Watermelon Team | 2020 CucCAP Progress Report

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: Fusarium wilt, Gummy stem blight, Powdery mildew, Phytophthora fruit rot, Papaya ringspot virus (PRSV) and Cucumber green motile mosaic virus (CGMMV).
Team Members: Amnon Levi, Patrick Wechter, Shaker Kousik, Kai-shu Ling (USDA, ARS).
Todd Wehner (NCSU) and Cecilia McGregor (UGA)

Genetic mapping of QTL associated with resistance to Fusarium wilt race 2

Pat Wechter, Sandra Branham, and Amnon Levi, USDA, ARS, U.S. Vegetable Laboratory (USVL), Charleston, SC

A genetic population of 220 F2:3 families derived from the cross USVL-252FR x PI 244019-PRSV-R(S3) was 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. This QTL co-locates on the same genomic region conferring resistance to Fon race 1 identified in our previous studies (Lamble et al. 2014; Branham et al. 2018). Also, a major QTL (qFon1-9) associated with resistance to Fon race 1 was identified on chromosome 9 of USVL246-FR2. These discoveries provide host-resistance source of resistance to Fon races 1 and 2 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). Sixty KASP markers were constructed and are being validated for utility of incorporating resistance to Fon races 1 and 2 into the genetic background of watermelon cultivars.

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 Fon races 1 and 2 into the genome background of watermelon cultivars.

Downy mildew resistance

Pat Wechter and Dennis Katuuramu

C. amarus PI have been assayed for downy mildew resistance and GWAS performed, identifying two putative QTL. Assays will be performed during 2020-2021 of the USVL246 x USVL114 RILs which segregate for Downy mildew resistance, and QTL analysis performed (Katuuramu et al. 2020). Assays will be performed during 2020-2021 of the USVL246 x USVL114 RILs which segregate for Downy mildew resistance, and QTL analysis performed.
What do you plan to do during the next reporting period to accomplish the goals?
Following the Covid19 aftermath we should complete development and validation of KASP markers. We have crossed USVL246-FR2 FR and USVL-252FR with watermelon cultivars and constructing genetic populations that will be used in marker assisted selection (MAS) to incorporate Fon races 1 and 2 resistance into the genomic background of watermelon cultivars (Wechter, Branham and Levi). BC1F1 have been generated for USVL246 x sugar baby, USVL246 x Charleston Grey, and USVL246 x Calhoun grey. BC2F1 and BC2 F2 will be generated during 2020-2021. Similar populations have also been constructed with USVL 252 and will be used for incorporating the Fon 1 and 2 resistance gene loci into watermelon cultivars.

Other Products

Product Type

BC2F2 and BC3F2 lines with resistance to FW races 1 and 2.
Students and Post-docs: A Post-doctoral researcher shared with Pat Wechter

Identifying QTL associated with Papaya ringspot virus (PRSV) and with Zucchini yellow mosaic virus (ZYMV) resistance

Amnon Levi, Kai-shu Ling, and Sandra Branham, USDA, ARS, U.S. vegetable Laboratory (USVL), Charleston, SC

Identifying QTL associated with Papaya ringspot virus (PRSV) and with Zucchini yellow mosaic virus (ZYMV) resistance- Several F2 and BC1 populations derived from the cross USVL-252FR x PI 244019-PRSV-R(S3) and a BC3F2 population [(PI 595203 x Charleston Gray) Charleston Gray] were analyzed 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 and at Sakata Seeds Station in Ft. Meyers, FL. The genetic analyses 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). A QTL-seq analysis of the BC3F2 population also identified a major QTL associated with ZYMV-resistance on chromosome 3 of PI 595203, counterparts to the genomic region on chromosome 3 of PI 244019. 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.

Plans 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 to ZYMV resistance in PI 595203 and use them to incorporate resistance into genome background of watermelon cultivars (Levi, Ling, Branham).

Other Products

Product Type

BC1F2 and BC2F2 lines with resistance to PRSV [(244019 x Charleston Gray) Charleston Gray]
BC6F2 and BC7F2 lines with resistance to ZYMV [(595203 x Crimson Sweet) Crimson Sweet].
Students and Post-docs: A Post-doctoral researcher.

