Watermelon Team members: Amnon Levi (USDA, ARS), Shaker Kousik (USDA, ARS), Kai-shu Ling (USDA, ARS), Cecilia McGregor (Univ. Georgia), Pat Wechter (USDA, ARS), and Todd Wehner (North Carolina St. Univ.) reported on team progress and work plans.
Overall objectives: Identifying quantitative trait loci (QTL) associated with resistance to major and emerging diseases, developing useful molecular markers and utilizing the genomic tools to incorporate resistance into watermelon cultivars.
Major diseases: Gummy stem blight, Fusarium wilt, Powdery mildew, Phytophthora fruit rot, Papaya ringspot virus (PRSV) and Cucumber green motile mosaic virus (CGMMV).
Work in progress and plans
1.2. Perform GBS analysis of PI collections, establish core populations of the four species, and provide community resource for genome wide association studies (GWAS)
1.2.1. GBS of cucurbit species, establish molecular-informed core populations (T.Wehner, T. Patel)
Increasing watermelon PI accessions and preparing leaf samples for DNA isolation and genome wide association study (GWAS).
We will collect and increase Citrullus PI accessions, heirloom cultivars, and gene mutant type lines. Seed increase of the 2000 PI accessions is being accomplished by seed companies, USDA scientists, and university researchers. Each is increasing 1 to 10 accessions per year using controlled pollination in greenhouse or field.
A list of the PI accessions and gene type lines is being developed to use for the DNA sampling. The sampling protocol is from Michigan State University, and will involve 1000 PI’s sampled at the seedling stage, with one plant per accession.
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).
A list of the 42 type lines is included in Appendix 3.
2.1 QTL map resistances
2.1.1. Watermelon
2.1.1.1 CGMMV
Evaluating the watermelon PI collection for resistance to Cucumber green mottle mosaic virus (CGMMC) and conducting genome-wide association mapping to identify SNPs associated with CGMMV resistance (KS Ling, A Levi)
CGMMV is a tobamovirus, similar to Tobacco mosaic virus (TMV). This virus was first discovered in Europe and has caused serious epidemics in several Asian countries, like China, Japan, and Korea, and more recently in Australia and the Middle East (Jordan and Israel). Due to its seed-borne nature and global seed trade CGMMV geographic distribution has been expanding rapidly and it became a major threat to all major cucurbit crops and the entire cucurbit industries in the U.S. and around world. The CGGMV has been identified as an emerging virus on cucumber, melon, watermelon and other cucurbit crops in Canada and the United States (Ling et al., 2014; Tian et al., 2014) and in Australia.
- Commercial watermelon cultivars are highly susceptible to CGMMV infection. With a start-up fund support from a USDA germplasm evaluation grant and a watermelon-CGMMV consortium, we have planned to screen the entire collections of USDA watermelon germplasm (~1600 accessions).
- Under a special USDA-APHIS permit we have designated an isolated greenhouse at the USDA, ARS, U.S. vegetable Laboratory for working specifically with CGMMV.
- We completed the first screening of 800 accessions using mechanical inoculation with an Asian CGMMV isolate.
- Most of the watermelon PIs evaluated were susceptible CGMMV while several PI plants that may have some level of resistance or tolerance were selected and are under further evaluation.
- Screening of the second half of germplasm collection (800 PI accessions) are underway and should be completed over the 2016 summer.
- Once the primary screening has been completed, a repeat screening with selected promising resistance accessions will be carried out in the second half of the year 2016, as well as for fruit and seed production on the selected resistant plants.
- Once the watermelon germplasm screening is completed. Single plant selection will be conducted. Genetic populations will be developed to generate F1, F2, BC1S, and BC1R populations for the genetic of inheritance study.
- The phenotypic data obtained from this disease resistance screening will be further used in genome-wide association study (GWAS) to identify putative SNPs associated with CGMMV resistance, in collaboration with CucCAP collaborators.
2.1.1.2- Fusarium race 1,2
Genetic mapping of QTL associated with resistance to Fusarium oxysporum races 1 and 2 in Citrullus lanatus var. citroides. (P Wechter, A Levi)
Fusarium oxysporum f. sp. niveum which causes fusarium wilt of watermelon, is considered one of the most important diseases of watermelon production in the United States. There are currently no economical or even viable chemical control strategies or methods that can control this soil-borne pathogen. To date, only a few watermelon lines have been identified and reported as resistant or tolerant to this pathogen. Unfortunately, although some of these lines were reported more than twenty-five years ago, no commercial cultivar is available with resistance to the most serious of the races of this fungus, race 2.
