Development and Phenotypic Evaluation of a Brassica oleracea Leafy Greens Diversity Panel

Progress report for GS22-263

Project Type: Graduate Student
Funds awarded in 2022: $16,500.00
Projected End Date: 08/31/2024
Grant Recipient: Clemson University
Region: Southern
State: South Carolina
Graduate Student:
Major Professor:
Dr. Sandra Branham
Clemson University
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Project Information


Collard greens are primarily produced in the Southern region where they are an important cultural heritage crop. Brassica oleracea leafy greens, including collard greens and kale, are an exceptional source of vitamins, minerals, and phenolic compounds. However, research on these crops is limited leading to reduced availability and use of genetic resources for Brassica leafy greens. Despite the increasing popularity of collard greens and kale, the number of cultivars available for their production remains low. In addition, production is challenged by high insect and disease pressure, and the emergence of new pathogenic strains of economically important diseases (Bacterial leaf blight, Alternaria leaf spot) emphasizing the need for improved cultivars. We propose to develop a B. oleracea leafy green diversity panel from public germplasm sources, including the USDA-NPGS collection and the Seed Savers Exchange (SSE). The developed diversity panel, BLG1, would be a valuable source of alleles for improved nutrition (high glucosinolates), biotic (diseases and pests) and abiotic (salinity and high temperature) stress resistance for B. oleracea breeding programs. Release of new, improved cultivars would increase sustainability and profitability of Brassica leafy greens production through reduction of pesticide usage combined with increased yields.

Project Objectives:

The main goal of this project is to develop a collection of B. oleracea leafy green accessions (collected from USDA-NPGS and the Seed Savers Exchange) to identify desirable alleles and facilitate their introgression into improved collard and kale cultivars for production in the Southeastern US. The transition of these accessions into more stable, homozygous lines will improve their utility for future breeding and genetics research projects. The long-term goals of these projects are to (i) genotype the diversity panel (BLG1) and map the genomic regions associated with biotic and abiotic stress resistance, and nutrition using genome-wide association studies (GWAS); (ii) develop new B. oleracea-leafy green cultivars with better horticultural traits and resistance; (iii) introgression of desirable alleles from these accessions to other economically important B. oleracea species like cabbage.
We have already self-pollinated 260 accessions (S0) once and have S1 seeds for each accession. Keeping this in mind our major objective will be to purify these accessions for one more season to obtain S2 seeds. Towards the fall 2023, phenotyping of each BLG1 individual will be done for different horticultural traits and the records will be maintained.

The proposed proposal has been formulated for these objectives:

Objective 1: Self-pollination and seed increase of the collected accessions during the first year, 2022-23
In fall 2022, single seeds of each accession (S1) will be sown to obtain S2 seeds for phenotyping to reduce segregation (i.e., to generate ‘true  breeding’ seed) within each accession. Manual pollination will be carried out to ensure selfing for all 260 accessions of the diversity panel. This will be done in greenhouses available at Clemson University Coastal Research and Education Center (CREC) in Charleston, SC.

Objective 2: Phenotypic evaluation of all the S2 genotypes for different horticultural traits and disease resistance during the second year, 2023-24
Two replications of 10 seeds for each S2 accession will be seeded for field trials in Fall 2023 and Spring 2024 to evaluate the morphological diversity of all the accessions (collard, kale and non-heading cabbage), including leaf shape, leaf texture, leaf color, days to bolting and days to flowering. In addition to the field trials, the S2 kale genotypes will be screened for resistance against Alternaria leaf spot (Alternaria sp.). Alternaria leaf spot is the most important fungus on organic kale in South Carolina esp. in fall and the cultivars ‘Darkibor’ and ‘Winterbor’ are susceptible to the new causal agent, A. japonica (Keinath et al. 2021). The kale accessions in BLG1 (N=50) will expand the germplasm available for disease screening increasing the likelihood of identifying resistance sources.


Materials and methods:

Objective 1
Self-pollination and seed increase of the collected accessions during the first year, 2022-23
The developed diversity panel (N=260 accessions), BLG1, will include four sub-populations, collards (Brassica oleracea var. viridis), non-heading cabbage (B. oleracea var. capitata), kale (B. oleracea var. acephala, B. oleracea var. sabellica, B. oleracea var. medullosa, B. oleracea var. palmifolia) and wild cauliflower (B. oleracea var. oleracea). The S1 seeds developed by one round of selfing of the obtained USDA-NPGS and SSE accessions are available at CREC, Charleston, SC. The heirloom cultivars from SSE were added to expand the available germplasm of the collection. A single individual of each accession will be used to maximize the genetic and phenotypic diversity, given the resources available. We will continue to try to find additional heirloom cultivars from other sources (small seed companies, seed savers, etc.) to represent most of the available genetic and phenotypic diversity in the US. Of the 260 accessions, 210 have known origin spanning different continents including, Asia, North America, Europe, and Australia.

