Watermelon Team | 2023 Progress Report

View tables and images in the PDF version of the 2023 Watermelon Team Report.

Team members:

Amnon Levi (USDA, ARS)
Sandra Branham (Clemson University)
Shaker Kousik (USDA, ARS)
Kai-Shu Ling (USDA, ARS)
Cecilia McGregor (University of Georgia)
Umesh Reddy (West Virginia St University)
Pat Wechter (Clemson University)

Seed multiplication of core collections

Cecilia McGregor (University of Georgia)

Forty-seven accessions were obtained from the USDA germplasm collection for inclusion in the watermelon core collection. These included 39 C. amarus, six C. lanatus and one C.

mucosospermus accessions. S₂ seed for 40 accessions were sent to MSU for DNA extractions.

Map resistances and identify QTL for key cucurbit diseases

The WPop GSB1 (PI 482276 x Crimson Sweet) F_2:3population used for identification of

Qgsb5.1 (syn. ClGSB5.1; Gimode et al., 2020) and Qgsb7.1 (syn. ClGSB7.1; Gimode et al., 2020) is being advanced to a RIL population. This RIL population will be used to identify QTL associated with additional Stagonosporopsis isolates.

We also developed a large (n = 2,000) F₂ population WPop GSB3 from a cross between Crimson Sweet and PI 482379 (resistant). The plant was validated as an F₁ and then vegetatively propagated to make 14 clones in order to obtain a large population from a single F₁ plant. QTL seq will be used to identify QTL associated with resistance in this population. PI 482379 has not been previously used for QTL mapping or breeding.

Develop and verify markers for marker assisted selection (MAS)

We previously developed and/or validated KASP marker assays for selection of Qgsb5.1 (syn. ClGSB5.1;

Gimode et al., 2020), Qgsb5.2 (Adams & McGregor, 2022), Qgsb7.1 (syn. ClGSB7.1; Gimode et al., 2020), Qgsb8.1 (Ren et al., 2020) and Qgsb8.2 (syn. qLL8.1 and qSB8.1; Lee et al. (2021). These QTL were currently being introgressed into elite backgrounds (Table 1). After the two generations of a backcross, recombinant markers and background markers for domestication genes were added to the selection process. Background markers were developed to select cultivar-type alleles for the fruit quality-related domestication traits; loss of bitterness (basic helix-loop-helix, bHLH), soluble sugar content (ClAGA2), and red flesh color (LYCB) (Gong et al., 2022; Ren et al., 2018; Wang et al., 2019). Backcross lines will be evaluated in the field in summer 2023.

Table 1. Progress of introgression of Gummy stem blight resistance loci into elite backgrounds.

Line	Generation	QTL introgressed	Source of resistance
5, 73, 80 &140	BC₃	Qgsb5.2, Qgsb8.1, Qgsb8.2	PI 189225
9, 35, 62, 177 & 262	BC₄	Qgsb7.1, Qgsb5.1	PI 482276
22	BC₃	Qgsb7.1, Qgsb5.1	PI 482276

We also evaluated fourteen genotypes obtained from North Carolina State University (RiveraBurgos et al., 2021a) in the field. These included four released lines, NC-GSB-530W, NC-GSB-
531W, NC-GSB-532W and NC-GSB-528W (Rivera-Burgos et al., 2021b). Sugar Baby and
Crimson Sweet were included as susceptible controls and UGA11 (selection from PI 482379), UGA81 (selection from PI 189225) and UGA 1081 (selection from PI 482276) were included as resistant controls. Plants were artificially inoculated with an S. citrulli isolate and leaf surfaces were kept wet using a mist system. NCSU-RIL-033, NCSU-RIL-002, NCSU-RIL-027, NCSURIL-204 and NCSU-RIL-117 had significantly lower gummy stem blight disease symptoms (AUDPC) than susceptible controls, but symptoms were more severe than resistant controls (Table 2).

