Team members: Yiqun Weng (USDA, ARS), Rebecca Grumet (Michigan St. Univ.), Todd Wehner (North Carolina St. Univ.)
1. Develop genomic approaches and tools
1.2 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. The rest have gone through at least one-generation of selfing. We also re-sequenced one plate of samples (96 lines) at >10× coverage.
Phenotyping of morphological traits and DM resistance in cucumber natural populations
Three hundred cucumber lines were grown in the University of Wisconsin Hancock Agricultural Research Station (HARS) for collection of morphological data. Meanwhile, 300 cucumber lines (2 reps, 6 plants per rep) were planted in North Carolina State University experimental field in summer 2018. Data for responses to DM natural infestation were collected. Unfortunately, only data form one rep were collected due to unforeseen natural disaster.
2019 work plan
- Continue selfing and seed increase of the GWAS panel lines.
- Seed increase of cucumber lines by self-pollination.
- Prepare GWAS panel lines for re-sequencing (partial).
- Approximately 250 lines in the GWAS panel will be planted in 2019 summer season in North Carolina State University fields for collecting data for responses to natural DM infestation.
2. Genomic assisted breeding
2.1 and 2.2: QTL mapping, marker development for DM and PFR resistances
Downy mildew (DM)
(Weng and Wehner Labs)
2018 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.
In 2018, through QTL analysis in the secondary F2 populations, the dm4.1 and dm5.3 loci have been delimited to ~60-80 kb intervals on chromosomes 4 and 5, respectively.
2019 work plan
1. Narrow down the QTL region (1.5 LOD interval) of target QTL regions through continued fine genetic mapping and GWAS
2. Identify candidate genes for dm4.1 and dm5.2.
3. Growth chamber and field evaluation of DM resistance of the NILs.
Phytophthora capsici fruit rot resistance in cucumber
(R Grumet lab – B Mansfeld, Y-C Lin)
Young fruit resistance to P. capsici
2018/19 progress:
QTL-seq analysis. 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:
i. An F2 population (n=397) of Gy14 X PI109483-53B from field grown plants, in summer 2017.
ii. An F2 population (n=222) of Gy14 X doubled haploid (DH) line A4-3 grown in the greenhouse in spring 2018. The DH lines were generously produced by Rijk Zwaan from three resistant breeding lines derived from PI109483. Based on tests in summer 2017, DH A4-3, was chosen for further population development.
iii. An F2 population (n=362) of Gy14 X DHA4-3 tested from field grown plant, summer 2018.
Three sets of harvests were performed for each experiment to provide replication in sampling dates, and at least 10 fruit per F2 individual. The populations exhibited a normal distributions for disease scores, consistent with a quantitative trait. Individuals from each end of the distribution, representing the most resistant and most susceptible plants were selected for bulk segregant QTL-seq analysis. Cleaned reads were aligned to the cucumber Gy14 version 2 reference genome (Weng et al., http://cucurbitgenomics.org/) and the Genome Analysis Toolkit pipeline (GATK; v3.6) (McKenna et al., 2010; https://software.broadinstitute.org/gatk/) used for identification of SNPs and indel variations. SNP-indices were calculated as described in Takagi et al. (2013) using QTL-seqr (Mansfeld and Grumet, 2018) and mapped across the cucumber genome. There was good correspondence between peaks observed on chromosomes 5 and 6 in the two field seasons. The greenhouse trial gave different peaks, suggesting possible environmental effects on response.
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.
View figure 1 on page 50 of the pdf
2019/20 work plan
Design KASP markers to validate and narrow the QTL regions found in association with resistance to P. capsici for field grown plants. Screen large F2 population, identify recombinants in regions of interest, and test recombinant individuals in the field.
Age-related resistance (ARR) to P. capsici
2018/19 progress:
Defense response in ARR+ fruit.
The transcriptomic analysis performed in 2017 using samples collected from susceptible- and resistant- age fruit (8 dpp and 16 dpp, respectively) at 0, 4, 24 and 48 hours post-inoculation (hpi) suggested that in ARR-expressing fruit, a successful defense is mounted within the first 24 hours. To understand the dynamics of infection during the first 24 hours, inoculated and control samples were collected from 8 and 16 dpp fruit peel for 3’RNAseq transcriptomic analysis and scanning electron microscopy at 0, 2, 4, 8, 12, 18, 24 hpi. SEM of resistant peels showed evidence for infection failure as early as 4 hpi, including aberrant long germ tubes, and un-germinated, deflated and/or disintegrated spores and hyphae, that were not observed on susceptible fruit. PCA of the transcriptome data showed strong transcriptional changes from 4 hpi and beyond for the inoculated 8 dpp. In contrast, marked changes occurred in the resistant samples by 2 hpi, suggesting an earlier response to infection in the resistant-aged fruit, with only minor changes after 4 hpi. Weighted co-expression networks identified several modules with differential, earlier response to infection at the resistant ages.
QTL mapping of ARR.
Doubled haploid (DH) lines derived from F1 progeny of ‘Gy 14’ (ARR-) X ‘Poinsett 76’ (ARR+) were kindly produced by Rijk Zwan and used for QTL-seq analysis. Seed from 79 lines were planted in the greenhouse in 5 replicated blocks along with the two parental lines and F1 progeny. Flowers were hand pollinated, and a single fruit per plant was harvested at 17 days post pollination. Fruits were inoculated with 12 equally spaced 30 μL droplets (105 zoospores/ml) and scored at 7 days post inoculation (dpi) using a 0-5 point disease score (0 – no symptoms, 5 – extensive sporulation).
A total of 424 fruit were phenotyped with a mean disease rating of 2.0+0.1. The parental lines were consistently either ranked resistant or susceptible with mean disease ratings of 0.3 and 3.0 for ‘Poinsett 76’ and ‘Gy 14’, respectively. Consistent with our prior studies suggesting a dominant major factor, the RIL population was bimodally distributed and F1 fruit were largely resistant (1.1). High within-line variability of disease rating was observed in lines showing intermediate susceptibility, highlighting the need for reproducibility made available by using a fixed DH population. Fifteen of most resistant and susceptible lines were selected for a second trial in the greenhouse. The disease rating distributions of the two groups separated (Welch’s T test, P = 0.003), with means of 2.1 (Resistant) and 4.0 (Susceptible). Eight resistant and susceptible lines which were consistently ranked in both experiments were selected for QTL-seq analysis.
A major ~9Mb QTL passing the 99% confidence interval and a Δ(SNP-index) maximum of 0.88, was identified on chromosome 3. This region also was identified in a prior screen of F2 progeny.
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 figure 2 on page 51 of the pdf
2019/20 work plan:
Prepare publication describing infection response to P. capsici and early expression of defense on ARR-expressing fruit.
Begin to examine QTL region on chromosome 3.
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 2017-2018 period, homozygous lines carrying both dm4.1 and dm5.2 were developed. In 2017 summer trial, these plants were grown in the University of Wisconsin Hancock Agricultural Research Station for preliminary observations. The plants were also tested for DM inoculation responses in controlled environments.
In 2019, we will:
- Continue marker-assisted backcrossing in Gy14 genetic background for pyramiding of dm4.1 and dm5.2 QTL from WI7120/PI 197088. Combine dm3 into Gy14+dm4.1+dm5.2 genetic background through marker assisted selection.
- Conduct field and greenhouse screening tests to evaluate DM resistance and performance of horticulture traits.
- Prepare 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
