Source: AGRICULTURAL RESEARCH SERVICE submitted to
CRACKING HARD SEED IN HAIRY VETCH: MARKER-ASSISTED COVER CROP IMPROVEMENT
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1015043
Grant No.
2018-67013-27570
Cumulative Award Amt.
$490,000.00
Proposal No.
2017-07673
Multistate No.
(N/A)
Project Start Date
Feb 15, 2018
Project End Date
Feb 14, 2023
Grant Year
2018
Program Code
[A1141]- Plant Health and Production and Plant Products: Plant Breeding for Agricultural Production
Project Director
Riday, H.
Recipient Organization
AGRICULTURAL RESEARCH SERVICE
1815 N University
Peoria,IL 61604
Performing Department
(N/A)
Non Technical Summary
Cover crops are powerful tools for improving agricultural productivity and sustainability. Hairy vetch (Vicia villosa) is a widely used a legume cover crop that excels in nitrogen production, weed suppression, and soil health improvement. Despite these benefits, V. villosa suffers from seed dormancy and shatter, traits that were eliminated in most crops thousands of years ago. We propose a modern approach to domesticate V. villosa. To achieve this goal, we will accomplish the following objectives: identify markers associated with seed dormancy and shatter, breed improved soft-seeded and low shatter varieties of hairy vetch, evaluate improved lines of hairy vetch with farmers, and develop a tool to guide seed companies in the emerging cover crop seed market. First, a genome-wide association study (GWAS) on diverse germplasm will pinpoint markers associated with seed dormancy and shatter. Second, classical breeding techniques will be applied to develop regionally adapted varieties of soft-seeded and non-shattering V. villosa. Third, participatory variety trialing will actively engage hundreds of stakeholders in the evaluation of top breeding lines for suitability to diverse soils, climates, and farming systems of the United States. Finally, we will develop seed market forecasting tools to facilitate the growing V. villosa industry. With the development of soft-seeded and non-shattering varieties of V. villosa, we anticipate remarkable shifts in adoption of this legume cover crop, boosting agricultural sustainability nationwide. The project's comprehensive approach is highly relevant to the goals of the Plant Breeding for Agricultural Production program, including genomics-enabled breeding, cultivar development, and participatory engagement.
Animal Health Component
40%
Research Effort Categories
Basic
20%
Applied
40%
Developmental
40%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011644108150%
2021644108150%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms; 202 - Plant Genetic Resources;

Subject Of Investigation
1644 - Winter annual legumes;

Field Of Science
1081 - Breeding;
Goals / Objectives
Our long-term goal is to improve agricultural sustainability by increasinglegume cover crop performance and adoption. In this proposal, we aim to use conventionalbreeding and a genomics approach to identify markers associated with hard seed and seedshattering in V. villosa. The results will contribute big data on functional genetics to improvefundamental flaws in V. villosa and related legume species. Our approach will also generateimproved varieties for farmers. Integrating farmers and seed companies during the variety testingand release phase will produce outcomes that directly serve stakeholder needs.To achieve ouraim, we propose the following research objectives:1. Identify markers associated with seed dormancy and seed shatter,2. Breed improved soft-seeded and low shatter varieties of hairy vetch,3. Evaluate improved lines of hairy vetch with farmers, and4. Develop a tool to guide seed companies in the emerging cover crop seed market.