References

Adams, L. and C. McGregor. 2022. QTL Associated with Resistance to Stagonosporopsis citrulli in Citrullus amarus. Sci Rep 12:19628.
Gimode, W., K. Bao, Z. Fei and C. McGregor. 2021. QTL Associated with Gummy Stem Blight Resistance in Watermelon. Theor Appl Genet 134:573–584.
Gong, C., B. Li, M. Anees, H. Zhu, S. Zhao, N. He, X. Lu and W. Liu. 2022. Fine-mapping Reveals that the bHLH Gene Cla011508 Regulates the Bitterness of Watermelon Fruit. Scientia Horticulturae 292:110626.
Lee, E.S., D.-S. Kim, S.G. Kim, Y.-C. Huh, C.-G. Back, Y.-R. Lee, M.I. Siddique, K. Han, H.-E. Lee and J. Lee. 2021. QTL Mapping for Gummy Stem Blight Resistance in Watermelon (Citrullus spp.). Plants 10:500.
Ren, R., J. Xu, M. Zhang, G. Liu, X. Yao, L. Zhu and Q. Hou. 2020. Identification and Molecular Mapping of a Gummy Stem Blight Resistance Gene in Wild Watermelon ( Citrullus amarus ) Germplasm PI 189225. Plant Disease 104:16–24.
Ren, Y., S. Guo, J. Zhang, H. He, H. Sun, S. Tian, G. Gong, H. Zhang, A. Levi, Y. Tadmor and Y. Xu. 2018. A Tonoplast Sugar Transporter Underlies a Sugar Accumulation QTL in Watermelon. Plant Physiol 176:836– 850.
Rivera-Burgos, L.A., E. Silverman, N. Sari and T.C. Wehner. 2021a. Evaluation of Resistance to Gummy Stem Blight in a Population of Recombinant Inbred Lines of Watermelon × Citron. HortScience 1:1–9.
Rivera-Burgos, L. A., E. J. Silverman and T. C. Wehner. 2021b. “NC-GSB-524W, NC-GSB-527W, NC-GSB528W, NC-GSB-530W, NC-GSB-531W, and NC-GSB-532W Watermelon Lines with Gummy Stem Blight Resistance and Good Fruit Quality.” HortScience 56(12): 1599-1604.
Wang, C., A. Qiao, X. Fang, L. Sun, P. Gao, A.R. Davis, S. Liu and F. Luan. 2019. Fine Mapping of Lycopene Content and Flesh Color Related Gene and Development of Molecular Marker–Assisted Selection for Flesh Color in Watermelon (Citrullus lanatus). Front. Plant Sci. 10.

Map and develop markers for disease resistance

Sandra Branham, Patrick Wechter and Amnon Levi Clemson University and USDA, ARS U.S. Vegetable Laboratory)

Developing populations (P), phenotyping (Ph), QTL mapping (Q), Fine mapping (F)

Fon race 2, AL/PW/SB, Ph
Completed QTL mapping in the USVL246-FR2xUSVL114 RIL population and narrowed the QTL intervals found in the F2:3 population from the same cross. Developed KASP markers for four QTL and validated them in an independent interspecific
(USVL246x’Sugar Baby’) population. Manuscript in preparation.
Completed disease screening (two replicated tests) of the C. amarus core collection for response to inoculation with Fon race 2 and used the phenotypes for GWAS. Manuscript under review at Plant Disease.
GSB, PW/AL/SB/AK, Ph
Completed disease screening (two replicated tests) of the C. amarus core collection for GSB resistance and used the phenotypes for GWAS. -Manuscript in preparation.
Downy mildew, PW/AL/SB, Ph
Completed disease screening (two replicated tests) of the C. amarus core collection for DM resistance and used the phenotypes for GWAS.
Katuuramu et al. 2022.

Develop marker (M), verify (V)

Fon race 2, AL/PW/SB, M
KASP markers for Fon race 2 resistance were developed in the C. amarus 246×114 RIL population. An F2:3 interspecific population of USVL246-FR2 by ‘Sugar Baby’ was evaluated for reponse to inoculation with Fon race 2 in two replicated tests and the phenotypes used for QTL mapping with the KASP markers. Manuscript under review.
Powdery mildew race 2w
XP-GWAS of powdery mildew race 2 resistance was completed for the USDA Citrullus core collection using historical data. KASP markers were designed for three regions of
the genome with a significant signal. They were validated in two hundred accessions from the extremes of the distribution. Manuscript in preparation.

Introgress, pyramid/stack resistances into advanced breeding lines

Develop breeding lines (B), introgress into cultivated (I), advanced lines (A), release to breeders (R)

Fon races 1 and 2, AL/PW/SB
Phenotypic and marker-based selections for Fon race 2 resistance were made from the interspecific population of USVL246-FR2 by ‘Sugar Baby’ and selfed and backcrossed to ‘Sugar Baby’. We are currently making seed for the F4 and BC1F4 generations from this cross. We have also crossed USVL246 to ‘All Sweet’ and ‘Crimson Sweet’ to begin introgression into a variety of elite backgrounds.