Powdery mildew of watermelon

Shaker Kousik, Patrick Wechter, Sandra Barnham, Amnon Levi; USDA, ARS, U.S. vegetable Laboratory (USVL), Charleston, SC

Powdery mildew (PM) of watermelon (Citrullus lanatus) and other cucurbits caused by Podosphaera xanthii is a major factor limiting production in greenhouses and open fields. In recent years, occurrence of PM has been increasing on watermelon across the United States, and commercial watermelon cultivars with resistance are rare. The disease continues to be a constant problem throughout the southeast. Our survey of watermelon researchers also indicated that powdery mildew was considered an important priority for research across the U.S.A.
Prior to the start of the project, not much information on PM of watermelon was available and not many highly developed sources of resistance were available. In 2018 we released four red fleshed lines with high levels of resistance to powdery mildew (USVL608-PMR, USVL313-PMR, USVL585-PMR and USVL225-PMR). A paper documenting the release of these four PM resistant watermelon lines was published in HortScience in 2018. Several of these lines will be used in the extension part of CucCAP2. We also released USVL531-MDR which is resistant to powdery mildew and Phytophthora fruit rot. Seeds of these resistant germplasm lines have been provided to most seed companies upon request (e.g. Syngenta, HM Clause, Sakata, Seminis).
Relative susceptibility of commercial watermelon varieties to powdery mildew
This study was conducted in 2014, 2015 and 2016 to determine the relative susceptibility of twenty six watermelon varieties (seeded and seedless) and three pollenizers to P. xanthii populations prevailing in Charleston, South Carolina. USVL677-PMS, which is highly susceptible to PM and USVL531-MDR, which is resistant to PM were included as controls. A randomized complete block design with three replications was used each year and plants were rated on a 0-10 scale of increasing disease severity. During all three years, USVL677-PMS was the most susceptible line with the highest values for area under disease progress curve (AUDPC). Disease severity at the last ratings for USVL677-PMS ranged from 61-82%. In comparison USVL531-MDR was very resistant to PM based on disease severity (1-3%) and AUDPC. The commercial pollenizers, SP5, SP6 and Lion were all resistant to PM (1-5% disease severity) and had significantly lower AUDPC compared to USVL677-PMS and most other varieties evaluated. Among the red fleshed varieties, Suprema (seedless variety), was relatively resistant compared to other seeded and seedless varieties. Most of the seeded varieties evaluated (e.g. Malali, Black Mama, Mickey Lee) were highly susceptible to PM, however, some were relatively less susceptible (e.g. Declaration) under field conditions. Currently very few to no varieties with high levels of resistance to PM are available and there is a need to develop newer resistant varieties as PM is occurring more frequently during the growing season.
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.

Inheritance studies on USVL531-MDR x USVL677-PMS

We 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 F8) using USVL531-MDR as the source of resistance as described below.

Identification of an NBS-LLR R gene ClaPMR2 and development of CAPS markers

To gain a better understanding of the innate and activated molecular defense mechanisms involved during compatible and incompatible PM-watermelon interactions, we inoculated PM susceptible (USVL677-PMS) and resistant (USVL531-MDR) watermelon plants with 105 conidia ml-1 of P.xanthii. RNA-seq profiling was done on leaf samples collected at 0, 1, 3, and 8 days post inoculation (DPI). A total of 2566 unique differentially expressed genes (DEGs) were identified between compatible and incompatible interactions with P. xanthii. The compatible interactions resulted in distinct plant gene activation (>2 fold unique transcripts, 335:191:1762 :: 1:3:8 DPI) as compared to incompatible interaction (>2 fold unique transcripts, 314:681:487 :: 1:3:8 DPI). Further, comparative whole-genome resequencing analysis of USVL531-PMR, USVL677-PMS and four introgressed PM resistant recombinant inbred lines (RIL, USVL531-PMR x USVL677-PMS) were performed to identify the region of PM resistance introgressed break points along with other traits inherent by USVL531-PMR by comparing the SNPs and InDels. Based on SNPs identification and CAPS markers, the resistance gene was identified as ClaPMR2, Citrullus lanatus PM Resistance gene 2 {Chr2 : 26750001 .. 26753327 (-)}, a NBS-LRR resistance protein (R) with homology to the Arabidopsis thaliana PM resistance protein, RPW8 (Figure 1). The CAPS marker was validated using the parents, four PM resistant RILs and susceptible and resistant F2 populations using DNA gel electrophoresis (Figure 2). The transcriptome data also revealed a complex regulatory network associated with the introgressed junctions mediated by PM resistance R proteins (R genes) that may involve multiple signal regulators and transducers, carbohydrate metabolism, cell wall modifications and the hormone-signaling pathway. The information presented here has been accepted for publication in Scientific Reports and the manuscript is entitled “Elucidation of Resistance Signaling and Identification of Powdery Mildew Resistant Mapping Loci (ClaPMR2) during Watermelon-Podosphaera xanthii interaction using RNA-Seq and Whole-Genome Resequencing Approach.”

