CucCAP#1 Final Annual Progress Report
Team members: Yiqun Weng (USDA, ARS), Rebecca Grumet (Michigan St. University), Todd Wehner (NC State University)
Develop genomic approaches and tools
GBS of PI collection, establish GWAS core
Personnel: Weng (Wang Y, Tan J, Dymerski R), Grumet (Grumet R, Hammar S.) and Wehner (Wehner T., Silverman EJ) Labs
GBS of PI lines and GWAS panel selection
GBS has been completed for 1234 cucumber accessions including plant introduction (PI) lines and historical cultivars or landraces of cultivated (Cucumis sativus var. sativus) and wild (C. sativus var. hardwickii) cucumber lines. Data analysis was been performed by the bioinformatics team to identify SNPs, determine minor allele frequency, perform phylogenetic, population genomic, and linkage disequilibrium (LD) analysis. A core collection consisting of 392 lines was constructed which captures >95% of allelic diversity as well combined with representation of key disease resistance, fruit quality and agronomic features. This part of work was recently published in the journal, Horticultural Research (Wang et al., 2018).
Seed increase and selfing was started for the GWAS panel. Among the 390 lines, we requested fresh seeds from USDA collection for 119 lines. All 390 lines have undergone at least at least two-generation of selfing. Seed increase for 188 lines is underway. We have also re-sequenced one and half plates of samples 144 lines) at ~10× coverage.
Phenotyping of morphological traits and DM resistance in cucumber core population.
Three hundred cucumber lines were grown in the University of Wisconsin Hancock Agricultural Research Station (HARS) for collection of morphological data. Meanwhile, 100 cucumber lines (2 reps, 6 plants per rep) were planted in North Carolina State University experimental field in summer 2019. Data for responses to DM natural infestation were collected. Unfortunately, the data were incomplete due to weather-related inoculation failure.
Genomic assisted breeding
QTL mapping, marker development for DM and PFR resistances
Downy mildew (DM)
(Weng and Wehner Labs)
2019 progress: We aim to conduct QTL mapping of DM resistance from two resistant sources: PI 330628 (WI7120) and PI 197088. We previously identified two major-effect QTL dm4.1 and dm5.2 for DM resistance from WI7120 (Wang et al. 2016). Using the PI 197088×Coolgreen RIL population, we also identified 4 major- or moderate-effect QTL, dm4.1, dm5.1, dm5.2, and dm5.3 for DM resistance in PI 190788; dm5.3 is co-localized with pm5.1 (syn. CsMLO1 or CsMLO8, pm-h), which is a major-effect QTL for PM resistance in cucumber (Wang et al. 2017). We focused on three major-effect DMR QTL, dm4.1, dm5.2 from WI7210 and dm5.3 from PI 197088 for fine mapping.
F2 and RIL plants carrying respective QTL regions were selected to backcross with the susceptible cucumber line 9930. Near isogenic lines (NILs) for each QTL were developed in the susceptible 9930 genetic background. We have completed marker-assisted development of NILs for dm4.1 and dm5.2. Secondary F2 populations from crosses between resistant and susceptible NILs were developed, which were genotyped for DM inoculation responses in both field and controlled environments. The development of NILs for dm5.3 has been advanced to BC2.
Through QTL analysis in the secondary F2 populations, the dm4.1 and dm5.3 loci have been delimited to ~33 and 68 kb intervals on chromosomes 4 and 5, respectively. Growth chamber and field evaluation of DM resistance of the NILs was conducted in collaboration of industry collaborators.
Phytophthora capsici fruit rot resistance in cucumber
(R Grumet lab – YC Lin, B Mansfeld )
Young fruit resistance to P. capsici
SNP-based linkage analyses are being performed to identify disease resistance QTL from crosses between the susceptible, sequenced pickling cucumber breeding line, Gy14, and two PI109483-derived breeding lines using three populations. Phenotyping of F2 populations exhibited normal distributions for disease scores. Individuals representing the most resistant and most susceptible plants were selected for bulk segregant QTL-seq analysis. There was good correspondence between peaks observed on chromosomes 5 and 6 from field grown plants in 2018 and 2019.
Figure 1. QTL-seq analysis of response of young cucumber fruit to P. capsici (data from field trial, summer 2018). Red and green lines – significance P,0.05, 0.01, respectively.
