Watermelon Team | 2021 Progress Report

cuccap

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.

Team members

  • Amnon Levi
  • Shaker Kousik
  • William Patrick Wechter
  • Sandra Branham
  • Kai-shu Ling
  • Umesh Reddy
  • Cecilia McGregor

CucCAP Postdocs and Graduate Students

  • Dennis Katuuramu-Postdoc at USDA, ARS, U.S. Vegetable Laboratory (Wechter, Levi)
  • Bidisha Chanda-Postdoc at USDA, ARS, U.S. Vegetable Laboratory (Kousik; Ling
  • Gabriel Rennberger -Postdoc at USDA, ARS, U.S. Vegetable Laboratory (Wechter)
  • Puru Natarajan -Postdoc at WVSU (Reddy)
  • Yan Tomason – Postdoc at WVSU (Reddy)
  • Venkata Rao Ganaparthi -Graduate Student at Clemson (Branham)
  • Samikshya Rijal -Graduate Student at UGA (McGregor)
  • Lincoln Adams – Graduate Student at UGA (McGregor)
  • Subhash Mahankali -Graduate Student at WVSU (Reddy)

Obj 1. Develop genomic, bioinformatic, mapping approaches and tools for cucurbits

Seed multiplication of core populations (Amnon Levi, Pat Wechter, Shaker Kousik and Cecilia McGregor)

We are preparing 384 Citrullus spp. accessions to be included in the core collection. Of these 305 are Citrullus lanatus, 23 are C. mucosospermus, 38 are C. amarus, 8 are C. colocynthis, and 10 are heirloom cultivars. We shipped to HM.Close 249 seed packs (accessions-PIs) with 50 S2 seeds in each pack. Prior to shipping the seeds to the HM.Close station in Davis, California, they were tested (Wechter’s Lab) for presence of Bacterial fruit blotch using an RT-PCR procedure. HM.Close conducted seed health testing in California and the lots were shipped to the HM.Close station in Thailand. The first 80 accessions are being increased there at this time. HM.Close are planning to provide us roughly 3-4 selfed seed lots per accession – enough to reach the 1,000 seed/accession target. The S1 and S2 seeds are being increased at the U.S.  Vegetable Laboratory in Charleston and by Cecilia McGregor and Team at University of Georgia, Athens, GA (UGA).

Obj 2. Map and develop markers for disease resistance: Sandra Branham, William Patrick Wechter, Shaker Kousik, Amnon Levi, and Dennis Katuuramu

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

  • CGMMV, KSL/AL, P
  • Fon race 2, AL/PW/SB, Ph
  • GSB, PW/AL/SB/AK, Ph
  • Downy mildew, DK/PW/AL/SB, Ph

2.2: Develop marker (M), verify (V)

  • Fon race 2, AL/PW/SB, M
  • PRSV-W, AL/KSL/SB, M
  • ZYMV, AL/KSL/SB, M

Marker development and validation for Fon race 2 resistance in Watermelon

Fusarium oxysporum f. sp. niveum (Fon) is a soil born disease responsible for seedling wilt and death in watermelon. Among the four known pathogenic races, race 2 is considered as the most important soil-born pathogen in US by the National Watermelon Association. Lack of effective fungicides necessitates development and release of resistant cultivars. A Fon race 2 resistant inbred line, USVL246-FR2, was developed in CucCAP1 and will be used as the donor parent for resistance breeding. Fon race 2 resistance of USVL246-FR2 is polygenic and is controlled by five loci on chromosomes 2, 5, 8, 9 and 10. KASP markers have been developed across all of five QTL and tested in the original C. amarus mapping population (USVL246-FR2xUSVL114). An interspecific F2:3 population is being generated to verify the effectiveness of the KASP markers and begin the breeding process. Commercial cultivar, ‘Sugarbaby’ was crossed with USVL246-FR2 to generate the interspecific population. The KASP markers were used to genotype the parents, and four markers were polymorphic for Q9, and 6 each for Q1, Q6 and Q8. The F2 population will be genotyped with the polymorphic KASP markers. F3 families from the self-pollinated, genotyped F2 plants (N=240) will be screened for Fon race 2 resistance in replicated growth chamber trials. Genotype and phenotypic data taken together will be instrumental in validating the KASP markers developed, haplotype block or blocks imparting maximum resistance and to understand the influence of Sugarbaby genetic background in expression of resistance. [True breeding F3 for Fon race 2 resistance will be utilized in further selections leading towards cultivar development]

