2018 CucCAP Squash Team Annual Report

Progress Reports and Work Plans

Team members: Michael Mazourek, (team leader, Cornell Univ.), Linda Beaver (Univ. Puerto Rico), Angel Linares (Univ. Puerto Rico), Chris Smart (Cornell Univ.)

Powdery mildew resistance in squash Marker development and verification (Mazourek lab –K. LaPlant)

Completed in 2017

Virus resistance in squash, Mapping resistance (M. Mazourek lab-K. LaPlant)

We are investigating resistance derived from C. ecuadorensis by locating genomic regions that contain C. ecuadorensis-specific alleles within a panel of 86 C. pepo cultivars. We have defined C. ecuadorensis-specific alleles in this study as a set of 13,274 GBS SNP markers that are fixed variants between C. ecuadorensis and a phenotypically diverse set of heirloom C. pepo cultivars. To aid in the identification of C. ecuadorensis introgressions, we have genotyped ‘Whitaker’ using GBS, a C. pepo summer squash from Cornell that is resistant to PRSV and CMV, as well as powdery mildew and ZYMV. The pedigree of ‘Whitaker’ contains C. ecuadorensis and C. okeechobeensis subsp. martinezii, and therefore it contains several introgressions from each species within its genome. By using ‘Whitaker’ as a guide to common introgressions from C. ecuadorensis, we have tentatively identified a genomic region on chromosome 16 with a length of approximately 1 Mb that may be associated with resistance to PRSV. The 20 C. pepo members of the cultivar panel that express PRSV resistance are homozygous for C. ecuadorensis-specific alleles within this genomic region. To bolster the cultivar panel, we are using GBS to genotype historical germplasm from the Cornell breeding program that has been phenotyped for PRSV and CMV resistance. ‘Whitaker’ has been used extensively in the breeding program, and thus these additional resources will aid in refining the genomic region associated with resistance. Additional sources of resistance have been incorporated to this historical germplasm, which will also be explored. Additionally, we are developing a ‘Whitaker’-based mapping population to further refine and validate any identified genomic regions associated with resistance.

Introgress resistance into advanced breeding lines

(L. Beaver, A. Linares labs – M. Miranda, W. Seda)

Inheritance of resistance to PRSV:

To date we have evaluated the following resistant x susceptible F2 populations: Nigerian Local x Taína Dorada (335 F2 plants evaluated), Menina x Taína Dorada (120 F2 plants evaluated) and Verde Luz x Menina (120 F2 plants evaluated). In addition, we have recently finished evaluating the F2 population cross between our two sources of PRSV resistance: Nigerian Local and Menina.  Plants of each population were mechanically inoculated with PRSV and then scored for disease severity and tested with ELISA. DNA samples were collected from most of the F2 plants. DNA samples will be lyophilized and will be used only in the event that other approaches to development of a molecular marker for PRSV are not successful (they could be then used for bulk segregant analysis) . The most resistant plants (based on both symptom severity and ELISA) have been transplanted to the field for self-pollination (see below).  Although data analyses of the inheritance studies are not complete, we can make some conclusions. Segregation ratios from the resistant x susceptible F2 populations varied, depending on the susceptible or resistant parent used in the cross. None of the populations segregated 1:3 (resistant:susceptible), indicating that there is not a single recessive gene for PRSV resistance as has been previously reported in the literature.  When Menina and Nigerian Local were crossed, the F2 population clearly segregated for resistance, indicating that at least some gene or genes for resistance in these two genotypes are not allelic. Based on symptom severity, we saw a segregation of 215 resistant to 23 susceptible in the F2 (combined data from 4 greenhouse trials). Based on ELISA readings, we saw a segregation of 166 resistant to 72 susceptible. We tested two common 2-gene models, 15:1 and 13: 3 for goodness-of-fit. The null hypothesis of goodness-of-fit was rejected in each case, but not by very much. However, each of these models proposes a single dominant gene in each resistant genotype. But susceptible x resistance crosses have not produced resistant F1 plants as would be expected if Menina and Nigerian Local carried a dominant gene for resistance. These traditional inheritance studies are being carried out as an auxiliary study to the search for molecular markers. Therefore, the fact that these two sources of resistance carry separate genes for resistance will need to be considered when developing markers for resistance to PRSV.