- We developed two Citrullus lanatus var. citroides (Clc) germplasm lines derived from United States plant introductions (PIs) with high levels of resistance to Fon race 1 and 2, USVL246-FR2 and USVL252-FR2. Genetic populations F1, F2, F3 BC1F2 are being generated by crossing each of these resistant lines with susceptible parents.
- Genetic populations F2:F3 are being generated from USVL246-FR2 crossed with the susceptible Clc parent PI 542114 (Pop 46-14); and for USVL252-FR2 crossed with the susceptible Clc parent PI 244017 (Pop 52-17) or PI 244019 (Pop 52-19).
- Inheritance studies are being performed using the populations generated from USVL246-FR2 and the susceptible Clc parent PI 542114 (Pop 46-14), while inheritance study and GBS analyses will be performed for Pop 52-17 and Pop 52-19 in early 2017.
- The first round of Fusarium race 2 assays have been performed on the F3 individuals of Pop 46-14.
- Additional rounds of phenotyping for tolerance to Fon race 2 will be performed during the next 4 months, with Fon race 1 assays beginning at that time.
- DNA has been isolated from 200 F2 plants and each of the F2 plants is being self-pollinated to generate an F3 families of Pop 46-14.
- Genotype-by-sequencing (GBS) will be performed during the next 4 months for Pop 46-14.
2.2.1.1.Converting a Fusarium wilt race 1-resistance QTL to a DNA marker (A Levi, P Wechter)
Fusarium wilt (FW) race 1 is a major disease of watermelon throughout the United States. In a recent study (Lambel et al. 2014), we identified on chromosome 1 of watermelon a major quantitative trait locus (QTL) associated with resistance to FW race 1. There is a need to develop a SNP marker(s) useful in marker assisted selection (MAS) to precisely predict the presence of resistance in large genetic populations.
We have been developing genetic populations F1, F2, BC1R, BCS [Calhoun Gray (R) x Sugar Baby (S); or Calhoun Gray (R) x Black Diamond (S)] segregating for resistance to FW race 1. Also, the bioinformatics team led by Dr. Zhangjun Fei at Boyce Thompson Institute developed for us a data set to compare SNP sequences within this specific QTL on Charleston Gray, Calhoun Gray, Sugar Baby, and Black Diamond.
To identify a tightly linked DNA marker, we will screen candidate SNPs using the DNA of the original F2 plants used to identify the QTL based on the phenotyping of their F3 families (Lambel et al. 2014). Also, we will validate the marker using 500 F2 plants of [Calhoun Gray (R) x Sugar Baby (S); or Calhoun Gray (R) x Black Diamond (S)] segregating for FW race 1 resistance.
2.1.1.3. Gummy stem blight
Develop molecular markers for high resistance to gummy stem blight (GSB) using genome-wide association studies (GWAS) approach in the USDA watermelon germplasm collection, and introgress GSB resistance into watermelon cultivars. (T Wehner, Luis Rivera)
Phenotyping: The WmGsb population was developed by intercrossing the most resistant accessions of Citrullus four times (I4), followed by crossing with elite cultivars of watermelon (I4F1), followed by intercrossing without selection, while maintaining wild and elite types in the populations (I4F1I4), followed by self-pollinations of plants at random (I4F1I4S1). The 296 lines will be screened in the MAF greenhouse and the field at Clinton NC. Resistance will be rated several times on each plot, in an experiment having 2 years, 4 replications, and 2 locations (greenhouse and field).
Genotyping: A group of 384 watermelon accessions are being selected to develop a core watermelon populations. The core watermelon population will be used to develop sequence-based molecular markers (SNPs) using the genotyping by sequencing (GBS) method at Cornell University. We expect to get several thousand of SNPs for the association analysis.
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.
QTL mapping, marker validation and trait introgression of Gummy Stem Blight resistance in watermelon. (C McGregor)
Our goal for this year was to develop two mapping populations for gummy stem blight resistance.