Using ‘genomic selection’ approach to incorporate Fusarium wilt race 2 resistance into watermelon cultivars

Constructing and utilizing training populations for ‘genomic selection’ experiments

USVL246 x USVL114 (RIL), USVL252 x USVL114 (F3)
USVL246 x Sugar Baby (F3), USVL 252 x Sugar Baby (F3; F4; BC1;F2;F3)
Developing small-seeded lines with multiple disease resistance (FW races 1, 2 and Potyviruses)

Genome Wide Association Analysis of Resistance to Downey Mildew in the USDA-ARS Citron Watermelon Germplasm Collection

Dennis Katuuramu, Sandra Branham, Amnon Levi and Patrick Wechter

We screened 122 Citrullus amarus accessions for resistance to Cucurbit downy mildew (CDM) over two tests (environments). The accessions were genotyped by whole-genome resequencing to generate 2,126,759 single nucleotide polymorphic (SNP) markers. A genome-wide association study was deployed to uncover marker-trait associations and identify candidate genes underlying resistance to CDM. Our results indicate the presence of wide phenotypic variability (1.1 – 57.8%) for leaf area infection, representing a 50.7-fold variation for CDM resistance across the C.
amarus germplasm collection (Table 1). Broad-sense heritability estimate was 0.55, implying the presence of moderate genetic effects for resistance to CDM. The peak SNP markers associated with resistance to P. cubensis were located on chromosomes Ca03, Ca05, Ca07, and Ca11. The significant SNP markers accounted for up to 30% of the phenotypic variation and were associated with promising candidate genes encoding disease resistance proteins, leucine-rich repeat receptor-like protein kinase, and WRKY transcription factor.

Watermelon resistance to CGMMV

Kai-Shu Ling, Bazgha Zia, and Amnon Levi (USDA, ARS, U.S. Vegetable Laboratory)

Cucumber green mottle mosaic virus (CGMMV) is an emerging tobamovirus in North America. Following its first detection in 2013 in California, the preventative measures, including quarantine, official control and certified seeds tested negative for CGMMV, have relatively restricted the virus in that state. However, severe CGMMV outbreaks have been reported in Asia, Australia and Europe, which resulted in severe yield losses on various cucurbit crops, including watermelon. The control of this virus through breeding for natural resistance requires the identification of a new source of genetic resistance. In screening of the USDA watermelon germplasm, we have identified a source of resistance to CGMMV in a wild watermelon relative (Citrullus colocynthis L.). A segregating population of F2 libraries was generated through a cross between resistance (USVL#157) and susceptible (USVL#138) C. colocynthis lines. Seedlings from these populations were inoculated with CGMMV and assessed for their resistance using serological assay for virus titer and their phenotypic reaction. Phenotypic analysis through mechanical inoculation of the F2 population revealed a genetic segregation, suggest the existence of two gene model controlling the CGMMV resistance. Bulked segregant analysis was conducted to identify SNPs that are associated with loci that are associated with the resistance to CGMMV infection in watermelon. A segregating F2 population was phenotyped. Resistant and susceptible bulks of F2 individuals were selected for whole genome resequencing. Bulk segregant analysis revealed a total of four tightly associated SNPs to the CGMMV resistance (Figure 1, Table 1). Further analysis revealed several resistance-associated candidate genes in the genome sequence region. The identified markers could be useful to accelerate breeding watermelon with CGMMV resistance through marker-assisted selection.

Plant material and phenotyping

Umesh Reddy (West Virginia State University)

Three hundred MAGIC derivatives of F8 generation have been created using intercrosses involving resistant accessions of Citrullus amarus (PI 482342, PI 189225, PI 526233, PI 482283, PI 482374) and susceptible accessions of Citrullus lanatus (Charleston Grey, Calhoun Gray,
Mickylee, Minilee, All sweet, Crimson Sweet, Petite Sweet). Genomic DNA was isolated from
30 GSB susceptible and 30 GSB resistant individuals using the E.Z.N.A. Plant DNA DS Kit (Omega Bio-Tek, USA). The quantity of genomic DNA was determined using a Qubit fluorimeter (Thermo Scientific, USA), and the quality was evaluated using agarose gel electrophoresis. The resistant and susceptible bulks were constructed by combining equimolar DNA from 30 extremely resistant RILs and 30 extremely susceptible RILs. The two bulks were subjected to whole-genome sequencing on the Illumina platform using paired-end sequencing chemistry (2×150 bp), generating more than one billion reads per bulk and attaining deep genome coverage. Subsequently, the reads were mapped to the parental genomes (USVL246 and Charleston Grey), and the mapping percentage was above 99%. By mapping to the USVL 246 genome, we identified 7,163,041 and 7,266,736 variants from the resistant and susceptible bulks, respectively. Furthermore, when mapping to the Charleston Grey genome, we found 2,205,134 and 517,389 variants from the resistant and susceptible bulks, respectively.