Project Methods
Objective 1.In the winter of 2017/2018, seed from 500 maternal breeding lines will be assayed for germination and hard seed. Logistic regression models will test whether maternal line is significantly associated with the log odds of germination. A subset of 80 mother plants representing the distinct categories of seed dormancy will form the trial set for a genome-wide association study (GWAS). Extra seed of the 80 mother plants in the test set will be frozen and stored for a validation trial.In the fall of 2018, the test set will be planted at two sites with low humidity summer conditions, where hard seed and shatter are most likely to be expressed: Corvallis, OR and Knox City, TX. Each site will plant 800 seed derived from 80 maternal parents. Seed will be mechanically scarified to effectively establish both soft and hard seeded genotypes. Genotypes within replicate will be further blocked by maturity timing, to facilitate phenotyping and harvest timing. The trial sites will maintain a minimum of 500' isolation from other V. villosa plants at the seed production sites. Data collected on each plant will include: two fall and spring vigor ratings, maturity rating when sites are in 10% bloom, and seed shatter. Fresh tissue from elongating meristems will be collected on each plant for DNA extraction. Fifty pods on each plant will be hand harvested, gently hand-threshed, and evaluated for hard seed. The remaining seed will be harvested by individual plant, cleaned, and frozen.From each phenotyping location, we will identify two divergent groups for dormancy and seed shatter: the hardest 5% and softest 5% of maternal families, and the least and most shattering 5% of maternal families. We will genotype 384 individuals from divergent groups using Genotype by Sequencing (GBS). DNA will be extracted and digested using the common restriction enzyme, ApeKI, and amplified with PCR. Illumina sequencing with five to ten duplicate coverage for the genome of V. villosa will ensure marker alignment near causative genes. GBS reads will be aligned to the assembled V. villosa genome, or if unavailable by 2020, the Lens culinaris genome. Using a set of random markers, we will assess the population structure of divergent groups using the program STRUCTURE.The divergent groups will be compared for differences in single nucleotide polymorphism (SNP) frequency. Correlations will check for potential confounding effects on seed dormancy and shatter, such as flowering time, seed pod maturity and environment in which the seed was grown. Mixed linear models will be used to account for population structure and kinship in the populations. A set of random markers will correct for population structure using program principal component analysis (PCA). Scree plots and Bayesian information criterion (BIC) will be used to determine the number of PCs to include in the model. A compressed kinship matrix will also be incorporated to account for relationship within subpopulations. Optimal models will be chosen based on goodness of fit using likelihood ratio tests. A chi-squared test will be used to evaluate the significance of the difference in allele frequency between the divergent groups. We will then use False Discovery Rate to correct for multiple testing errors. Odds ratios will be visualized on Manhattan plots.Objective 2.In addition to identifying markers linked to seed dormancy and shatter, we intend to create soft-seeded experimental varieties through selection. In the fall of 2020, remnant halfsib seed from genotypes with the softest seed and least shattering phenotypes from the marker identification program will enter a distinct crossing block in Wisconsin forming a first generation synthetic (Syn 1). In fall of 2021 a Syn 2 seed increase will generate larger quantities of seed.In fall of 2020, remnant halfsib seed from soft-seeded genotypes from the marker identification program will also be planted in Maryland and Wisconsin along with regionally-adapted advanced breeding populations from the OREI program in a selection nursery. This nursery will facilitate integration of the soft-seeded germplasm with regionally adapted lines. Data collected on individual plants will include those listed in objective 1.We will incorporate identified and validated markers from objective 1 in our ongoing field-based selection program using marker-assisted recurrent selection. We will design an assay to efficiently amplify the targeted region(s) of the genome around markers for soft seed and non-shattering. If multiple QTL are identified, we will develop a weighted index for marker-assisted recurrent selection. We will then screen advanced breeding material for marker of interest prior to flowering. We will only allow those with the markers of interest to cross and contribute to the next generation. After fixing the soft seed and non-shattering traits in breeding lines, we will conduct further testing in more environments, and prepare for variety release.Objective 3.In the fall of 2021, improved material will be evaluated by farmers at twenty sites across the U.S. The variety evaluations will include the increased Syn 1 soft seed and low shatter population, regionally adapted breeding lines from objective 2, advanced breeding lines from six OREI sites, and commonly grown check varieties. Plants will be grown and evaluated in one meter row plots in a randomized complete block design. Each site will assess mean values for each plot of the measurements listed in Objective 1.Around 50% flowering, when farmers typically incorporate V. villosa, each site will host a participatory variety trial field day. Farmers and seed company representatives will visit and rate the varieties at their respective regional location. Participants will rate their most and least preferred five lines. The line identity will be masked as participants complete ratings. Hosts will facilitate a discussion among participants on their most and least preferred lines. The participatory variety rating will be accompanied by presentations on cover crop management from one farmer innovator and one researcher of the region.The results of our trait-based evaluations will be analyzed for genotype by environment interactions across sites. Results will indicate whether variety preference by farmers, and overall variety performance for traits of interest varies among sites. The results of the farmer evaluation will help inform which varieties are released. Seed companies will be informed of any regions that have specifically adapted lines. We will also assess correlations between all evaluated traits in the variety trials. Correlations and farmer ratings could reveal potential deleterious effects linked to soft seed and non-shattering alleles.Objective 4.As a final aspect of the project, we will develop a method to forecast seed demand for the emerging cover crop seed market. The Agricultural Research Service (ARS) will host a meeting of seed companies selling V. villosa seed in 2019. At the facilitated meeting, researchers will work with seed company representatives to develop an action plan for sustainable growth of the cover crop seed market. As part of this initiative, ARS researchers will gather predictive data on how many acres of V. villosa are expected to be grown by farmers in the coming year, using the annual Sustainable Agriculture Research and Education (SARE) cover crop survey and 2017 NASS Census of Agriculture. ARS researchers will also gather data from seed companies and the Oregon State University Forage Seed Survey to estimate existing and projected supply of V. villosa seed. The information will be directly provided to seed companies, allowing for needed adjustments in seed supply to meet grower demand. For the final three years of the project, we will evaluate the effectiveness of the projections and seed company communications to meet cover crop seed demand.