Phenotypic screening for CDM resistance and marker-trait associations testing via GWAS
in watermelon

Cucurbit downy mildew (CDM), caused by Pseudoperonospora cubensis, is an emerging threat to watermelon production. We screened 122 C. amarus accessions for resistance to CDM over two tests. The accessions were genotyped with 2,126,759 single nucleotide polymorphic (SNP) markers. A genome-wide association study approach 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. Broad-sense heritability estimate was 55%, implying the presence of moderate genetic effect 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 candidate genes including disease resistance proteins, leucine-rich repeat receptor-like protein kinase, and WRKY transcription factor. This information will be useful in understanding the genetic architecture of the P. cubensis–Citrullus spp. patho-system as well as development of resources for genomics-assisted breeding for resistance to CDM in watermelon.

Fig. 1: Distribution of CDM leaf area infection (A); Manhattan plot depicting genome-wide marker-trait
associations for CDM leaf area infection (B) across 122 Citrullus amarus genotypes across two combined
screening tests.

View Figure 1 on page 23 in the PDF version of this report.

Marker development and validation for Powdery mildew resistance

Historical data (Tetteh et al. 2010) for the USDA-NPGS Citrullus collection’s (N=1148) response to inoculation with powdery mildew were used to choose resistant and susceptible bulks (N=50 each) for bulked segregant analysis. Whole-genome resequencing data of the bulks was input for an extreme phenotype-genome wide association study (XP-GWAS). Three QTL (chromosomes 2, 4 and 7) were significantly associated with powdery mildew resistance. The QTL on chromosome 2 was previously associated with resistance in a traditional QTL mapping study (Kim et al. 2015), while the other two QTL are novel. Sixteen KASP markers were designed across these QTL. We are currently testing the KASP markers in 300 of the most and least resistance accessions for validation of the markers.

Marker development and validation for ZYMV resistance

A BC4F2 Citrullus lanatus population (N=183) segregating for resistance to ZYMV was developed from a cross of PI595203 (R) and ‘Charleston Gray’ (S). The population was evaluated for response to ZYMV and 25 each of the most and least resistant individuals were bulked for QTL–seq analysis. QTL–seq using whole–genome resequencing data identified a single significant region on chromosome 3 that had been identified in previous studies. KASP markers (N=22) were developed across the QTL with 13 successful amplification in the population. The peak SNP was a non–synonymous SNP in the eIF4E gene. An independent population (BC6F2; PI 595203 x Charleston Gray–recurrent parent) have been developed for verification.

Marker development and validation for PRSV-W resistance

An F2:3 C. amarus population segregating for resistance to PRSV-W and Fon race 2 resistance was derived from the cross of USVL252-FR2 by USVL019. The population was genotyped with GBS and phenotyped for both diseases. A single QTL (chromosome 3) was associated with resistance to PRSV-W and KASP markers were developed across the region. Twenty-two KASP amplified in the population. An independent F2 population (PI 244019-PRSV-R x PI 596665) has been developed (by Dennis Katuuramu) for verification of the markers.

