Team members:
- Michael Mazourek (Cornell University)
- Linda Beaver (University of Puerto Rico)
- Angela Linares (University of Puerto Rico)
- Chris Smart (Cornell University)
Objectives: Develop common genomic approaches and tools for squash; Perform GBS analysis of PI collections, establish core
populations, provide community resource for genome wide association studies (GWAS); GBS of squash, establish molecular-informed core
populations
Powdery mildew resistance in squash
Marker development and verification
(Mazourek lab –K. LaPlant)
Powdery mildew is a major fungal disease on squash and pumpkins (Cucurbita spp) in the USA and throughout the world. Genetic resistance to the disease is not known to occur naturally within Cucurbita pepo and only infrequently in Cucurbita moschata, but has been achieved in both species through the introgression of a major resistance gene from the wild species Cucurbita okeechobeensis subsp. martinezii. Today, this gene, Pm-0, is used extensively in breeding, and is found in nearly all powdery mildew-resistant C. pepo and C. moschata commercial cultivars. In this study, we mapped C. okeechobeensis subsp. martinezii-derived SNP marker alleles in a set of taxonomically and morphologically diverse and resistant C. pepo and C. moschata cultivars bred at Cornell University that, by common possession of Pm-0, form a shared trait introgression panel. High marker density was achieved using genotyping-by-sequencing, which yielded 266,913 de novo SNP markers in the three Cucurbita species genotyped. A single 516.4 kb, wild-derived introgression was present in all of the resistant cultivars and absent in an equally diverse set of heirlooms that predated the Pm-0 introgression (See Figure 1). The contribution of this interval to powdery mildew resistance was confirmed by association mapping in a C. pepo cultivar panel that included the Cornell lines, heirlooms, and 68 additional C. pepo cultivars. The region containing the resistance allele was refined to a final candidate interval of 76.4 kb. Studies are currently underway to validate markers in this region and explore gene candidates.
Virus resistance in squash
Mapping resistance
(M. Mazourek lab)
We are using an association mapping approach similar to that for powdery mildew resistance, above, to identify introgressions from the sources of resistance: ‘Nigerian Local’ and ‘Menina’. This goal will be accomplished by utilizing a genome-wide association study (GWAS), with a focus on C. pepo, for mapping the resistance genes to Cucumber mosaic virus (CMV), Papaya ringspot virus (PRSV), Watermelon mosaic virus (WMV), and Zucchini yellow mosaic virus (ZYMV). We selected 95 cultivars as the basis of the GWAS panel. The market classes represented in the panel include zucchini, summer squash, crookneck, acorn squash, and pumpkin. The cultivars in the panel include 43 cultivars with resistance to ZYMV, 27 cultivars with reported resistance to WMV, 13 with reported resistance to PRSV, and 10 with reported resistance to CMV. Genotyping-by-sequencing (GBS) was used to genotype the panel. 96-plex GBS libraries were prepared using the restriction enzyme ApeK1 for digestion.
We grew the cultivars in the greenhouse for inoculation screens to verify the resistance phenotype for each virus. We performed an initial inoculation screen on a single replicate of the cultivar panel for each of the four viruses. Plants were inoculated with a 1:10 ratio of disease leaf tissue to phosphate inoculation buffer at the first true leaf stage, and then reinoculated a week later to prevent escapes. Viral symptom severity was recorded several times after the second inoculation. The viral titer in each plant, as indicated by concentration of viral coat protein, was determined using an enzyme-linked immunosorbent assay (ELISA). Young leaf tissue was collected from each plant, and samples were prepared using a 1:10 ratio of leaf tissue to extraction buffer. Analysis of the ELISA revealed that the majority of plants, regardless of symptom severity, accumulate the viruses. We are currently performing inoculation screening on additional replicates for each virus and evaluating the viral titer of each plant using ELISA, which confirms successful inoculation.