Progress 02/15/18 to 02/14/23

Outputs
Target Audience:Seed companies, organic farmers, researchers. Changes/Problems: The Noble Research Institute, major collaborator on this grant, dissolved plant sciences research. All staff and PIs associated with the project were no longer able to continue. We were able to reallocate their pending funding to complete objectives 1 and 2 through the USDA-ARS and USDA-NRCS-PMCs. Disease was so intense in the two mapping population fields that the plants were lost. We were unable to gather enough seed to phenotype for shatter and hard seed. We were unable to host in-person participatory field days due to the global pandemic. After extensive research and outreach (see accomplishments), we concluded that lack of information and transparency will prevent us from accomplishing this objective. The highly repetitive and heterozygous genome required more coverage in Nanopore sequencing to create a reliabile assembly. Hard seed (and pod shatter) are environmentally dependent, so certain field trials had limited information due to extreme (no shatter or complete shatter) phenotypes. What opportunities for training and professional development has the project provided? Monteros mentored one post-doc. Kissing Kucek mentored four undergraduates (experimental design, R, randomization, scientific manuscript writing), two PhD students and two post-docs(error correction using spatial relationships, multi-environment trial analysis). Kissing Kucek taught a section on plant breeding for sustainable agriculture for a University of Minnesota undergraduate class. Riday mentored two post-docs, one PhD student, and one undergraduate.? How have the results been disseminated to communities of interest? Multiple manuscripts are in preparation for peer-reviewed journals. We presented at scientific meetings (PAG, ASA-CSSA-SSSA), grower meetings (MCCC, MOSES, USDA-ARS-DFRC seminar series), and seed company meetings (ASTA, upper Midwest Seed Summit). A website (www.covercropbreeding.com) was created for the project to increase visibility and to facilitate future variety release. Collaborator Kissing Kucek developed a standardized Participatory Variety Testing sheet to gather feedback on advanced breeding lines from farmers and seed companies. Feedback on breeding lines of vetch was gathered at field days in Columbia, MO, May 2019. Seed companies selling cover crops, including two grant advisory board members, were invited to the 2019 annual Cover Crop Breeding meeting in Raleigh, NC. The meeting included a tour of a hairy vetch breeding nursery. Team members gathered feedback from companies on breeding objectives and market needs. PD Riday hosted a legume cover crop seed company to view cover crop breeding work in Prairie du Sac, WI. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1. Identify markers associated with seed dormancy and seed shatter, • Our team completed a draft assembly of the hairy vetch genome. Collaborator Bickhart ran various assembly methods, and found the optimal method and draft assembly. Scaffolding of 7 chromosomes was completed. • PI Bhamidimarri and Collaborator Ali conducted time sulfuric acid assays to assess the degree of hard seededness in diverse hairy vetch lines. They made crosses between hard and soft seeded lines to determine inheritance of the trait. They also assessed correlations between hard seed and seed size. • PI Bhamidimarri and Collaborator Wiering (Univ of MN) developed clonal propagation methods for hairy vetch. • Collaborator Monters (NRI) developed a tissue culture method for hairy vetch. Using this protocol, Collaborator Monters is increasing 'AU Merrit' genotypes for sequencing. • Collaborator Ali completed Genotype by Sequencing (GBS) of all 1,140 individual genotpes from the GWAS test set. • Collaborator Kissing Kucek and PD Riday coordinated the phenotyping of the 1140 individual genotypes of the GWAS test set for hard seed and pod shatter. • Collaborator Tilhou completed a GWAS for hard seed and seed shatter and found a large-effect locus for hard seed in chromosome 1. Results are drafted and will be submitted to a journal in 2023. • An inexpensive, rapid size-based marker is being created to screen for individuals with the hard seed reducing locus from the GWAS. • Collaborator Ali phenotyped and partially harvested a mapping population of an extreme hard seed line and a soft seed line, AU Merit. In order to construct a genetic linkage map, generate SNP markers and identify QTL governing the seed hardness traits, we have generated a bi-parental mapping population by crossing NF21 (hard seed) x AU Merit (soft seed). ?