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 F7 and further advancement to F8 is in progress. We are currently evaluating 12 red fleshed F8 lines that were homozygous for resistance. We completed assessment of fruit quality from F6 and F7 progenies that were homozygous for resistance to PM and had red flesh and brix >7 in 2019.

Inheritance of PM resistance, identification of QTL and RNAseq in USVL608-PMR

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.

Phytophthora fruit rot of watermelon

Shaker Kousik; USDA, ARS, U.S. vegetable Laboratory, Charleston, SC

Phytophthora fruit rot of watermelon has been a major problem in watermelon growing areas in the Southeastern U.S. (FL, GA, SC, NC and VA). In recent years it has also become a problem in watermelon growing areas in Maryland (MD), Delaware (DE) and Indiana (IN). The National Watermelon Association considered Phytophthora fruit rot as its top research priority in 2017 as well. At the U.S. Vegetable Laboratory (USDA, ARS) in Charleston we have 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 are completed phenotyping the populations from USVL003-MDR x USVL677-PMS for resistance to Phytophthora fruit rot in 2019. DNA was extracted from the most susceptible and resistant F2 plants and bulked. Bulked DNA was sequenced by Novogene. Data analysis is in progress.

We completed experiments to determine the transcriptomic profile during P. capsici infection of resistant and susceptible genotypes. 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). 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 have identified three red fleshed (plants) with tolerance to Phytophthora fruit rot and high level of resistance to Powdery mildew. These will be screened for resistance to both the diseases and advanced further. Once we have adequate seeds of these we hope to use these in CucCAP2.

Project metrics (timeline) 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 QTL in watermelon: In progress.
  • Introgress powdery mildew resistance into cultivated type watermelon: Completed
  • Introgress Phytophthora fruit rot resistance into cultivated type watermelon: In Progress
  • Develop markers for powdery mildew resistance: Completed
  • Develop and Validate Markers for Phytophthora fruit rot: In Progress.
  • Participation in outreach to stakeholder groups per year via industry events and field days. Completed

Increasing watermelon PI accessions and preparing leaf samples for DNA isolation and genome wide association study (GWAS)

Todd C. Wehner

Increasing watermelon PI accessions and preparing leaf samples for DNA isolation and genome wide association study (GWAS)

GBS of cucurbit species, establish molecular-informed core populations

Objective: Develop molecular markers for high resistance to gummy stem blight (GSB) using genome-wide association studies (GWAS) in the USDA watermelon germplasm collection, and introgress GSB resistance into watermelon cultivars.
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.

Association analysis: Collected phenotypic and genotypic data will be analyzed using R packages: SNPassoc, snpMatrix, GenABEL and pbatR. The result of the analysis will allow us to locate and identify SNP markers associated with GSB resistance.

PI accessions

Of the 1408 accessions of Citrullus that are available, we were able to grow 1365, which were sampled for leaf tissue, and shipped to Michigan State University for DNA sequence. 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).

Watermelon gummy stem blight resistance

Luis Rivera and Todd C. 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 plant 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.
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.

Watermelon Breeding Lines

NC-524

Breeder: T.C. Wehner, L. Rivera and E.J. Silverman. Vendor: North Carolina State University, Raleigh. Parentage: S7 inbred selected from NC GSB resistant population. The population was developed as follows: 5 Citrullus amarus PI accessions (189225, 482283, 482342, 482374, 526233) intercrossed 4 generations, then crossed with 7 cultivars (Allsweet, Calhoun Gray, Charleston Gray, Crimson Sweet, Mickylee, Minilee, Petite Sweet) and the progeny intercrossed 4 generations before self pollinating. Previous number 18GH-031@ (RIL-039). Characteristics: monoecious watermelon with GSB resistance, elongate fruit shape, wide-stripe pattern, tough rind, coral red flesh, sweetness of 11 °brix, medium-size black seeds, high quality (8 on 1-9 scale), hollowheart resistant. Resistance: gummy stem blight. Similar: Allsweet. Adaptation: southern U.S. 2020.