Obj. 3. Introgress, pyramid/stack resistances into advanced breeding lines

  • Develop breeding/germplasm lines (B), introgress resistance into cultivars (I), Develop and validate advanced lines (A), release lines to breeders (R)
  • Fon races 1 and 2, AL/PW/SB, I
  • PRSV-W, AL/KSL/SB, B
  • ZYMV, AL/KSL/SB, B
  • Pyramiding Fon1, Fon2, PM, PRSV, ZYMV, into advanced breeding lines are ongoing projects at the USDA, ARS, USVL (AL/PW/SK/KL/SB)
  • RILs and MAGIC populations derived from crossing disease resistant C. amarus PIs and watermelon cultivars with desirable fruit quality are being developed and will be used to identify and select advanced lines having resistance and sufficient fruit quality. The MAGIC-RILs should be a useful germplasm resource with diverse allelic combinations to be exploited by the cucurbit/watermelon community for mapping quantitative trait loci (QTLs) and for watermelon varietal development.

Cecilia McGregor and Team at University of Georgia, Athens, GA (UGA)

2.1 Map resistances and identify QTL for key cucurbit diseases

The WPop GSB1 (PI 482276 x Crimson Sweet) F2:3 population 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.

2.2. Develop and verify markers for MAS

KASP marker assays were developed for Qgsb5.1 (syn. ClGSB5.1; Gimode et al., 2020), Qgsb5.2 (Adams and McGregor, unpublished), and Qgsb7.1 (syn. ClGSB7.1; Gimode et al., 2020) identified in our lab. In addition, KASP marker assays were developed and/or tested for Qgsb8.1 (Ren et al., 2020) and Qgsb8.2 (syn. qLL8.1 and qSB8.1; Lee et al. (2021) (Fig. 1).

F2:3 or F2:4 plants were selected from WPop GSB1 (PI 482276 x Crimson Sweet) population based on the presence of resistant alleles for Qgsb5.1 and Qgsb7.1. These lines were backcrossed to Crimson Sweet.

F2:3 plants were selected from WPop GSB2 (PI 189225 x Sugar Baby) population based on the presence of resistant alleles for Qgsb5.2 and Qgsb8.1 or Qgsb8.2. The lines were backcrossed to Sugar Baby.

Umesh Reddy, Todd Wehner, Padma Nimmakayala, Puru Natarajan, Yan Tomason, and
Subhash Mahankali (WVSU)
Using historical data (Gusmini 2005; Todd Wehner) to identify QTL associated with
resistance to gummy stem blight (GSB)

We have sequenced resistant and susceptible bulks (bulks of 30 individual RIL progeny) of the gummy stem blight phenotyped individuals from the MAGIC population. This sequencing produced 1346984333 (RB), 1693263106(SB), 1344506187 (RB) and 1681510931 (SB) mapped reads for resistant bulk (RB) and susceptible bulk (SB) upon mapping with USVL-246 and Charleston Grey (CG) reference genome sequences. These reads produced 6001242 (RB with USVL), 6119469 (SB with USVL), 1821568 (RB with CG) and 401496 (SB with CG) polymorphic SNPs between the bulks.  QTL-seq was performed separately with USVL-246 and CG genomes (Fig 1, 2).

Fig 1: QTL-seq analysis using USVL-246 genome as reference

QTL-seq and G’ analysis detected 16 homozygous loci in susceptible bulk and a mix of R and S loci in resistant bulk when counted respective BAM reads.

Fig 2: QTL-seq analysis using CG as reference

QTL peaks were detected for chromosomes 1, 2, 3, 4, 5, 7, 8, 10 and 11 chromosomes. Known
resistant genes like NBS LRR motif containing, lipoxygenase, several kinases and other important transcription factors were noted in the Delta peaks. A GWAS was conducted to detect robust associations with GSB resistance among 1345 collections using the publicly available GBS generated SNPs.

View figures 1 & 2 on page 25 of the PDF version of this report

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

Powdery mildew of watermelon.

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.

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 in 2021
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.

Development of KASP markers for powdery mildew resistance in watermelon.