• Collaborator Monteros assessed divergent lines from 2017 and 2018 screenings for candidate genes for pod shatter in Medicago sativa L. • Collaborator Tang and progressed on a gene expression atlas for annotation of the Vicia villosa reference genome, including gene expression from 26 different samples out of 7 tissue types. We have identified 2,763 to 7,361 constitutively expressed genes from these tissue samples. Differential expression analysis identified number of candidate genes that are expressed specifically in seed coats and/or immature pods. These resources provide hairy vetch specific sequences for primer design of key target genes involved in seed-related traits (hard vs. soft, shatter vs. non-shattering) and an initial framework of genes expressed in different tissues. These sequences will inform a hairy vetch 'gene atlas' and provide the foundation for the annotation of the hairy vetch genome. • Advisor Smykal completed histological analyses of four extreme lines for hard seed. 2. Breed improved soft-seeded and low shatter varieties of hairy vetch • Collaborator Kissing Kucek conducted a pedigree analysis on 26 site-years of data, including over 3500 genotypes to find the softest seeded and lowest shatter genotypes to enter into breeding nurseries. • PD Riday, PI Mirksy, and collaborator Englert (ORPMC) established, collected data, and harvested seed from nurseries of soft-seeded and low shatter vetch in 2021. PD Riday established a nursery of soft-seeded and low shatter vetch in October of 2021. • Collaborator Kissing Kucek coordinated the evaluation of hard seed and shatter on over 1000 breeding lines from 2021 hairy vetch nurseries. • Collaborator Kissing Kucek analyzed data to measure gain in selection for hard seed and shatter in five years of a hairy vetch breeding program. 3. Evaluate improved lines of hairy vetch with farmers • Collaborator Kissing Kucek coordinated Participatory Variety Testing of vetch varieties at 13 sites across the country. Farmer feedback was gathered at a field day in Lockeford, CA, March 2020. Due to COVID-19, the remaining field days were cancelled in 2020. • Due to COVID-19, no participatory field days were hosted in 2021. • Collaborators Kissing Kucek and Bastos Martins are exploring virtual variety rating platforms that farmers and seed companies could use through the Cover Crop Breeding website (www.covercropbreeding.com). 4. Develop a tool to guide seed companies in the emerging cover crop seed market • Collaborators Kissing Kucek and Bastos met with Professor Roderick M. Rejesus (NCSU Dept of Agricultural and resource Economics) to create a strategy and action plan for cover crop seed market forecasting. For the analysis of cover crop seed demand, we sought county-level sales data from seed companies. For the analysis of cover crop seed supply, we sought production estimates from growers or public surveys (e.g. the Oregon State University Forage Seed Production report) and pricing information. • Collaborator Bastos completed a search of the scientific literature concerning cover crop seed markets. Little to no information has been published. • Collaborator Bastos, Riday, Kissing Kucek, and Reberg-Horton (NCSU) conducted one-on-one interviews with cover crop seed companies concerning willingness to share necessary data for cover crop seed market forecasting. • Notes were found and explored from a 2-day meeting with 10 seed companies in 2018. This meeting served as a baseline for seed company needs, rather than recreating the meeting at ARS in 2019, as initially proposed in the grant. • Collaborator Moore presented to the American Seed Trade Association about the cover crop breeding program and soft seed development in Vetch in November of 2020. • The Oregon State University has stopped publishing legume cover crop seed production reports. Consequently, we lack seed production and sales data from both seed companies and public tracking. • After extensive research and outreach, we concluded that lack of information and transparency will prevent us from accomplishing this objective.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2023 Citation: Ali, S., L. K. Kucek, H. Riday, N. Krom, S. Krogman et al., 2023 Transcript profiling of hairy vetch (Vicia villosa Roth) identified interesting genes for seed dormancy. The Plant Genome 16: e20330.