NC-527

Breeder: T.C. Wehner, L. Rivera and E.J. Silverman. Vendor: North Carolina State University, Raleigh. Parentage: S7 inbred selected from NC GSB resistant population. The population was developed as follows: 5 Citrullus amarus PI accessions (189225, 482283, 482342, 482374, 526233) intercrossed 4 generations, then crossed with 7 cultivars (Allsweet, Calhoun Gray, Charleston Gray, Crimson Sweet, Mickylee, Minilee, Petite Sweet) and the progeny intercrossed 4 generations before self pollinating. Previous number 18GH-221@ (RIL-267). Characteristics: monoecious watermelon with GSB resistance, round fruit shape, solid light green pattern, tough rind, coral red flesh, sweetness of 10 °brix, medium-size black seeds, high quality (8 on 1-9 scale), hollowheart resistant. Resistance: gummy stem blight. Similar: King & Queen. Adaptation: southern U.S. 2020.

NC-528

Breeder: T.C. Wehner, L. Rivera and E.J. Silverman. Vendor: North Carolina State University, Raleigh. Parentage: S7 inbred selected from NC GSB resistant population. The population was developed as follows: 5 Citrullus amarus PI accessions (189225, 482283, 482342, 482374, 526233) intercrossed 4 generations, then crossed with 7 cultivars (Allsweet, Calhoun Gray, Charleston Gray, Crimson Sweet, Mickylee, Minilee, Petite Sweet) and the progeny intercrossed 4 generations before self pollinating. Previous number 18GH-222@ (RIL-268). Characteristics: monoecious watermelon with GSB resistance, round fruit shape, solid light green pattern, tough rind, coral red flesh, sweetness of 11 °brix, medium-size black seeds, high quality (8 on 1-9 scale), hollowheart resistant. Resistance: gummy stem blight. Similar: King & Queen. Adaptation: southern U.S. 2020.

NC-530

Breeder: T.C. Wehner, L. Rivera and E.J. Silverman. Vendor: North Carolina State University, Raleigh. Parentage: S7 inbred selected from NC GSB resistant population. The population was developed as follows: 5 Citrullus amarus PI accessions (189225, 482283, 482342, 482374, 526233) intercrossed 4 generations, then crossed with 7 cultivars (Allsweet, Calhoun Gray, Charleston Gray, Crimson Sweet, Mickylee, Minilee, Petite Sweet) and the progeny intercrossed 4 generations before self pollinating. Previous number 18GH-049@ (RIL-066). Characteristics: monoecious watermelon with GSB resistance, round fruit shape, solid light green pattern, tough rind, coral red flesh, sweetness of 9 °brix, small black seeds, high quality (8 on 1-9 scale), hollowheart resistant. Resistance: gummy stem blight. Similar: King & Queen. Adaptation: southern U.S. 2020.

NC-531

Breeder: T.C. Wehner, L. Rivera and E.J. Silverman. Vendor: North Carolina State University, Raleigh. Parentage: S7 inbred selected from NC GSB resistant population. The population was developed as follows: 5 Citrullus amarus PI accessions (189225, 482283, 482342, 482374, 526233) intercrossed 4 generations, then crossed with 7 cultivars (Allsweet, Calhoun Gray, Charleston Gray, Crimson Sweet, Mickylee, Minilee, Petite Sweet) and the progeny intercrossed 4 generations before self pollinating. Previous number 18GH-099@ (RIL-125). Characteristics: monoecious watermelon with GSB resistance, oval fruit shape, medium-wide stripe pattern, tough rind, scarlet red flesh, sweetness of 11 °brix, large black seeds, high quality (8 on 1-9 scale), hollowheart resistant. Resistance: gummy stem blight. Similar: Crimson Sweet. Adaptation: southern U.S. 2020.