The seeds for all the 260 accessions will be sown in 50-cell plug trays containing soil mix (Metro- Mix 360). One month old seedlings will then be transplanted into pots greenhouses at CREC, Charleston, South Carolina. A single plant per accession (S1) (N=260) will be grown under standard greenhouse conditions and upon maturity, these plants will be manually self-pollinated to reduce variability within the accessions. Single seed will be sown to develop S2 seed for phenotyping. We would keep the number of pollinations high to obtain enough seeds from each accession, which necessitates a part-time undergraduate student to assist in manual pollinations. The seed will be collected from each plant, labelled, and stored for morphological characterization in the next fall, 2023. To increase the number of seeds for the self-compatible S2 accessions, pollination cages will be used. This will be done by placing two to four S2 plants in a cage and flies will be released inside each cage to serve as pollinators. This will help us to attain more seed for these accessions enabling seed distribution, when requested.

Objective 2
Phenotypic evaluation of all the S2 genotypes for different horticultural traits and disease resistance during the second year, 2023-24
The S2 genotypes will be sown for phenotypic characterization in field trials in Fall 2023 and Spring 2024. The experimental design for each field trial will consist of ten plants of each accession in two replicates planted in a randomized complete block design. The accessions will be evaluated for leaf color, leaf texture, leaf shape, bolting, and days to flowering. These horticultural traits will aid selection of desirable genotypes within the sub-populations. Simultaneously, the morphological characters for commercial collard (‘Top Bunch 2.0’ and ‘Champion’) and kale (‘Winterbor’ and ‘Darkibor’) cultivars will also be assessed. The recorded observations will be analyzed using the aov package in R (R Core Team 2022) for variability within the sub-populations on basis of their phenotypic characters.

The screening of S2 kale genotypes for resistance against Alternaria leaf spot (Alternaria sp.) will be done in collaboration with Dr. Keinath (Plant Pathologist, Clemson University). The S2 kale accessions (N=50) will be screened in two replications against both, A. brassicae (conventional causal agent) and A. japonica (new causal agent, first report in Southeastern US in 2021). The three oldest leaves of kale S2 genotypes will be inoculated with a 5 x 105 conidia/ml suspension, while the control plants will be sprayed with water. The S2 genotypes will be kept at 100% relative humidity for 48 hours in a dew chamber after which they will be moved to a growth chamber at 21°C day and 16°C night temperature. Thirteen days post inoculation, the second and third oldest leaves will be rated based on the disease severity (0-100%). Kale cultivars ‘Winterbor’ and ‘Darkibor’ will be used as susceptible controls. Any genotype with less than 25% disease severity will be retested against Alternaria sp. and will be prioritized in our future kale breeding programs.

All the S2 genotypes with important horticultural traits will be shown to the growers and major stakeholders as a part of field demonstrations during both field trials (November 2023 and April 2024) at CREC, Charleston. Their feedback will be used to guide selection of accessions with ideal horticultural traits in our breeding program. The graduate student, Khushwinder Kaur, will present the phenotypic observations and evaluations at National Association of Plant Breeders (NAPB) annual meeting in August 2024. The results will also be published in plant breeding or horticultural journals. Additionally, the developed and characterized BLG1 will be maintained at Clemson’s Coastal Research and Education Center, Charleston and will be distributed with material transfer agreement (MTA) to other Brassica breeders upon request.

Research results and discussion:

Brassica leafy greens in the vernalization chamber.

One seed of all the accessions (N=210) were planted in October and vernalized at 5°C for a minimum of 6 weeks to initiate flowering. For twenty accessions, no germination was observed and twelve plants were lost to black rot in February this year. Please note that we were not able to continue with eighteen accessions as no more seed is available from USDA-NPGS.

The plants were vernalized in two sets of one hundred as we have only one vernalization chamber. Half of the plants started flowering and are being self-pollinated in the greenhouse now. Data collection is not part of the timeline until Fall 2023.

An image of plants in the vernalization chamber and greenhouses has been attached.

Participation Summary

Educational & Outreach Activities

Participation Summary:

Education/outreach description:


Project Outcomes

Project outcomes:

This grant year, the graduate student and a part-time person are focused on the development of S2 genotypes for field trials this fall and Spring 2024. During the field trials this year and Spring 2024, farmers will be invited to select the different genotypes for desirable horticultural traits. This will help us in selecting horticulturally preferable genotypes in our breeding program that will benefit the farmers. In addition, the kale genotypes will also be screened this fall for resistance to an emerging pathogen, Alternaria japonica, that was first reported in South Carolina in 2021 (Keinath et al., 2021).

The overall benefit of this project will be (i) consolidation and purification of the available germplasm, (ii) grouping of germplasm according to its preferable horticultural characteristics, (iii) disease screening for kale to sustainably manage Alternaria leaf spot on kale farms in South Carolina, (iv) reduced dependence of farmers on pesticide/fungicides in future if disease/pest resistant genotypes are identified.

Knowledge Gained:


Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.