  • Type: Journal Articles Status: Submitted Year Published: 2023 Citation: Fuller, T., L. M. Koch, L. K. Kucek, S. Ali, H. Mangelson et al., 2023 A reference assembly for the legume cover crop, hairy vetch (Vicia villosa). 2023.03.28.534423.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Advances in hairy vetch breeding for cover crop use (NAAIC 2022 June 7-9)


Progress 02/15/21 to 02/14/22

Outputs
Target Audience:Farmers, seed companies, researchers Changes/Problems:The Noble Research Institute, major collaborator on this grant, dissolved plant sciences research. All staff and PIs associated with the project were no longer able to continue. We were able to reallocate their pending funding to complete objectives 1 and 2 through the USDA-ARS and USDA-NRCS-PMCs. Disease was so intense in the two mapping population fields that the vast majority of plants were lost. We were unable to gather enough seed to phenotype for shatter and hard seed. We were unable to host in-person participatory field days due to the global pandemic. After extensive research and outreach (see accomplishments), we concluded that lack of information and transparency will prevent us from accomplishing this objective. What opportunities for training and professional development has the project provided?Monteros mentored one post-doc. Kissing Kucek mentored three undergraduates, one PhD student and one post-doc. Kissing Kucek taught a section on plant breeding for sustainable agriculture for a University of Minnesota undergraduate class.Riday mentored one post-doc, and one undergraduate. How have the results been disseminated to communities of interest?Multiple manuscripts are in preparation for peer-reviewed journals. We presented at scientific meetings (PAG, ASA-CSSA-SSSA), grower meetings (MCCC, MOSES, USDA-ARS-DFRC seminar series), and seed company meetings (ASTA, upper Midwest Seed Summit) ?A website (www.covercropbreeding.com) was created for the project to increase visibility and to facilitate future variety release. What do you plan to do during the next reporting period to accomplish the goals?Ali plans to complete analysis, manuscript writing, and publication of Genome Wide Association Study for hard seed and pod shatter. Collaborators Bickhart and Monteros plan to publish the reference genome of Vicia villosa. We plan to host participatory field days

Impacts
What was accomplished under these goals? 1. Identify markers associated with seed dormancy and seed shatter, Our team generated a draft assembly of the hairy vetch genome. Collaborator Bickhart ran various assembly methods, and found the optimal method and draft assembly. Scaffolding of 7 chromosomes was completed. Collaborator Ali completed a draft GWAS of the test set using Golden Helix software. Collaborator Ali drafted a manuscript of RNAseq data. Collaborator Ali phenotyped and partially harvested a mapping population of an extreme hard seed line and a soft seed line, AU Merit.In order to construct a genetic linkage map, generate SNP markers and identify QTL governing the seed hardness traits, we have generated a bi-parental mapping population by crossing NF21 (hard seed) x AU Merit (soft seed). DNA was isolated from F1 progenies and 215 F1s were identified as a result of confirm crosses in SSR marker analysis. GBS libraries were constructed from these progenies and sequenced in Illumina NextSeq machine using single read with 75bp run setting. Collaborators Ali and Ibrahim worked with CSU to complete metabolic profiling of hard and soft seed coats of hairy vetch. Data has been received, but is not yet analyzed. Collaborator Monteros assessed divergent lines from 2017 and 2018 screenings for candidate genes for pod shatter in Medicago sativa L. Collaborator Ali completed Genotype by Sequencing (GBS) of all 1,140 individual genotpes from the GWAS test set. Collaborator Kissing Kucek and PD Riday coordinated the phenotyping of the 1140 individual genotypes of the GWAS test set for hard seed and pod shatter. Collaborator Tang and progressed on a gene expression atlas for annotation of the Vicia villosa reference genome, including gene expression from 26 different samples out of 7 tissue types. We have identified 2,763 to 7,361 constitutively expressed genes from these tissue samples. Differential expression analysis identified number of candidate genes that are expressed specifically in seed coats and/or immature pods. These resources provide hairy vetch specific sequences for primer design of key target genes involved in seed-related traits (hard vs. soft, shatter