NC-532

Breeder: T.C. Wehner, L. Rivera and E.J. Silverman. Vendor: North Carolina State University, Raleigh. Parentage: S7 inbred selected from NC GSB resistant population. The population was developed as follows: 5 Citrullus amarus PI accessions (189225, 482283, 482342, 482374, 526233) intercrossed 4 generations, then crossed with 7 cultivars (Allsweet, Calhoun Gray, Charleston Gray, Crimson Sweet, Mickylee, Minilee, Petite Sweet) and the progeny intercrossed 4 generations before self pollinating. Previous number 17GH-154@ (RIL-131). Characteristics: monoecious watermelon with GSB resistance, round fruit shape, solid light green pattern, tough rind, coral red flesh, sweetness of 12 °brix, large black seeds, high quality (8 on 1-9 scale), hollowheart resistant. Resistance: gummy stem blight. Similar: King & Queen. Adaptation: southern U.S. 2020.

QTL Mapping, Marker Validation and Trait introgression of Gummy Stem Blight resistance in Watermelon.

Cecilia McGregor, University of Georgia, Athens, GA

Phenotyping

WPop GSB1: PI 482276 x Crimson Sweet population of 178 F2:3 lines (2 x 3 reps x 4 plants/rep x 178 lines = 4,272 plants) was phenotyped in a humidity tunnel in the greenhouse using C. citrilli isolate 12178A (GA). Disease symptoms for each seedling were scored on a 0 – 5 scale and disease severity calculated. Parents and F1 and 4 other control genotypes were also included. Complete

WPop GSB2: PI 189225 x Sugar Baby. PI 189225 x Sugar Baby population of 114 F2:3 lines (4 x 4 plants/rep x 114 lines = 1,824 plants) was phenotyped in a humidity tunnel in the greenhouse using C. citrilli isolate 12178A (GA). Disease symptoms for each seedling were scored on a 0 – 9 scale and disease severity calculated. Parents and F1 and 4 other control genotypes were also included. Complete

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. lanatus Charleston Gray reference genome. A genetic map consisting of 1,525 SNP markers with a 1.2 cM average distance between markers and a total length of 1,744 cM was created. Three QTL for GSB resistance was identified: ClGSB3.1 (R2=14.1%; 76-79.1 cM); ClGSB5.1 (R2=10.2%; 135.3-147.1 cM) and ClGSB7.1 (R2=21.1%; 103.1-116.3 cM) (Fig.1a). Complete

WPop GSB2

DNA from the 12 most resistance and most susceptible F2 lines of PI 189225 x Sugar Baby population was bulked to form a R-bulk and S-bulk, respectively. The bulks sequenced and aligned to the 97103_V2 watermelon genome. Δ-SNP index was then used with a 1Mb sliding window to calculate a smoothed Δ-SNP index as well as 95% and 99% confidence using the R package QTLseqr (Mansfeld & Grumet, 2018). Four significant (95% confidence interval) QTL were detected: ClGSB2.1, ClGSB5.1, ClGSB9.1, ClGSB11.1 (Fig.1b). The QTL on chromosome 5 of the two populations partially overlap. Complete

KASP markers have been developed for selection of QTL. In progress

Remaining Goals: Validate KASP makers for selection and start introgression of QTL in elite backgrounds.

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

Remaining goals

Cucumber green mottle mosaic virus. 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.

Other Products

Product Type

A germplasm line tolerant to CGMMV.

Research materials

Advanced S2 single plant selection from the most promising tolerance line have been generated, are being advanced to S3 and S4 generation before sharing with seed companies. Several F1 progenies for CGMMV resistance in watermelon will be advanced to segregating F2 and BC1 populations.

Watermelon Core Collection

Our group is increasing 384 Citrullus spp. lines (including 300 C. lanatus and C. mucosuspermus, 50 C. amarus and 10 C. colocynthis PIs and 24 heirloom cultivars) to S2 generation. Leaves for DNA extraction have been collected from the S1 plants and stored for shipment to Michigan State University. Fifteen S1 seeds of each of the 384 PIs are being collected and tested for presence of bacterial fruit blotch (BFB) using real time PCR (Wechter’s Lab). The S2 seeds of each PI will be increased to 1000 S3 seeds by HM.CLAUSE.

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