The QTLs associated with GSB resis”ance’were Identified using the qtlseqr (Mansfeld and Grumet, 2018) R package with single-nucleotide polymorphism-index (SNP-index) and Gprime methods (Figure 1). We have identified statistically significant variants/loci on chromosomes 1, 2, 3, 5, 7,8, and 10 associated with GSB resistance. QTL-seq analysis identified several key candidate genes for GSB resistance based on their physical location in the important QTL regions on these chromosomes (Table 1). Significant loci associated with GSB resistance were used to develop PACE genotype markers. Allele-specific primers were designed for the QTLseq SNP and INDEL markers. Polymerase chain reaction (PCR) allelic competitive extension (PACE) genotyping chemistry constituting FAM, HEX, and ROX fluorophores was used to analyze the SNPs (3CR Bioscience, Essex, UK). The polymorphic PACE SNP/INDEL markers were used for genotyping the mapping populations used for QTLseq (N = 60) with contrasting phenotypes.

Gimode, W., K. Bao, Z. Fei and C. McGregor. 2021. QTL Associated with Gummy Stem Blight Resistance in Watermelon. Theor Appl Genet 134:573–584.
Rivera-Burgos, L. A., N. Sari and T. C. Wehner. 2021. Evaluation of resistance to gummy stem blight in a population of recombinant inbred lines of watermelon x citron. HortScience 56: 380-388.
Mansfeld B.N. and Grumet R, QTLseqr: An R package for bulk segregant analysis with next-generation sequencing. 2018. The Plant Genome. 11:2.

Powdery mildew of watermelon.

Shaker Kousik, Amnon Levi (USDA, ARS, U.S. Vegetable Laboratory (USVL), Charleston, SC)

Assembled seeds of commercial seedless cultivars for powdery mildew resistance screening. Sent seeds to CucCAP2 collaborator in Raleigh, NC in 2021 and 2022.
Completed evaluation of commercial seedless varieties for resistance to powdery mildew in 2021 and 2022. Several resistant seedless watermelon lines were identified, and detailed results are presented in Extension section of the report.
Evaluated and collected data on powdery mildew development on 190 RIL lines in the field (Spring 2022). Data analysis is in progress.
Evaluated and collected data on powdery mildew development on RIL lines in the field (summer 2022). Five plants of each RIL line was planted per plot and each RIL line had two replications. The experiment will be conducted again in summer 2023 and currently the seedlings are being grown in the greenhouse and will be transplanted during the last week of April.
Advanced RIL lines with PM resistance and red flesh to develop useable resistant germplasm lines. The advanced RIL lines had the KASP marker based on the NBS-LRR gene in watermelon Chr02, ClaPMR2 that is tightly linked with PM resistance.
Crossed Advanced RIL lines with red flesh to the cultivar Dixie Lee to generate F1 seed. F1 plants were sown in February 2023 to generate F2 seed for phenotyping for resistance to powdery mildew.
Backcrossed USVL608-PMR with Dixie Lee to develop powdery mildew resistant germplasm lines with good horticultural traits. Dixie Lee is a watermelon cultivar with good horticultural traits including high brix and uniform red flesh. Backcross populations will be evaluated phenotypically by inoculating with powdery mildew and genotypically with KASP makers in Fall 2023.
Evaluated multiple disease resistant (MDR) lines (powdery mildew and Phytophthora fruit rot) in the field. (Fall 2022). Of these 36 lines display high levels of resistance to powdery mildew and have been advanced from various PI. Of the 36, 13 are also resistant to Phytophthora fruit rot and can be considered as multiple disease resistant.
Publicly released USVL531-MDR. Manuscript submitted to HortScience has been accepted and was published in the April 2023 issue. The cover Page for the April 2023 issue displays resistance to Powdery mildew in USVL531-MDR compared to USVL677PMS.
Continued development of KASP markers for powdery mildew resistance in watermelon. We utilized the KASP marker developed based on ClaPMR2 in watermelon Chr02 to identify resistant F2 plants in a cross of USVL608-PMR X USVL677-PMS. Based QTLseq analysis done in first SCRI CucCAP grant we had identified one region in Chr02 that was tightly linked to powdery mildew resistance in USVL608-PMR. Time course RNAseq experiments conducted on powdery mildew inoculated plants in a growth chamber (23° ± 1 C) on USVL608-PMR and USVL677-PMS identified the same region in Chr02 with the NBS-LRR gene ClaPMR2 being highly up regulated compared to USVL677-PMS at three and eight days after inoculation. No visible powdery mildew development was observed on USVL608-PMR eight and 14 days after inoculation. As expected USVL677-PMS displayed severe powdery mildew development. Individual F2 plants were genotyped using KASP markers in Chr02. A strong correlation between the resistant phenotype and genotype was observed (Figure 1)

