CucCAP researchers and\u00a0stakeholders<\/a> met on October 27, 28 & 29, 2022 to present and discuss the grant\u2019s accomplishments, ongoing research, plans and expectations. View all tables and figures in pages 21 \u2013 28 of the pdf version of this report<\/a><\/strong>.<\/p>\n We are preparing 384 Citrullus<\/em> spp. accessions to be included in the core collection. Of these 305 are Citrullus lanatus<\/em>, 23 are C. mucosospermus<\/em>, 38 are C. amarus<\/em>, 8 are C. colocynthis, <\/em>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\u2019s 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 \u2013 enough to reach the 1,000 seed\/accession target. The S1 and S2 seeds are being increased at the U.S.\u00a0 Vegetable Laboratory in Charleston and by Cecilia McGregor and Team at University of Georgia, Athens, GA (UGA).<\/p>\n Fusarium oxysporum <\/em>f. sp. niveum (Fon) <\/em>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<\/em> race 2 resistant inbred line, USVL246-FR2, was developed in CucCAP1 and will be used as the donor parent for resistance breeding. Fon<\/em> 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<\/em> mapping population (USVL246-FR2xUSVL114). An interspecific F2:3 <\/sub>population is being generated to verify the effectiveness of the KASP markers and begin the breeding process. Commercial cultivar, \u2018Sugarbaby\u2019 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<\/sub> population will be genotyped with the polymorphic KASP markers. F3<\/sub> families from the self-pollinated, genotyped F2<\/sub> plants (N=240) will be screened for Fon<\/em> 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<\/sub> for Fon<\/em> race 2 resistance will be utilized in further selections leading towards cultivar development]<\/p>\n Cucurbit downy mildew (CDM), caused by Pseudoperonospora cubensis, <\/em>is an emerging threat to watermelon production. We screened 122 C<\/em>. amarus<\/em> 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<\/em>. amarus<\/em> 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<\/em> 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<\/em>–Citrullus<\/em> spp. patho-system as well as development of resources for genomics-assisted breeding for resistance to CDM in watermelon.<\/p>\n View Figure 1 on page 23 in the PDF version of this report.<\/a><\/strong><\/p>\n Historical data (Tetteh et al. 2010) for the USDA-NPGS Citrullus<\/i> collection\u2019s (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.<\/p>\n A BC4F2 Citrullus lanatus<\/em> population (N=183) segregating for resistance to ZYMV was <\/span>developed from a cross of PI595203 (R) and \u2018Charleston Gray\u2019 (S). The population was <\/span>evaluated for response to ZYMV and 25 each of the most and<\/span> least resistant individuals were <\/span>bulked for QTL<\/span>–<\/span>seq analysis. QTL<\/span>–<\/span>seq using whole<\/span>–<\/span>genome resequencing data identified a <\/span>single significant region on chromosome 3 that had been identified in previous studies. KASP <\/span>markers (N=22) were developed across the Q<\/span>TL with 13 successful amplification in the <\/span>population. The peak SNP was a non<\/span>–<\/span>synonymous SNP in the eIF4E gene. An independent <\/span>population (BC6F2; PI 595203 x Charleston Gray<\/span>–<\/span>recurrent parent) have been developed for <\/span><\/span>verification.<\/span><\/span><\/p>\n An F2:3 C. amarus<\/i> 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.<\/p>\n 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 Stagonosporopsi<\/em>s isolates.<\/p>\n 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).<\/p>\n 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.<\/p>\n 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.<\/p>\n 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.\u00a0 QTL-seq was performed separately with USVL-246 and CG genomes (Fig 1, 2).<\/p>\n QTL-seq and G\u2019 analysis detected 16 homozygous loci in susceptible bulk and a mix of R and S loci in resistant bulk when counted respective BAM reads.<\/p>\n QTL peaks were detected for chromosomes 1, 2, 3, 4, 5, 7, 8, 10 and 11 chromosomes. Known<\/span> View figures 1 & 2 on page 25 of the PDF version of this report<\/strong><\/a><\/p>\n Powdery mildew (PM) of watermelon (Citrullus lanatus<\/em>) and other cucurbits caused by Podosphaera xanthii<\/em> 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.<\/p>\n 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 During the previous project we had identified as ClaPMR2, Citrullus lanatus<\/em> PM Resistance gene 2 {Chr2 : 26750001 .. 26753327 (-)}, a NBS-LRR resistance protein (R) with homology to the Arabidopsis thaliana<\/em> 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\u22640.0001) 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.