Figure 3. (a) Distribution of disease ratings of young fruit harvested from F2 individuals [Gy14 x PI109483-53B] non-recombinant for the putative resistance QTL on chromosome 5 and/or 6. . Ratings at 5 days post inoculation. (Population size=82; 10-30 fruits were sampled from each plant over three harvests to provide replication in sampling dates). (b) Frequency of A4-3 allele at chromosome 5 QTL as a function of disease rating.
View figures on page 56 of the PDF version
2019/2020. KASP markers flanking the putative QTL on chromosomes 5 and 6 were designed to verify QTL and narrow the QTL intervals. A population of F2 seedlings (n=768) was planted in the greenhouse in late spring 2019. Non-recombinant individuals for the QTL on either chromosome 5 or 6 were selected for QTL verification. Plants were grown in the field in summer 2019 and inoculated in the laboratory with P capsisi, isolate Bartley’s 1.
Disease screening of F2s supported a role for disease resistance for the QTL on chromosome 5. The frequency of the A4-3 was very high (0.78) in the most resistant plants and dropped to 0.19 in the most susceptible plants (Figure 3b). The QTL on chromosome 6 did not appear to correlate with resistance.
Recombinant individuals within the QTL regions were selected to refine the QTL interval. We are currently producing F3:4 seed for testing this summer.
To enable future GWAS analysis, seed for 267 accessions of the cucumber core population that were available, were tested for response to P. capsisi. Three plants per line were grown in a completely randomized design in the field in summer 2019; 20-40 fruit were sampled per line. Depending on restrictions, the remainder of the population will be tested in 2020 or 2021.
Age-related resistance (ARR) to P. capsici
QTL mapping of ARR. F2 progeny and doubled haploid (DH) lines derived from F1 progeny of ‘Gy 14’ (ARR-) X ‘Poinsett 76’ (ARR+) were tested for ARR for QTL-seq analysis. In both populations a single QTL was identified on chromosome 3.
Figure 2. QTL-seq analysis of ARR of cucumber fruit to P. capsici. Red and green lines – significance, P,0.05, 0.01, respectively.
View the figure on page 57 of the PDF version
2019/2020. KASP markers flanking the QTL on chromosome 3 were designed to initiate fine mapping. A population of F2 seedlings (n=768) was screened to identify recombinants in this region. F3:4 seed has been produced from 41 recombinant families. These families will be genotyped with an expanded set of KASP markers to identify points of recombination. Pending permits, the F4 families will be phenotyped in the greenhouse in summer 2020.
Phenotyping the cucumber core collection.
Phenotyping of the core collection for fruit traits and resistance to P. capsici was initiated with 267 lines (S2 or S3 generation, 3 plants/accession) in summer 2019 as a prelude to future GWAS analysis. The core accessions exhibited variation for response to P. capsici.
Figure 3. Phenotypic analysis of the cucumber core collection for fruit morphology and resistance to P. capsici. 30-50 fruits were tested for P. capsici/PI
View images of the core collection on page 58 of the pdf
2.3 Advanced line development for downy mildew resistance
Marker-assisted QTL pyramiding
(Weng and Wehner Labs)
Our objective is to develop a new version of the elite pickle cucumber inbred line Gy14 with improved DM resistance to the post-2014 DM strain. We focused on marker-assisted pyramiding of the two major-effect QTL (dm4.1 and dm5.2) of DM resistance from WI7120 into Gy14 genetic background. Crosses were made between Gy14 and plants carrying dm4.1 and dm5.2 QTL from WI7120/PI 197088. In 2019, homozygous lines carrying dm4.1, dm5.2 and both in Gy14 background (dm1) were selected from BC3F2. Plants of the three NILs (dm4.1, dm5.2, dm4.1+dm5.2) and control (Gy14) plants were gown in the University of Wisconsin Hancock Agricultural Research Station for evaluation of horticultural traits. These lines plants were also tested for DM inoculation responses in controlled environments (growth chambers, climate control rooms, artificial inoculation) and open fields (natural infection). Single aNIls and NIL carrying both QTL consistently performed better for downy mildew resistances in oth field and control environments. No linkage drag was found for either QTL on other horticultural traits. A manuscript is being prepared for public release of the introgression lines carrying dm4.1, dm5.2, and dm4.1+dm5.2 (in Gy14 background).
Breeding line development for DM resistance
(Wehner lab: T Wehner, EJ Silverman)
RIL development and evaluation of DM resistance.