vs. non-shattering) and an initial framework of genes expressed in different tissues. These sequences will inform a hairy vetch 'gene atlas' and provide the foundation for the annotation of the hairy vetch genome. Advisor Smykal completed histological analyses of four extreme lines for hard seed. 2. Breed improved soft-seeded and low shatter varieties of hairy vetch Collaborator Kissing Kucek conducted a pedigree analysis on 26 site-years of data, including over 3500 genotypes to find the softest seeded and lowest shatter genotypes to enter into breeding nurseries. PD Riday, PI Mirksy, and collaborator Englert (ORPMC) established, collected data, and harvested seed from nurseries of soft-seeded and low shatter vetch in 2021. PD Riday established a nursery of soft-seeded and low shatter vetch in October of 2021. Collaborator Kissing Kucek coordinated the evaluation of hard seed and shatter on over 1000 breeding lines from 2021 hairy vetch nurseries. Collaborator Kissing Kucek analyzed data to measure gain in selection for hard seed and shatter in five years of a hairy vetch breeding program. 3. Evaluate improved lines of hairy vetch with farmers Collaborator Kissing Kucek coordinated Participatory Variety Testing of vetch varieties at 13 sites across the country. Farmer feedback was gathered at a field day in Lockeford, CA, March 2020. Due to COVID-19, the remaining field days were cancelled in 2020. Due to COVID-19, no participatory field days were hosted in 2021. Collaborators Kissing Kucek and Bastos Martins are exploring virtual variety rating platforms that farmers and seed companies could use through the Cover Crop Breeding website (www.covercropbreeding.com). 4. Develop a tool to guide seed companies in the emerging cover crop seed market Collaborators Kissing Kucek and Bastos met with Professor Roderick M. Rejesus (NCSU Dept of Agricultural and resource Economics) to create a strategy and action plan for cover crop seed market forecasting. For the analysis of cover crop seed demand, we sought county-level sales data from seed companies. For the analysis of cover crop seed supply, we sought production estimates from growers or public surveys (e.g. the Oregon State University Forage Seed Production report) and pricing information. Collaborator Bastos completed a search of the scientific literature concerning cover crop seed markets. Little to no information has been published. Collaborator Bastos, Riday, Kissing Kucek, and Reberg-Horton (NCSU) conducted one-on-one interviews with cover crop seed companies concerning willingness to share necessary data for cover crop seed market forecasting. Notes were found and explored from a 2-day meeting with 10 seed companies in 2018. This meeting served as a baseline for seed company needs, rather than recreating the meeting at ARS in 2019, as initially proposed in the grant. Collaborator Moore presented to the American Seed Trade Association about the cover crop breeding program and soft seed development in Vetch in November of 2020. The Oregon State University has stopped publishing legume cover crop seed production reports. Consequently, we lack seed production and sales data from both seed companies and public tracking. After extensive research and outreach, we concluded that lack of information and transparency will prevent us from accomplishing this objective.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Wiering, N. 2021. Cover crop breeding & estimating biomass in situ with remote sensing. National Association of Plant Breeders Conference.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Kissing Kucek, L., et al. 2020. The Cover Crop Breeding Project Midwest Cover Crop Council, Kansas City, MO, February 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Kissing Kucek, L. 2020 Cover Crop Variety Performance US Dairy Forage Research Center Webinar Series, December 2020. https://www.ars.usda.gov/ARSUserFiles/50901500/Archives/Presentations/201202_Kissing-Kucek_web_sm.mp4
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Wiering, N., G. Gardner, C.C. Sheaffer, and N.J. Ehlke. 2020. Root and axillary shoot development of hairy vetch stem cuttings and cessation of flower development under a short photoperiod. Crop Science 60(5): 23862393. doi: https://doi.org/10.1002/csc2.20238.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Moore, V. 2021. Cover Crop Breeding Network. New York Seed Growers Meeting, July 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Moore, V. 2021. Cover Crops for NY Dairy Farms. Forage Congress, March 2021.