Introgress resistance into advanced breeding lines
(L. Beaver, A. Linares labs – M. Miranda, W. Seda)
The PR portion of the project emphasizes developing resistance to potyviruses in tropical pumpkin (Cucurbita moschata), with primary emphasis on Papaya ringspot virus (PRSV), although work will be done on Zucchini yellow mosaic virus (ZYMV) as well. In Puerto Rico and other parts of the tropics, tropical pumpkin is used like a winter squash, that is, in its mature state. It is the most important local vegetable crop in Puerto Rico. Many people consume it daily as part of a traditional “rice and beans” dish, where pieces of pumpkin are added to the bean sauce. Summer squash (C. pepo) and other types of winter squash (butternut types of C. moschata and C. maxima winter squash) are very seldom grown (nor consumed) in Puerto Rico and most Cucurbita cultivars developed for temperate areas are not adapted to the humid tropics. The local market also expects tropical pumpkin (“calabaza”) to have a certain appearance, and most mainland U.S. winter squash cultivars would not be acceptable in the Puerto Rican market. Thus, it is important to incorporate potyvirus and other resistances into germplasm adapted to local conditions. The primary local tropical pumpkin cultivars in Puerto Rico are ‘Taína Dorada’, ‘Soler’ and ‘Verde Luz’. We also would like to incorporate potyvirus resistance into an important breeding line, TP411. These genotypes range from mildly to very susceptible to both PRSV and ZYMV. Two very good sources of resistance to PRSV are known to exist: ‘Nigerian Local’ (NL) and ‘Menina’.
Development of Biparental Populations in Puerto Rico
C. moschata: We have developed F2 populations from crosses between the resistant parents (‘Nigerian Local’ and ‘Menina’) and two susceptible genotypes (‘Taína Dorada’ and ‘Verde Luz’), and are in the process of developing F2 populations between the resistant parents and another two genotypes we will work with (‘Soler’ and ‘TP411’). We are also developing F2 populations between the highly susceptible ‘Waltham Butternut’ and the two resistant genotypes. A previous study concerning the inheritance of resistance to PRSV was done using ‘Waltham Butternut’ x NL, and resistance was reported to be a single recessive gene. We are moving ahead on the assumption that there is a major gene for PRSV resistance. Considerable seed has been or will be produced of each F2 population (at least 400 seeds). Inheritance studies of PRSV will be carried out in at least some of the F2 populations (in all populations if time and resources permit). That work will be done over the next 12 months. Each plant will be phenotyped for symptom severity and tested with ELISAIf markers are identified from the association panel study, then the DNA samples and associated phenotype data could be used to begin validating the those markers in collaboration with Cornell. Further marker validation will be done by self-pollinating a subset of F2 plants (resistant and susceptible types with good horticultural characteristics) to the F4, and then testing those lines for the presence/absence of PRSV resistance markers while at the same time evaluating PRSV symptoms and line performance for horticultural traits.
Phenotyping potyvirus resistance
Our experience suggests that PRSV resistance may be controlled by more than a single recessive gene. Type of symptoms and symptom severity varies among susceptible genotypes. It is possible, even likely, that the two resistant genotypes do not carry the same alleles for resistance (populations for an allele test of ‘Nigerian Local’ and ‘Menina’ are also being developed). Therefore, one of the challenges to working with PRSV resistance will be to effectively phenotype plants in a segregating population. We are using both ELISA and symptom severity to phenotype plants. We have completed a preliminary study that looked at variability in ELISA readings of tissue taken from different parts of a plant. A paper on that topic has been submitted for the Cucurbitaceae 2016 meeting this summer in Poland. Currently we are working on developing a screening system that will likely have 3 to 5 phenotypic classes (not just susceptible vs. resistant) for both symptom severity and ELISA readings. We will test this system when we begin screening the first biparental population (Taína Dorada x Nigerian Local) this month (May 2016) using both of these phenotyping methods.
Field study of effect of PRSV and ZYMV on yield and other horticultural traits
In early April we initiated a field study using seven tropical pumpkin genotypes that are known to have a range of resistance from highly resistance (Nigerian Local, Menina) to intermediately susceptible (Taina Dorada, Soler, Verde Luz) to highly susceptible (Waltham, Mos166). Plants were inoculated in the greenhouse with PRSV or ZYMV, or not inoculated (control), evaluated in the greenhouse for symptom severity and ELISA. About 3 weeks post-inoculation the plants were transplanted to the field (single plant plots, 5 reps per genotype-inoculation type combination). We are documenting how symptoms continue to develop in the field (Do symptoms weaken? Strengthen? Does it depend on the genotype?). Tissue will be collected from each plant on two occasions for ELISA testing. Horticultural traits (flowering date, fruit size, number and weight, flesh thickness, color [*L, chroma, hue], °Brix, %dry matter). We know of no similar study documenting the impact of potyvirus infection in tropical pumpkin.