The RILs population was developed in 2007 by a cross PI 197088 (HR) × Coolgreen (S). A total of 200 F2 lines were generated and self pollinated in the greenhouse in 2009. The RILs have been tested in 7 years of field evaluations under high disease intensity. The 2017 population contains 146 lines; 71 at S12 generation, 35 at S11 generation, 32 at S10 generation, and 8 at the S9 generation. Several lines are being recovered and advanced for use in genetic studies.
In 2016, we evaluated for high resistance to the new downy mildew in the field in North Carolina. The design was a randomized complete block with 3 replications and 4 disease ratings. Lines were also rated for fruit traits such as skin type (smooth, cracked, netted) and spine color (black, white).
In 2017, we evaluated for high resistance to the new downy mildew in the field in North Carolina. The design was a randomized complete block with 3 replications and 4 disease ratings. Lines were also rated for fruit traits such as skin type (smooth, cracked, netted) and spine color (black, white). The RILs ranged from 2.0 to 8.0 for best rating (0-9 scale) for DM resistance. The RILs ranged from 2.7 to 8.0 for fruit quality rating (9-1). Five RILs had DM resistance of 2.0 to 4.3 and fruit quality of 5.0 to 8.0, making them suitable for use in crossing with elite breeding lines to develop resistant cultivars.
In 2018, we evaluated the 127 sublines in S8 to S13 for high resistance to the new downy mildew in the field in North Carolina. The design was a randomized complete block with 3 replications and 4 disease ratings. Sublines were rated for fruit traits such as skin type (smooth, cracked, netted) and spine color (black, white). The RILs ranged from 2.0 to 7.7 for best rating (0-9 scale) for DM resistance. The RILs were tested for traits that make them suitable for use in crossing with elite breeding lines to develop resistant cultivars.
In 2019, we will evaluate sublines for high resistance to the new downy mildew in the field in North Carolina. The design will be a randomized complete block with 3 replications and 4 disease ratings. Sublines will be rated for fruit traits such as skin type (smooth, cracked, netted) and spine color (black, white). The RILs usually range from 2.0 to 8.0 for best rating (0-9 scale) for DM resistance. The RILs will be tested for traits that make them suitable for use in crossing with elite breeding lines to develop resistant cultivars. We will also advance nine sublines that had high resistance and good fruit quality for use by industry.
Inbreds with resistance and quality
The population PI 197088 (HR) × Poinsett 76 (MR) contains 72 lines. The plants have been self- pollinated in the greenhouse 8 generations and tested in the field for evaluation of yield, quality and resistance. We recovered 9 lines of the 72 that did not advance to S8 in the past greenhouse cycle. We were not able to recover 3 lines last greenhouse cycle and these lines are in the S7 generation. Lines in S6 and S7 are being tested in the field for yield, earliness and quality for release to the industry.
We selected and self-pollinated sub-lines from 41 lines that are at the S8 to S9 generation in the greenhouse in 2016. The lines were evaluated for high resistance to the new downy mildew, as well as fruit quality, in the field in North Carolina. The most resistant lines were crossed in the greenhouse using parents that had intermediate fruit quality, with the objective of improving fruit quality among the highly resistant lines.
In 2017, we evaluated sublines for high resistance to the new downy mildew as well as fruit quality in the field in North Carolina. A total of 38 sublines were evaluated in a randomized complete block with 3 replications and 4 disease ratings. The RILs ranged from 2.0 to 8.0 for best rating (0-9 scale) for DM resistance. The RILs ranged from 2.7 to 8.0 for fruit quality rating (9-1). Five RILs had DM resistance of 2.0 to 4.3 and fruit quality of 5.0 to 8.0, making them suitable for use in crossing with elite breeding lines to develop resistant cultivars.
In 2018, we evaluated sublines for high resistance to the new downy mildew as well as fruit quality in the field in North Carolina. Lines were evaluated in a randomized complete block with 3 replications and 4 disease ratings. The RILs were selected for traits that make them suitable for use in crossing with elite breeding lines to develop resistant cultivars.
In 2019, we will evaluate sublines for high resistance to the new downy mildew as well as fruit quality in the field in North Carolina. Lines will be evaluated in a randomized complete block with 3 replications and 4 disease ratings. The RILs will be selected for traits that make them suitable for use in crossing with elite breeding lines to develop resistant cultivars.
Develop inbred cucumber populations.