Tropical pumpkin lines with resistance to PRSV and ZYMV:

Self-pollination of lines derived from the various resistant x susceptible F2 populations has continued. After each self-pollinated, the lines that have been advanced a generation are tested for resistance to PRSV and ZYMV via mechanical inoculations in the greenhouse. Highly and moderately susceptible lines are eliminated, and a single most resistant plant is transplanted to the field for another generation of self-pollination.  This process was somewhat slowed because of Hurricane Maria in Sept 2017. All field plantings were destroyed. But to date we have 54 F3 families, 13 F5 families and 10 F6 families that have been selected for resistance to PRSV. For ZYMV we have 5 F3 families, 6 F5 families and 12 F6 families. In general, we have observed that it has been easier to select for resistance to ZYMV (which is known to be controlled by dominant genes). In the case of PRSV, many families identified as resistant in the F2, F3 or F4, later appear to be completely susceptible or continue segregating for resistance (again, suggesting that inheritance of resistance to PRSV is somewhat complex).  The resistant families generated to date will be used to validate any PRSV markers that are developed.

Effect of PRSV and ZYMV on tropical pumpkin development and production:

A field trial in summer 2017 in Lajas, Puerto Rico was conducted to document the effect of PRSV and ZYMV on development and growth of tropical pumpkin. The study included resistant genotypes Nigerian Local and Menina along with 4 susceptible cultivars. There was a general trend for infected plants to flower later, although this effect was significant only for plants infected with both PRSV+ZYMV (double infection). Control plants produced an average of 3.4 fruits per plant, while plants infected with PRSV produced only 2.15 fruits on average.  Plants infected with ZYMV or PRSV+ZYMV produced fewer fruits per plant compared to control plants, although the difference was not significant. Fruit yield (weight) was strongly impacted by PRSV, ZYMV and PRSV+ZYMV (double infection). Yields were almost 50% less in infected compared to control plants.  We noted that fruit production in Menina was unaffected by virus infection (infected plants produced the same yield as control plants). However, yields in control plants of Nigerian Local were double that of PRSV or ZYMV infected plants, despite the fact that we never observed virus symptoms in this supposedly resistant genotype. Pulp thickness and % dry matter were unaffected by the presence or absence of virus. This is the first study that we know of to document the effects of these two potyviruses in tropical pumpkin at the field level.

“Resistant” lines as a source of virus inoculum:

Lines with a low score for symptom severity (score of 0 or 1, and thus classified as “resistant” for PRSV or ZYMV) can sometimes have high ELISA readings (we consider readings of >0.400 as positive for virus). We conducted a series of studies to determine whether sap from “resistant” lines (based on the line having few or no foliar symptoms) can infect susceptible genotypes. Our results indicate that plants of Nigerian Local and Menina inoculated with PRSV or ZYMV are not capable of infecting susceptible plants. In contrast, several of the “resistant” lines that we have developed are capable of infecting susceptible plants. These results support the practice of considering both foliar symptoms and virus titer (as indicated by ELISA) when evaluating for virus resistance.

Phytophthora blight resistance in butternut squash

Mapping resistance and breeding new butternut squash with resistance to Phytophthora blight and Introgress resistance into advanced breeding lines

(M. Mazourek)

We are self-pollinating F2’s between a mildew resistant bush butternut breeding line and Phytophthora blight resistant C. moschata accessions PI 211996, PI 483347. Crosses with PI 634693 and this breeding line were unproductive and this population has been put on hold accordingly. Last summer we will screened F2 individuals and found them to be asymptomatic when inoculated with P. capsici on the blight farm. We will repeat this screen in the greenhouse with F2:3 populations for QTL analysis. Work in the greenhouse will allow us to have more controlled inoculations with more aggressive strains of the pathogen.