Cucurbit Coordinated Agricultural Project Annual Progress Report | Status of core panels (seed stocks; resequencing) & Breeding for disease resistance (powdery mildew, downy mildew, CYSDV, Fusarium)
CucCAP researchers and stakeholders met on October 27, 28 & 29, 2022 to present and discuss the grant’s accomplishments, ongoing research, plans and expectations.
Team members:
- Jim McCreight (USDA, ARS)
- Shaker Kousik (USDA, ARS)
- Pat Wechter (USDA, ARS),
- Bill Wintermantel (USDA, ARS)
CucCAP Affiliated Postdocs and Graduate Students
- Shaonpius Mondal, USDA-ARS, Salinas (McCreight, Wintermantel)
- Prabin Tamang, postdoc, USDA-ARS, Salinas (McCreight, Wintermantel)
- Obj. 2. Map and develop markers for disease resistance
Objectives: Map and develop markers for disease resistance in Melon and introgress, pyramid/stack resistances into advanced breeding lines
Powdery mildew
The F2:3 PI 313970 x Top Mark developed in CA will be planted in a greenhouse at Charleston in November to evaluate for resistance to powdery mildew.
CYSDV
Two QTL for resistance in were found in F2:3 PI 313970 x Top Mark, on chromosomes 3 and 5. The QTL on chromosome 5 was observed in naturally infected field tests in 2018 and 2019, and explained 16 % and 35 % of the variation in CYSDV titer, respectively. The QTL on Chromosome 3 explained 20 % of virus titer variation in 2018 but was undetected in 2019 (Tamang et al. 2021). Single gene recessive, Mendelian resistance was previously reported in PI 313970 (McCreight and Wintermantel, 2011).
One or both of the two markers flanking the gene on Chromosome 5 were present in six of 10 other putative CYSDV resistance sources. Eight F2:3 lines with low virus titer resembled PI 313970 for the two flanking markers, which can, therefore, be utilized in marker assisted breeding of CYSDV-resistant melons.
One of the eight F2:3 lines with low virus titer in 2019 has been evaluated to date for resistance reaction in a controlled inoculation, growth chamber test. Plants will be selfed and backcrossed with CYSDV-susceptible ‘Top Mark’. The other seven F2:3 families are in preparation for screening. Testing was initially delayed due to a permit modification issue, but is now in progress. QTL mapping of these lines will be evaluated concurrently with evaluation of resistance reactions.
Marker development and verification in Melon
Powdery mildew and Fusarium
Melon production is threatened by Fusarium wilt, powdery mildew, and downy mildew. Fusarium wilt is caused by Fusarium oxysporum f. sp. melonis (Fom) and four races have been identified so far in North America. Resistance to races 1 and 2 were identified and well characterized. Powdery mildew (PM) and downy mildew (DM) are caused by biotropic pathogens Podosphaera xanthii and Pseudoperonospora cubensis, respectively. Resistances to PM and DM were identified and characterized. PM resistance identified is oligogenic in nature with a single QTL responsible for the majority of resistance. DM resistance is complex, and all the significant loci together explained lesser than 50 % of resistance. Sulfur, effective against PM, is also effective against DM, and QTL imparting sulfur tolerance were identified. Sulfur application is a feasible strategy to control DM on sulfur-tolerant melons. Thus, sulfur tolerance is an important trait for breeding.
Recombinant inbred line (RIL) 206 is resistant to Fom races 1 and 2, PM and is sulfur-tolerant; its flesh is light-orange in color with low sugar content. Orange flesh, western shippers like ‘Top Mark’ are commonly grown in North America; their flesh is orange colored and fruit is heavily netted. ‘Charentais’ is a French melon with deep orange or salmon color flesh and little to no netting on the fruit surface.
With the objective to develop disease-resistant and sulfur-tolerant commercial melons (orange flesh, western shipper and Charentais), the following crossing scheme was designed to produce four-way [F1(206 x TM) x F1(206 x Charentais)] progenies for genetic analyses.