Phytophthora fruit rot of watermelon

Completed studies and published manuscript on broad resistance to post-harvest fruit rot in USVL watermelon germplasm lines. U.S. vegetable Laboratory (USVL) developed germplasm lines USVL020-PFR, USVL203-PFR, USVL782-PFR, USVL489-PFR, and USVL531-MDR and two susceptible cultivar Sugar Baby and Mickey Lee were evaluated against 20 isolates of Phytophthora capsici collected from different states and crops in the USA. All five resistant germplasm lines were significantly more resistant
than the two susceptible checks to all 20 P. capsici isolates. Among the five resistant germplasm lines, USVL020-PFR, USVL782-PFR and USVL531-MDR had broad resistance. Some P. capsici isolates induced minor lesions and rot on USVL489-PFR compared to the other resistant lines. Variation in virulence and genetic diversity among the 20 P. capsici isolates was also observed. The five watermelon germplasm lines will be useful for developing commercial watermelon cultivars with broad resistance to P. capsici. We have made crosses of the germplasm lines with susceptible cultivars and have developed breeding populations (F1, F2 and back cross populations). NIFA SCRI CucCAP grants were acknowledged in this publication.
Crossed Advanced RIL lines (F10-F11) that displayed Phytophthora fruit rot resistance and with red flesh to the cultivar Dixie Lee to generate F1 seed (Fall 2022). Resistance in each of the advanced RIL plants was confirmed prior to making the cross to generate the F1 seed. F1 plants were sown in February 2023 to generate F2 seed for phenotyping for resistance. The F2 population will be grown in a hoop house in September 2023, selfpollinated and the fruit from each individual F2 plant will be evaluated for resistance to Phytophthora fruit rot in a walk-ingrowth chamber. Leaf samples will be collected from all the F2 plants for use in QTLseq and marker analysis.
Collected leaf samples for extraction of DNA from 190 advanced RIL lines.
Completed phenotyping 190 RIL lines for resistance to Phytophthora fruit rot in summer 2022. We will phenotype the RIL lines again in 2023. Data analysis from 2022 is in progress.
Evaluated F2 and F2:3 population of USVL003-MDR (Citrullus mucosospermus) X Dixie Lee (C. lanatus, cultivated type with good horticultural traits). QTLseq analysis indicated significantly associated QTLs with Phytophthora fruit rot resistance in Chr04, Chr07 and Chr10.
Advanced, red-fleshed resistant Phytophthora fruit rot resistant lines (USVL003-MDR x Dixie Lee) after screening and selection.
Advanced F11 RIL lines with res flesh and fruit rot resistance and increased seeds. Another round of seed increase is in progress (March-June 2023) in the hoop house in Charleston, SC.
We are currently evaluating six advanced lines (F11) with Phytophthora fruit rot resistance for horticultural traits and fruit rot resistance at the U.S. Horticultural Research Laboratory FL and in Charleston, SC. One to three of the advanced lines that display good horticultural traits (uniform red flesh and decent brix) will be evaluated again in the fall in Fort Pierce, FL and Charleston prior to public release.

Developing a Multi-Parent Advanced Generation Intercross (MAGIC) Population Useful for Enhancing the Watermelon Germplasm and for identification of gene loci associated with Disease Resistance Watermelon CucCAP2 Team in Collaboration with Seed Companies

Amnon Levi, Shaker Kousik, Cecilia McGregor, Sandra Branham, Patrick Wechter, Zhangjun Fei, Umesh Reddy, and Dennis Katuuramu

Two MAGIC populations are under construction
MAGIC populations are at F2 stage and will be continued to F8/F9 generations in collaboration with seed companies, with the objective to have 500 F8/F9 RIL lines for each population.