Squash Team | 2020 CucCAP Progress Report

Final Progress Report for the CucCAP#1 Squash Team

Team members:

• Michael Mazourek (Cornell University)
• Linda Wessel-Beaver (University of Puerto Rico)
• Angela Linares (University of Puerto Rico)
• Chris Smart (Cornell University)

Objective. 1. Develop common genomic approaches and tools for cucurbits

Establish core GWAS populations

GBS of cucurbit species, establish molecular-informed core populations and 1.2.2. Population genetics and GWAS analysis

The core set of accessions representing Cucurbita pepo diversity in the NPGS has been self-pollinated and is being combined with heirloom cultivars to anchor market classes and enrich for cultivar genetics. Sources of resistance and other representatives of diversity in other species to extend the utility of the panel and being selfed pollinated. The goal of representatives from other species is both because squash improvement often involves crosses between species and to extend the benefits of CucCAP investments to those that work with other species such as C. moschata (Puerto Rico). The second round of self pollination will likely take place with a subcontractor. The process is now including strain purification within the stocks for accessions that do not match their descriptors for hull-less seeded accessions that aren’t, we are creating new selfed stocks from hull-less segregants.

Three projects are already taking advantage of the GWAS population. For CucCAP, given the lack of phenotypic data, we have phenotyped the collection for qualitativetraits of bush growth habit and hull-less seeds. Markers were created with this material validated in breeding populations to include as part of the MS. A separate study is mapping cotyledon cucurbitacin content to support results from biparental populations.

PI collection genomics

Roughly 4,000 -40,000 quality SNPs were called in each of the species collections (C. pepo., C. moschata and C. maxima), which ranged in size from 314 to 829 accessions. Filtered SNPs were used for population structure analysis. Available geographical, phenotypic, and other metadata were retrieved from GRIN and were used to help interpret structure results. These data support five ancestral groups in each of the species. Population structure was driven mostly by geography, except in where the presence of different subspecies was responsible for some of the structure. Filtering all available historical data from GRIN traits with at least 100 entries resulted in 21 traits for C. pepo, 5 for C. moschata and 16 for C. maxima for further analysis. Traits spanned fruit and agronomic-related characteristics, as well as pest resistances. Fruit traits included fruit width, length, surface color and texture, and flesh color and thickness. Agronomic data included plant vigor and vining habit, andseveral phenotypes related to maturity. Pest-related traits included susceptibility to cucumber beetle and squash bug in C. pepo and Watermelon mosaic virus (WMV) and powdery mildew (PM) in C. maxima. Marker-based narrow-sense heritability was calculated for each trait and ranged from 0.12 to close to 1. Most traits had moderate to high heritabilities (≥0.4). Regression of trait data on the Q matrix obtained from structure analysis was used to determine the amount of phenotypic variation explained by population structure. In C. pepo, traits related to fruit morphology tended to have high correlations with population structure. Genome wide association was conducted for all traits using standard mixed-model analysis. No significant signals were detected in C. moschata. A weak signal was detected in C. maxima for fruit set on chromosome 12 and fruit ribbing on chromosome 17. Three phenotypes were significantly associated with SNPs in C. pepo: bush/vine plant architecture on chromosome 10, fruit flesh color on chromosome 5, and fruit width on chromosome 3.

PRSV resistance – Construction of RIL population for mapping

Unlike powdery mildew resistance, virus resistance has not been as widely deployed in commercial breeding lines. Therefore, while we were able to implicate a genomic region in PRSV resistance using a cultivar based association mapping approach, this was not definitive. To validate this region, we created a biparental F2:3 mapping population from a cross between Whitaker (C. pepo subsp pepo) and Success PM (C. pepo subsp ovifera). One hundred three of these families were inoculated with PRSV and phenotyped for the recessive single generesistance derived from C. equadorensis. We designed SNP markers that we can test in this population once campus reopens to research.

Insect resistance

Lauren Brzozowski, a recent PhD in the Mazourek group, used the genotype data from the C. pepo collection to do targeted phenotyping of cucurbitacin content. She currently has a paper in review describing the mapping and candidate genes for the Bi-4 locus in squash which drives striped cucumber beetle herbivory in cotyledons mediated by the presense of cucurbitacins (Brzozowski et al, in review). Striped cucumber beetles are a vector of Squash mosaic virus. There is no known genetic resistance to this virus in Cucurbita and the neonicotinoid chemistry used to control these insects have increased regulatory scrutiny in the US and usage restrictions in Europe.

University of Puerto Rico, Mayagüez, Annual Report –May 2020

In this report we aggregate data from 2019-2020 with data collected in previous years concerning inheritance of resistance to Papaya ringspot virus (PRSV) in tropical pumpkin (Cucurbita moschata). Some of the conclusions made in previous annual reports have been modified after taking into consideration the additional data. For most of the populations, the new data doubles the number of individuals tested in F₂ families resulting in much more robust inheritance tests.