WPop GSB 1: PI 482276 x Crimson Sweet population of at least 100 seeds per line for 184 F2:3 lines. The resistant source for this population is a selection from PI 482276 (Citrullus amarus; previously C. lanatus var. citroides). C. amarus is a crop wild relative (CWR) of the sweet, edible watermelon types (C. lanatus; previousely C. lanatus var. lanatus). Severe segregation distortion and pollen fertility issues have been observed in crosses between C. amarus and C. lanatus. These issues as well as possible chromosomal re-arrangements make population development and QTL mapping challenging in this population.
WPop GSB 2: PI 526233 x Sugar Baby population of at least 100 seeds per line for 92 F2:3 lines. The resistance source is a selection from PI 526233. PI 526233 is C. lanatus which should simplify mapping and trait introgression. However, the level of resistance in PI 526233 is not a high as PI 482276.
Progress Year 1:
For WPop GSB 1, 149 F2:3 lines are complete (100 seeds per line). An additional 53 plants have fruit set and fruit will be harvested once mature.
For WPop GSB 2, 50 F2:3 lines are complete (minimum 100 seeds per line). An additional 50 plants will be transplanted in June.
Leaf samples of all parental, F1 and F2 plants were collected and are currently stored at -80°C.
A Ph.D. Student, Winnie Gimode has been appointed to carry out research on this project.
Conclusion and Future Work
We are on course to complete these 2 populations by the end of 2016. After discussion with the project leader, Amnon Levi, an additional population was initiated. WPop GSB 3 will be developed from a cross between PI 482276 and a susceptible C. amarus accession. This will be a backup population that can be used if the segregation distortion and chromosomal rearrangements in the inter-specific cross (WPop GSB 1) hampers its use.
Phenotyping will be the biggest challenge of this project. Syngenta has agreed to supply us with control lines with known, confirmed (field and greenhouse) phenotypes for Gummy Stem Blight resistance. The inclusion of such controls will ensure consistent results across experiments.
2.1.1.4. Phytophthora fruit rot of watermelon (S. Kousik)
Inheritance of resistance.
We conducted a study to determine inheritance of resistance to Phytophthora fruit rot using the the segregating population derived from the cross of USVL531-MDR x PI 269677. Fruit from parents, F1, F2 and back cross populations were harvested when mature and placed on wire shelves in a walk-in-humid chamber. Each fruit was inoculated with a 7-mm agar plug from an actively growing colony of Phytophthora capsici as described (Kousik et al., 2014). Data on fruit rot was recorded five days after inoculation. We are currently compiling and analyzing the data from this study.
2.1.1.5 Powdery mildew (S Kousik)
Inheritance of resistance.
Inheritance of resistance to powdery mildew of watermelon caused by Podosphaera xanthii was conducted on the segregating population derived from the cross of USVL531-MDR x PI 269677. A total of 713 plants were evaluated. Of these 66 plants were of the resistant parent (USVL531-MDR derived from PI 494531) and 81 plants of susceptible parent (PI 269677). Of the segregating population, 112 were F1, 311 F2, 64 BCF1S and 80 BCF1R. All the plants were inoculated using a suspension (105 conidia-ml) of powdery mildew conidia in sterile water plus 0.02% tween 20 as described before (Kousik et al., 2011). Powdery mildew ratings on a 0-10 scale of increasing disease severity was recorded for hypocotyl, cotyledons and true leaves. Resistance to powdery mildew in cotyledons and true leaves appears to be a dominant trait in USVL531-MDR. Leaf samples from the segregating population and parents were collected for DNA extraction and further analysis by GBS. Of the F2 plants we self-pollinated 186 plants kept in a net house to generate F2:3 populations for further evaluation. Fruit from F2 plants with powdery mildew resistance, uniform red flesh and decent brix (>7) were also collected for further advancement.
2.1.1.6 PRSV-W (A Levi, K-S Ling)
Identification of QTL associated with papaya ringspot virus (PRSV) in watermelon
Genetic populations F2:F3 are being generated using PRSV-susceptible Clc parent USVL252-FR2 crossed with the PRSV-resistant Clc parents PI 244017 (Pop 52-17) or PI 244019 (Pop 52-19) mentioned above. In early 2017 these genetic populations will be evaluated for PRSV-resistance (as described by Ling et al. 2009)