Three populations (PI 197088 × Gy14, NC-25, or Poinsett 76) are being developed for inbred development of pickling and slicing type. Eight to 10 lines each have been selected with yield, earliness, quality and resistance. They will be released to industry for use cultivar development. In 2016, we advanced the most resistant families that also had acceptable fruit quality by self pollination in the greenhouse. There were 3 populations of 8, 9 or 10 families each (S1 to S4 generation) to make 1 or 2 sublines each. The resulting 50 families were tested for high resistance to the new downy mildew in the field in North Carolina. The design was a randomized complete block with 3 replications and 4 disease ratings. Lines were also evaluated for fruit quality. Lines were evaluated for fruit quality on a 1 to 9 scale (1=poor, 9=excellent). A total of 3 lines were selected based on field data collected in 2016. The selected lines were self pollinated and also cross pollinated in pairs in fall 2016 to develop more highly resistant cucumber populations with better fruit quality.
In 2017, 54 lines (including checks) from the three populations (PI 197088 x Gy14, NC-25, or Poinsett 76) were tested for DM resistance (0-9 scale) and fruit quality (9-1 scale). The design was a randomized complete block with 3 replications and 4 disease ratings. Of those, 4 lines from Gy14, 3 lines from NC-25, and 2 lines from Poinsett 76 were advanced since they had resistance of 3 to 5 and quality of 5 to 7.
In 2018, lines (including checks) from the three populations (PI 197088 x Gy14, NC-25, or Poinsett 76) were tested for DM resistance (0-9 scale) and fruit quality (9-1 scale). The design was a randomized complete block with 3 replications and 4 disease ratings. The most resistant lines with high fruit quality were advanced.
In 2019, lines (including checks) from the three populations (PI 197088 x Gy14, NC-25, or Poinsett 76) were tested for DM resistance (0-9 scale) and fruit quality (9-1 scale). The design was a randomized complete block with 3 replications and 4 disease ratings. The most resistant lines with high fruit quality and high yield were advanced. Those were 2 lines of Gy14, 4 lines of NC-25, and 2 lines of Poinsett 76.
Identify new sources of resistance.
A new population derived from PI 605996 (HR) × ‘Poinsett 76’ is being developed to provide new sources of high resistance to downy mildew. The F2 progeny will be self-pollinated and the S1 lines tested in the field for high resistance to natural disease incidence of downy mildew at the Clinton, NC research station. In addition to resistance, lines will be selected for yield, earliness and quality.
In 2017, we produced sublines (S2) and backcross lines (BC1S1) from PI 605996 x Poinsett 76 that will be tested for high resistance to DM as well as fruit quality.
In 2018, we produced sublines (S4) and backcross lines (BC1S3) from PI 605996 x Poinsett 76 for testing for high resistance to DM, as well as fruit quality.
In 2019, we produced eight sublines (S4) and backcross lines (BC1S3) from PI 605996 x Poinsett 76 for testing for high resistance to DM, as well as fruit quality.
Cucumber Breeding Lines Developed
- NC-148 – Breeder: T.C. Wehner and E.J. Silverman. Vendor: North Carolina State University, Raleigh. Parentage: inbred self-pollinated past the S11 generation from a family (PI 197088 x Coolgreen), previous number 17GHFL-950-1@. Characteristics: monoecious pickling cucumber, tall indeterminate vines, with rapid growth, medium-green leaves, and early maturity; high resistance to downy mildew; medium quality fruit with medium-green color. Resistance: anthracnose, new downy mildew (high resistance). Similar: Clinton, M 17. Adaptation: southern U.S. 2020.
- NC-149 – Breeder: T.C. Wehner and E.J. Silverman. Vendor: North Carolina State University, Raleigh. Parentage: inbred self-pollinated past the S10 generation from a family (PI 197088 x Coolgreen), previous number 17GHSP-602-1@. Characteristics: monoecious pickling cucumber, tall indeterminate vines, with rapid growth, medium-green leaves, and early maturity; high resistance to downy mildew; medium quality fruit with medium-green color. Resistance: anthracnose, new downy mildew (high resistance). Similar: Clinton, M 17. Adaptation: southern U.S. 2020.
