2018 CucCAP Cucumber Team Annual Report

cuccap

Progress Reports and Work Plans

CucCAP Cucumber Team members; Yiqun Weng (USDA, ARS), Rebecca Grumet (Michigan St. Univ.), and Todd Wehner (North Carolina St. Univ.) reported the progress from each of their labs for 2017.

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

  1. GBS of PI lines: GBS has been completed for 1234 cucumber accessions including plant introduction (PI) lines andhistorical cultivars or landraces of cultivated (Cucumis sativus var. sativus) and wild (C.  sativus var. hardwickii) cucumber lines. Data analysis has been performed by the bioinformatics team to identify SNPs, determine minor allele frequency, perform phylogenetic, population genomic, and linkage disequilibrium (LD) analysis. This information is being used to establish a functionalcore population to capture ~99% of allelic diversity as well combined with representation of key disease resistance, fruit quality and agronomic features.
  2. Phenotyping of morphological traits and DM resistance in cucumber natural populations Four hundred and twelve cucumber lines were grown in the University of Wisconsin Hancock Agricultural Research Station (HARS) for collection of morphological data. Meanwhile, 352 cucumber lines (2 reps, 6 plants per rep) were planted in North Carolina State University experimental field in summer 2017. Data for responses to DM natural infestation were collected.
2018 work plan
  1. A core collection (384) are being selected using multiple criteria including GBS results to
    construct the GWAS panel.
  2. Seed increase of cucumber lines by self-pollination for those without enough seeds or with high degree of heterozygosity.
  3. Prepare GWAS panel lines for re-sequencing.
  4. Approximately 300 lines in the GWAS panel will be planted in 2018 field season at HARS for phenotypic data collection. The same set of materials will be grown in North Carolina State University fields for collecting data for responses to natural DM infestation.
  5. Prepare a manuscript describing cucumber diversity and establishment of functional panel.

QTL mapping, marker development for DM and PFR resistances

Fine mapping of DM resistance in cucumber

(Weng and Wehner Labs)

2017 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 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 ~100 kb intervals on chromosomes 4 and 5, respectively.

2018 work plan
  1. Narrow down the QTL region (1.5 LOD interval) of target QTL regions through continued fine genetic mapping and GWAS; identify candidate genes for dm4.1 and dm5.2. 2.
  2. Growth chamber and field evaluation of DM resistance of the NILs.

QTL mapping of Phytophthora capsici resistance in cucumber

R Grumet lab – B Mansfeld, Y-C Lin; in collaboration with C. Smart

Young fruit resistance to P. capsici

2017 progress:
  1. Initiate introgression and genetic analysis.
    a. Population development BC1 individuals showing resistance were backcrossed to Gy14; BC2 progeny were screened in the greenhouse in the fall 2017. Doubled haploid families derived from four PI 109483-53 lines were produced by Rijk Zwaan Seed from these plants were grown in the greenhouse and field in 2017and fruit tested for response to inoculation. Selected DH individuals were crossed with Gy14 to provide a second population for QTL analysis.
    b. Phenotyping. An F2 population (n=400) of GY14 X PI109483-53B was tested in summer 2017. Fruit were harvested from each plant at approximately 4-6 day post-pollination (4-8 mm), and inoculated with P. capsici. Three sets of harvests were performed on the full population to provide replication in sampling dates, and at least 10 fruit per F2 individual. The population exhibited a normal distribution for disease score; ~60 potentially resistant and potentially susceptible individuals were chosen for further testing. Three additional harvests were performed on those plants; the two groups of individuals performed as predicted, verifying the initial ratings as resistant or susceptible. DNA will be prepared from the 15-20 most resistant and most susceptible individuals for sequencing and subsequent QTL-seq analysis.
  2. Field trial in P. capsici infested field.
    Field trials were performed in 2017 in a heavily P. capsici- infested site maintained in Geneva, New York by Dr. Chris Smart’s group. Two methods of inoculation were tested: spray at fruiting time with zoospore suspensions, and incorporation of infested vermiculite into the soil between the rows. Overall infection rates were 22% and 39% of fruit harvested for the vermiculite and zoospore inoculations, respectively. Future work will test the breeding lines under development.
2018 work plan
  1. Phenotype young fruit of F2 progeny from Gy14 x DH 109483-53-derived resistant lines for
    response to P. capsici.
  2. Perform QTL-seq analysis on selected F2s from the summer 2017 (Gy x 109483-53) and spring 2018
    (Gy14 x DH 109483-53) experiments.
  3. Intercross BC progeny to increase resistance levels in a background with better fruit type,
    more uniform germination, and earlier female flower production.
  4. Perform GWAS analysis for resistance to P. capsici using GBS data for cucumber PI accessions and data from prior P. capsici screening of the cucumber PI collection.

Age-related resistance (ARR) to P. capsici

2017 progress:
  1. QTL seq analysis. In preparation for QTL-seq, B. Mansfeld developed an R package, QTLseqr (no R packages are currently available), using two statistical approaches, QTL-seq and a tricube smoothed G statistic, G’, to identify and assess statistical significance of QTL. QTLseqr, can import and filter SNP data, calculate SNP distributions, relative allele frequencies, G’ values, and log10(p-values). The source code is available at https://github.com/bmansfeld/QTLseqr. Since October it has been downloaded >600 times with an altmetric score in the top 5%. A manuscript describing the program has been submitted to Plant Genome.
  2. Transcriptomic and metabolomic analysis of peels from ARR+ and ARR- cultivars. Work describing the transcriptomic and metabolomic analysis was published in 2017 (Mansfeld et al., Hort Res): Transcriptomic and metabolomics analyses of cucumber fruit peels reveal a developmental increase in terpenoid glycosides associated with age-related resistance to Phytophthora capsici.
  3. Transcriptomic and microscopic analysis of the infection process is underway to determine the point at which infection is inhibited and compare responses between ARR+ (Poinsett) and ARR- (Gy14). The use of a second ARR+ line also helps to narrow candidate genes associated with ARR.
2018 work plan:
  1. Phenotype Gy14 x Poinsett-derived DH population. The DH population provides a second population and season for QTL-seq analysis and also allows for replicated screening not possible with F2 plants, as only a single fruit per plant can be set per plant to avoid competition effects on development.
  2. Perform QTL seq analysis for ARR. Compare results of transcriptome analysis with QTL seq analysis to help identify genomic regions of greater interest.
  3. Complete transcriptomic and microscopic analysis of infection process.

Advanced line development for downy mildew resistance

2017 progress:

 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.

Breeding line development for DM resistance

RIL development and evaluation of DM resistance

(Wehner lab: T Wehner, EJ Silverman)

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 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 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 ranged 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.

Inbreds with resistance and quality

(Wehner lab: TC Wehner and EJ Silverman)

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 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.

2018 work plan

(Weng and Wehner Labs)

  1. 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. 2. 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.
  2. 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) will be tested for DM resistance (0-9 scale) and fruit quality (9-1 scale). The design will be a randomized complete block with 3 replications and 4 disease ratings. The most resistant lines with high fruit quality will be advanced.
  3. 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 will produce sublines (S4) and backcross lines (BC1S3) from PI 605996 x Poinsett 76 for testing for high resistance to DM, as well as fruit quality.
  4. Field screening of downy mildew resistance for the 300-line GWAS panel.