During the previous project we had 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 and developed CAPS markers that were validated using the parents, four PM resistant RILs and susceptible and resistant F2 populations using conventional DNA gel electrophoresis. Based on SNPs in Chr2we developed and evaluated eight KAPS markers on a limited set of DNA samples from 40 F2 plants of the cross between USVL531-MDR X USVL677-PMR. Based on this evaluation we narrowed down our choices to four KASP markers and evaluated DNA from 179 F2 plants. Significant correlation (P≤0.0001) between observed phenotype (PM rating) and genotype was observed. This was in relation to powdery mildew ratings recorded on hypocotyl (r=0.72), cotyledons (r=0.84) and true leaves (r=0.81). One of the KASP markers identified the correct resistant or susceptible Phenotype with 97.2% accuracy.

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 (>8) were collected and have been advanced till F8. These lines were homozygous for resistance to PM. They will be further evaluated in the field in 2022 for PM resistance and fruit quality traits.

Phytophthora fruit rot of watermelon

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 it’s top research priority in 2017 as well.

Broad resistance to post-harvest fruit rot in USVL watermelon germplasm lines.

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. These germplasm lines were developed by phenotyping using a local isolate(RCZ-11)  of P. capsici from South Carolina. The present study was undertaken to determine if these resistant lines had broad resistance to diverse P. capsici isolates collected from different states and crops.  Five resistant germplasm lines (USVL020-PFR, USVL203-PFR, USVL782-PFR, USVL489-PFR and USVL531-MDR) and two susceptible cultivars Sugar Baby and Mickey Lee used as checks were grown in a field in 2014 and 2015 to produce fruit for evaluation. Mature fruit were harvested and placed in a walk-in growth chamber and inoculated with 20 different P. capsici isolates. The chamber was maintained at 26±2°C and high relative humidity (>95%) using a humidifier. 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. A publication describing this research was recently published in Plant Disease.

QTLseq analysis of USVL003-MDR X Dixie Lee

We are completed phenotyping the populations from USVL003-MDR x USVL677-PMS for resistance to Phytophthora fruit rot and DNA was extracted from the most susceptible and resistant F2 plants and bulked. Bulked DNA was sequenced by Novogene. QTLseq identified a major QTL in Chr4 significantly associated with resistance. Several other QTL’s significantly associated with resistance were also identified.  We are currently developing KASP Markers based on SNP’s in these regions to conduct marker analysis. In addition a recombinant inbred line developed based on the cross of USVL531-MDR X USVL677-PMS has also been completed and seeds have been increased. These will be evaluated in 2022.

Ling and Levi:
Characterizing watermelon for CGMMV resistance

Previously in CucuCAP1, we had identified a watermelon germplasm (Citrullus colocynthis, PI 537300) with resistance to CGMMV and another line (PI 195927) with susceptibility.  Using these two parental lines, we generated a F2 population of 600 seeds. In 2022, we will conduct phenotyping analysis through bioassay, symptom observation and confirmation through virus testing to evaluate the properties of genetic resistance in watermelon to CGMMV.  In addition, in 2020, we evaluated a list of over 20 chemicals and identified several disinfectants that are effective against the mechanical transmission of CGMMV in watermelon.

Levi, Kousik, Wechter, McGregor, Branham and Dennis Katuuramu
Progress in developing a multi-parent advanced generation inter-cross (MAGIC)
population in collaboration with seed companies

Table 1. Multi-parent advanced generation inter-crosses

View table 1 on page 28 of the PDF version of this report

We have completed the inter-crosses in the table above and are proceeding to the final inter- crosses generation prior to self-pollinating and generation of F2 through F8/F9 RIL generations.

Wehner, Reddy, McGregor, Levi
Advancing a MAGIC-RIL population described in the Table below and derived from crossing
resistant C. amarus lines with watermelon cultivars was developed by Todd Wehner and team at
NCSU.

Table 2. Generation, Breeding approach, and Description.

View table 2 on page 28 of the PDF version of this report.

We have a few seeds of the F8 RILs and we currently advancing them to F9 generation.