocynthis line to S3. Those seeds will be provided with the sponsoring seed companies to make crosses to your elite materials. In addition, once the F2, BC1 seeds are generated, materials from segregating populations will be used for Genotyping-by-sequencing or similar study to identify SNPs in association with the tolerance to CGMMV.