<\/p>\n 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.<\/p>\n 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\u2019s top research priority in 2017 as well.<\/p>\n 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)\u00a0 of P. capsici<\/em> from South Carolina. The present study was undertaken to determine if these resistant lines had broad resistance to diverse P. capsici<\/em> isolates collected from different states and crops.\u00a0 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<\/em> isolates. The chamber was maintained at 26\u00b12\u00b0C 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<\/em> isolates. Among the five resistant germplasm lines, USVL020-PFR, USVL782-PFR and USVL531-MDR had broad resistance. Some P. capsici<\/em> 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<\/em> isolates was also observed. The five watermelon germplasm lines will be useful for developing commercial watermelon cultivars with broad resistance to P. capsici<\/em>. A publication describing this research was recently published in Plant Disease.<\/p>\n 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\u2019s significantly associated with resistance were also identified.\u00a0 We are currently developing KASP Markers based on SNP\u2019s 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.<\/p>\n Previously in CucuCAP1, we had identified a watermelon germplasm (Citrullus colocynthis<\/em>, PI 537300) with resistance to CGMMV and another line (PI 195927) with susceptibility.\u00a0 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.\u00a0 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.<\/p>\nTeam members<\/h2>\n
\n
CucCAP Postdocs and Graduate Students<\/h3>\n
\n
Obj 1. Develop genomic, bioinformatic, mapping approaches and tools for cucurbits<\/h2>\n
Seed multiplication of core populations (Amnon Levi, Pat Wechter, Shaker Kousik and Cecilia McGregor)<\/h3>\n
Obj 2. Map and develop markers for disease resistance: Sandra Branham, William Patrick Wechter, Shaker Kousik, Amnon Levi, and Dennis Katuuramu<\/h2>\n
2.1: Developing populations (P), phenotyping (Ph), QTL mapping (Q), Fine mapping (F)<\/h3>\n
\n
2.2: Develop marker (M), verify (V)<\/h3>\n
\n
Marker development and validation for Fon race 2 resistance in Watermelon<\/h3>\n
Phenotypic screening for CDM resistance and marker-trait associations testing via GWAS
\nin watermelon<\/h3>\nFig. 1: Distribution of CDM leaf area infection (A); Manhattan plot depicting genome-wide marker-trait
\nassociations for CDM leaf area infection (B) across 122 Citrullus amarus genotypes across two combined
\nscreening tests.<\/h4>\nMarker development and validation for Powdery mildew resistance<\/h3>\n
Marker development and validation for ZYMV resistance<\/h3>\n
Marker development and validation for PRSV-W resistance<\/h3>\n
Obj. 3. Introgress, pyramid\/stack resistances into advanced breeding lines<\/h2>\n
\n
Cecilia McGregor and Team at University of Georgia, Athens, GA (UGA)<\/h3>\n
2.1 Map resistances and identify QTL for key cucurbit diseases<\/h3>\n
2.2. Develop and verify markers for MAS<\/h3>\n
Umesh Reddy, Todd Wehner, Padma Nimmakayala, Puru Natarajan, Yan Tomason, and
\nSubhash Mahankali (WVSU)
\nUsing historical data (Gusmini 2005; Todd Wehner) to identify QTL associated with
\nresistance to gummy stem blight (GSB)<\/h3>\nFig 1: QTL-seq analysis using USVL-246 genome as reference<\/h4>\n
Fig 2: QTL-seq analysis using CG as reference<\/h4>\n
resistant genes like NBS LRR motif containing, lipoxygenase, several kinases and other\u00a0<\/span><\/span>important transcription factors were noted in the Delta peaks. A GWAS was condu<\/span>cted to detect <\/span>robust associations with GSB resistance among 1345 collections using the publicly available <\/span>GBS generated SNPs.<\/span><\/span><\/p>\nShaker Kousik, Patrick Wechter, Sandra Barnham, Amnon Levi; USDA, ARS, U.S. vegetable
\nLaboratory (USVL), Charleston, SC<\/h3>\nPowdery mildew of watermelon.<\/h3>\n
Identifying and developing multiple disease resistant lines from accessions<\/h3>\n
\nand 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.<\/p>\nDevelopment of KASP markers for powdery mildew resistance in watermelon.<\/h3>\n
Advancing Powdery mildew resistant inbred lines.<\/h3>\n
Phytophthora fruit rot of watermelon<\/h3>\n
Broad resistance to post-harvest fruit rot in USVL watermelon germplasm lines.<\/h3>\n
QTLseq analysis of USVL003-MDR X Dixie Lee<\/h3>\n
Ling and Levi:
\nCharacterizing watermelon for CGMMV resistance<\/h3>\nLevi, Kousik, Wechter, McGregor, Branham and Dennis Katuuramu
\nProgress in developing a multi-parent advanced generation inter-cross (MAGIC)
\npopulation in collaboration with seed companies<\/h3>\nTable 1. Multi-parent advanced generation inter-crosses<\/h4>\n