View an image of the crossing scheme on page 30 of the PDF version of this report
Four hundred, four-way progenies (see above) and their parents were planted, and total genomic DNA was extracted from 2-week-old seedling leaves following the modified Triticarte Pty.Ltd protocol. Quality and concentration of extracted DNA was checked using nano drop. DNA concentration was later adjusted to 10-20 ng/ul. Markers flanking and markers within the QTL region of targeted resistances were utilized for genotyping. Three markers for Fom race 1, five markers for Fom race 2, and three markers for PM race 1 were used as proxies for the respective resistance QTL. PCR reactions (5 μl volume) consisted of 0.07 μl of primer mix (IDT technologies; fluorophore-labeled, allele-specific forward primers and a reverse primer), 2.5 μl of 2× master mix (IDT technologies) and 10-20 ng of sample DNA. A standard thermal cycler was used for a touchdown PCR reaction with a 94°C hot-start activation step for 15 min, then 10 cycles of 94°C (20 s) and a starting annealing temperature of 61°C that dropped by 0.6°C each cycle. Twenty-six additional cycles of 94°C for 20 s and 55°C for 60 s followed the touchdown steps. Fluorescence was quantified with a Stratagene Mx3005P (Agilent Technologies, Santa Clara, CA) quantitative PCR system at 25°C. Fluorescence values were used to cluster samples into genotypes with MxPro v4.10 software associated with the qPCR machine. Selected genotypes were transplanted into 12-inch pots and moved to a greenhouse for crossing.
Two plants homozygous for all the targeted resistance loci were selected, one was crossed with ‘Charentais’ and other with ‘Top Mark’. Five to 10 plants from each cross will be backcrossed with their respective recurrent parent. KASP markers will be used to select desired BC1F1 population and BC1F2 population. Disease screening and sulfur tolerance testing will be done on BC1F3 and BC1F4 based on availability of seeds. Horticultural trait assessment will be done on BC1F5 families.
Fom race 2 resistance
The Fom-1 gene identified and cloned in ‘Védrantais’ imparts resistance against Fom races 1 and 2. Resistance from this source was not quantified. MR-1 is a treasure trove of disease resistance QTL in melon; it showed strong resistance against Fom race 2. Initial mapping with high density SNP map showed one strong peak explaining about 35% of phenotypic variation with Spanish genome as reference. Further, increasing marker density around the identified peak with same reference genome showed another peak, stronger than the first identified QTL and explained about 40% variation; on whole, phenotypic variation explained was 85% as opposed to 40% in the initial mapping experiment. These two peaks seem to be linked very strongly, and physical length of this whole region is 300 kb. Fom-1 was located inside of the peak explaining 30-35 % of phenotypic variation. Oumouloud et al., (2010) in their inheritance study mentioned two independent genes controlling resistance, i.e., one dominant and one recessive in ‘Tortuga’.
To better understand the genetic nature of resistance in MR-1, a large segregating four-way F1 population developed with RIL-206, ‘Top Mark’ and ‘Charentais’ (see above) was phenotyped, and 170 selected individuals were genotyped with KASP markers on both side of the two peaks. Two genotypes showing evidence of crossover were selected and are being grown in a greenhouse. Further cohort phenotyping of F2 families from selected plants, F1 progenies with both resistant and susceptible parents along with parents will reveal the genetic nature of Fom race 2 resistance in MR-1.
Literature Cited
- McCreight, J.D. and W.M. Wintermantel. 2011. Genetic resistance in melon PI 313970 to Cucurbit yellow stunting disorder virus. HortScience 46:1582–1587.
- Oumouloud, A., M.S. Arnedo-Andrés, R. G. Torres, and J.M. Alvarez. 2010. Inheritance of resistance to Fusarium oxysporum f. sp. melonis races 0 and 2 in melon accession Tortuga. Euphytica 176(2):183–189 DOI:10.1007/s10681-010-0201-4
- Tamang, P., K. Ando, W.M. Wintermantel, and J.D. McCreight. 2021. QTL mapping of Cucurbit yellow stunting disorder virus resistance in melon accession PI 313970. HortScience 56:424–430. doi: https://doi.org/10.21273/HORTSCI15495-20.