Progress 02/15/20 to 02/14/21

Outputs
Target Audience:Seed Industry, Organic Farmers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?1. Complete genome wide association study. 2. Begin evaluating experimental softseeded hairy vetch varieties. 3. Accomplish another cycle of selection for softseeded and low shattering hairy vetch germplasm.

Impacts
What was accomplished under these goals? 1. Initiated seed production on first softseeded hairy vetch experimental varieties. 2. Initial hairy vetch genome assembly created. 3. Completed genotyping of softseeded hairy vetch genome wide association panel.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Kucek, L.K., M.D. Azevedo, S.S. Egan, N.J. Ehlke, R.J. Hayes, S.B. Mirsky, C. Reberg-Horton, M.R. Ryan, S. Wayman, N.P. Wiering, and H. Riday. 2020. Seed dormancy in hairy vetch (Vicia villosa Roth) is influenced by genotype and environment. Agronomy 10:1804.


Progress 02/15/19 to 02/14/20

Outputs
Target Audience:farmers, seed companies, researchers Changes/Problems:The highly repetitive and heterozygous genome required more coverage in Nanopore sequencing. Some of the pods from the Knox City, Texas site were harvested too early. Collaborator Kissing Kucek requested a duplicate set of pods be pulled at NRI to rerun shattering and hard seed tests for these immature lines. What opportunities for training and professional development has the project provided?Collaborator Kissing Kucek mentored one undergraduate (experimental design, R, randomization, scientific manuscript writing), one PhD student and one post-doc (error correction using spatial relationships, multi-environment trial analysis). PD Riday mentored one post-docoral researcher (heritability analyses, non-parametric analyses, selection theory) and one PhD student (DNA extraction from seed, SSR development, and identification of selfing). PI Monteros mentored one post-doctoral researcher in transcriptomics. How have the results been disseminated to communities of interest?Field days and winter meetings communicated results and sought feedback from farmers and seed companies Collaborator Kissing Kucek developed a standardized Participatory Variety Testing sheet to gather feedback on advanced breeding lines from farmers and seed companies. Feedback on breeding lines of vetch was gathered at field days in Columbia, MO, May 2019. Seed companies selling cover crops, including two grant advisory board members, were invited to the 2019 annual Cover Crop Breeding meeting in Raleigh, NC. The meeting included a tour of a hairy vetch breeding nursery. Team members gathered feedback from companies on breeding objectives and market needs. PD Riday hosted a legume cover crop seed company to view cover crop breeding work in Prairie du Sac, WI. Collaborator Kissing Kucek discussed cover crop breeding progress to farmers, seed companies, and researchers during the Upper Midwest Seed Summit in Madison, WI, October, 2019. Collaborator Kissing Kucek participated in a discussion with farmers, researchers, and seed companies during the Midwest Cover Crop Council Annual Meetings, Kansas City, MO, February 2020. Collaborator Kissing Kucek facilitated a roundtable with farmers, researchers, and seed companies during the Midwest Organic Sustainable Education Service Annual Conference, La Crosse, WI, February 2020. Publications and meeting presentations communicated results to researchers see "products" reporting section What do you plan to do during the next reporting period to accomplish the goals?• Complete hard seed assays from the GWAS test set. • SNP calling and GWAS analysis of the test set. • Mine RNA sequences of candidate genes for seed coats (SNP discovery and differential transcripts) • Validate the differential expression of ~20 genes using qPCR • Develop linkage map, phenotype and QTL analysis • Evaluate inbreeding, heterosis and general/specific combining ability analysis from the diallel crosses • Explore value and feasibility of developing a MAGIC population in hairy vetch

Impacts