- NC-154 – Breeder: T.C. Wehner and E.J. Silverman. Vendor: North Carolina State University, Raleigh. Parentage: inbred self-pollinated past the S2 generation from a family (PI 197088 x Poinsett 76), previous number 17GHFL-988-1@. Characteristics: monoecious pickling cucumber, tall indeterminate vines, with rapid growth, medium-green leaves, and early maturity; high resistance to downy mildew; medium quality fruit with medium-green color. Resistance: anthracnose, new downy mildew (high resistance). Similar: Sumter. Adaptation: southern U.S. 2020.
- NC-155 – Breeder: T.C. Wehner and E.J. Silverman. Vendor: North Carolina State University, Raleigh. Parentage: inbred self-pollinated past the S3 generation from a family (PI 605996 x Poinsett 76), previous number 17GHFL-1084-2@. Characteristics: monoecious pickling cucumber, tall indeterminate vines, with rapid growth, dark-green leaves, and early maturity; high resistance to downy mildew; medium quality fruit with dark-green color. Resistance: anthracnose, new downy mildew (high resistance). Similar: Addis. Adaptation: southern U.S. 2020.
- NC-156 – Breeder: T.C. Wehner and E.J. Silverman. Vendor: North Carolina State University, Raleigh. Parentage: inbred self-pollinated past the S3 generation from a family (PI 605996 x Poinsett 76), previous number 17GHFL-1088-1@. Characteristics: monoecious pickling cucumber, tall indeterminate vines, with rapid growth, dark-green leaves, and early maturity; high resistance to downy mildew; medium quality fruit with dark-green color. Resistance: anthracnose, new downy mildew (high resistance). Similar: Sumter. Adaptation: southern U.S. 2020.
- NC-165 – Breeder: T.C. Wehner and E.J. Silverman. Vendor: North Carolina State University, Raleigh. Parentage: inbred self-pollinated past the S10 generation from a family (PI 197088 x Coolgreen), previous number 18GHFL-200-1@. Characteristics: monoecious pickling cucumber, tall indeterminate vines, with rapid growth, medium-green leaves, and early maturity; high resistance to downy mildew; medium quality fruit with medium-green color. Resistance: anthracnose, new downy mildew (high resistance). Similar: Clinton, M 17. Adaptation: southern U.S. 2020.
- NC-177 – Breeder: T.C. Wehner and E.J. Silverman. Vendor: North Carolina State University, Raleigh. Parentage: inbred self-pollinated past the S10 generation from a family (PI 197088 x Poinsett 76), previous number 17GHFL-1010-2@. Characteristics: monoecious pickling cucumber, tall indeterminate vines, with rapid growth, medium-green leaves, and early maturity; high resistance to downy mildew; medium quality fruit with medium-green color. Resistance: anthracnose, new downy mildew (high resistance). Similar: Clinton, M 17. Adaptation: southern U.S. 2020.
- NC-178 – Breeder: T.C. Wehner and E.J. Silverman. Vendor: North Carolina State University, Raleigh. Parentage: inbred self-pollinated past the S10 generation from a family (PI 197088 x Poinsett 76), previous number 17GHFL-1043-3@. Characteristics: monoecious pickling cucumber, tall indeterminate vines, with rapid growth, medium-green leaves, and early maturity; high resistance to downy mildew; medium quality fruit with medium-green color. Resistance: anthracnose, new downy mildew (high resistance). Similar: Clinton, M 17. Adaptation: southern U.S. 2020.
- NC-179 – Breeder: T.C. Wehner and E.J. Silverman. Vendor: North Carolina State University, Raleigh. Parentage: inbred self-pollinated past the S10 generation from a family (PI 197088 x Poinsett 76), previous number 17GHFL-1089-3@. Characteristics: monoecious pickling cucumber, tall indeterminate vines, with rapid growth, medium-green leaves, and early maturity; high resistance to downy mildew; medium quality fruit with medium-green color. Resistance: anthracnose, new downy mildew (high resistance). Similar: Clinton, M 17. Adaptation: southern U.S. 2020.
- NC-180 – Breeder: T.C. Wehner and E.J. Silverman. Vendor: North Carolina State University, Raleigh. Parentage: inbred self-pollinated past the S10 generation from a family (PI 197088 x Coolgreen), previous number 18GHFL-214-1@. Characteristics: monoecious pickling cucumber, tall indeterminate vines, with rapid growth, dark-green leaves, and early maturity; high resistance to downy mildew; medium quality fruit with dark-green color. Resistance: anthracnose, new downy mildew (high resistance). Similar: Clinton, M 17. Adaptation: southern U.S. 2020.