Two sources of resistance are well known in C. moschata: ‘Nigerian Local’ and ‘Menina’. The inheritance of resistance from ‘Nigerian Local’ has been previously studied, but prior to the CucCAP project inheritance studies have not been reported for ‘Menina’, nor was it known if resistance to PRSV in ‘Nigerian Local’ is allelic to that in ‘Menina’. In our inheritance study susceptible genotypes were ‘Verde Luz’, ‘Taina Dorada’ and ‘TP411’. The third to fifth leaf of inoculated seedlings were rated on a 0 to 4 scale for disease severity and scores were combined to convert to a 0 to 12 scale. Resistant x susceptible F₂ populations using ‘Nigerian Local’ as the source of resistance (distributions on the left-hand side of Figure 1) had somewhat normal distributions with an average disease severity of 5.385 (n=231) in Taína Dorada x Nigerian Local and 6.02 (n=208) in Verde Luz x Nigerian Local. In contrast, F2populations with ‘Menina’ (distributions on the right-hand side of Figure 1) were strongly skewed towards resistance with an average severity of 3.52 (n=220) in Taína Dorada x Menina, 3.34 (n=214) in Verde Luz x Menina and 2.80 (n=111) in TP411 x Menina. The Nigerian Local x Menina (resistant x resistant) F₂ population was very strongly skewed toward resistance, with an average combined severity of 0.840 (Figure 2).

To carry out chi-square goodness of fit tests, we grouped plants with an overall severity rating of <4 as resistant and plants with an overall severity rating of >5 as susceptible. The best fit for segregation in F₂ crosses made with ‘Nigerian Local’ was to a 7:9 (R:S) genetic model, although the fit was very poor for Verde Luz x Nigerian Local (Table 1). Goodness-of-fit to other two-class models were much worse than for 7:9. All three crosses using ’Menina’ fit a 3:1 model. The resistant x resistant cross (Nigerian Local x Menina) fit a 15:1 model. These segregations suggest that at least two genes are involved in the inheritance of resistance to PRSV for ‘Nigerian Local’ while a single dominant gene might be responsible for the resistance of ‘Menina’. The data clearly indicate that at least some of the genes for resistance in ‘Nigerian Local’ and ‘Menina’ are different. Our data indicates that the resistance to PRSV conferred by ‘Menina’ is superior to that of ‘Nigerian Local’. If the resistance of ‘Menina’ is a single dominant gene as this data suggests, then it will likely be easier to identify resistance markers in ‘Menina’ than in ‘Nigerian Local’.

Figure 1. (below) Distributions of symptom severity in F₂ populations of tropical pumpkin (Cucurbita moschata) inoculated with Papaya ringspot virus (PRSV). Populations developed with resistant parent ‘Nigerian Local’ are shown on the left; populations developed with resistant parent ‘Menina’ are shown on the right. For each plant, disease severity in leaf position 3, 4 and 5 was evaluated on a 0 to 4 scale (0 = no symptoms). Values were summed to produce an overall severity index of 0 to 12.

Figure 2. Distribution of combined severity ratings of plants (n=238) from the Nigerian Local x Menina F₂ population inoculated with Papaya ringspot virus (PRSV). For each plant, disease severity in leaf position 3, 4 and 5 was evaluated on a 0 to 4 scale (0 = no symptoms). Values were summed to produce an overall severity index of 0 to 12

View the figures on pages 67 & 68 of the pdf version of this report.

Table 1. Number of plants evaluated and observed segregations in parental, F₁ and F₂ populations. ‘Nigerian Local’ was the resistant parent in the F₁ and F₂ crosses. Goodness-of-fit in F₂ populations was tested with chi-square.

Genotype	Population	Observed segregation			χ2	Prob.
		R¹	S¹
Nigerian Local	Res. Parent	34	0
Menina	Res. Parent	50	0
Taina Dorada	Sus. Parent	2	18
Verde Luz	Sus. Parent	4	16
TP411	Sus. parent	0	9
Resistant x susceptible crosses with Nigerian Local as resistant parent:
Taína Dorada x Nigerian Local	F1	8	2
Verde Luz x Nigerian Local	F1	10	0
Taína Dorada x Nigerian Local	F2	88	143	7:9	3.019	0.0823
Verde Luz x Nigerian Local	F2	74	134	7:9	5.646	0.0175
Resistant x susceptible crosses with Menina as resistant parent:
Taína Dorada x Menina	F1	10	0
Verde Luz x Menina	F1	10	0
TP411 x Menina	F1	10	0
Taína Dorada x Menina	F2	156	64	3:1	1.964	0.1611
Verde Luz x Menina	F2	152	62	3:1	1.801	0.1796
TP411 x Menina	F2	91	20	3:1	2.886	0.0894
Cross between two resistant parents:
Nigerian Local x Menina	F1	20	0
Nigerian Local x Menina	F2	224	14	15:1	0.055	0.8147

View the pdf version of this report