What was accomplished under these goals? 1. Identify markers associated with seed dormancy and seed shatter, Reference genome sequencing Our team progressed toward sequencing the genome of hairy vetch. Collaborator Monteros conducted a screening for tissue culture, and selected a genotype that performed well in tissue culture: 'HV-30' derived from the cultivar 'AU-Merit.' The University of Minnesota (Ehlke) provided clones of an individual named 'V01' from a contrasting norther population for sequencing. Advisor Bickhart and staff at the DFRC conducted Oxford Nanopore sequencing of two reference individuals: 'V01' northern genotype and 'HV-30' southern genotype. 95x coverage was provided for the 'V01,' and slightly less coverage for the 'HV-30' genotype. Staff at DFRC is preparing Illumina libraries for short-read error correction. These libraries will be sent to the University of Wisconsin Biotech Center. PI Monteros and staff at NRI progressed on PacBio and Hi-C sequencing approaches to Nanopore. Hi-C sequencing is near completion. Genetic material has been sent to Sean Sullivan at Phase Genomics for PacBio sequencing. Collaborator Kissing Kucek consulted with researchers who have sequenced the related pea, lentil, and faba genomes. Researchers shared experiences with the best assembly and scaffolding techniques for repeat sequences and large genome size. GWAS Test Set Seed of the test set was scarified and sent to Corvallis, OR. Staff established transplants of each line in April of 2019. NRI, TXPMC, FSCRU, ORPMC collaborators tissue sampled test set trials. TXPMC and ORPMC collected phenotypic data on test sets. Collaborator Ali started Genotype-by-Sequencing of 1140 individuals of the test set. Other PI Monteros assessed divergent lines for dehiscence from 2017 and 2018 for candidate genes for pod shatter. Orthologous genes SHAT1, SHP2, and Expansin A like protein (from soybean) were associated with differential pod shattering in hairy vetch pods. A mapping population of hard and soft parents was generated using the extreme hard seeded parent NF21 (developed at NRI) and soft seeded parent 'AU Merit.' 215 F1 progeny were clonally propagated and evaluated in 2 locations in 3 replicates, then phenotyped and genotyped. Collaborator Ali and PI Monteros used RNA Seq to identify tissue-specific gene expression associated with soft vs. hard seed. Up-regulated genes associated with hard seed were identified in seed coats and cotyledons. Down-regulated genes associated with ahrd seed were identified in pods, leaves, and flowers. 2. Breed improved soft-seeded and low shatter varieties of hairy vetch, Collaborator Kissing Kucek and PD Riday coordinated the evaluation of pod shatter and hard seed on 615 breeding lines from 2018 hairy vetch nurseries and pod shatter on 608 breeding lines from 2019 hairy vetch nurseries. Collaborator Kissing Kucek generated Best Linear Unbiased Predictors and Estimates of maternal line performance for hard seed and shatter in 2017 and 2018 hairy vetch breeding lines. Regionally appropriate lines with the softest 5% of seed and lowest shattering lines were chosen for crossing blocks in Beltsville, MD; Prairie du Sac, WI; and Ardmore, OK. Seed was shipped from collaborators across the country, and transplants were established at all sites. The Ardmore, OK site established two separate crossing blocks: one for soft seed and low shatter and another for hard seed and high shatter. Collaborator Kissing Kucek developed a protocol for establishment, data collection, selection, and seed harvest for the crossing blocks. Collaborator Ali phenotyped 69 GRIN Plant Introductions using an acid scarification protocol for hard seededness. The most extreme individuals are being bred. Collaborator Ali developed populations with differing degrees of hard seededness, identified through an acid scarification protocol. A diallel cross was completed with 7 parents to determine inbreeding depression, heterosis, GCA/SCA, dominance/additivity, and narrow sense heritability of hard seed. 3. Evaluate improved lines of hairy vetch with farmers, and Collaborator Kissing Kucek developed a standardized Participatory Variety Testing sheet to gather feedback on advanced breeding lines from farmers and seed companies. Feedback on breeding lines of vetch was gathered at field days in Columbia, MO, May 2019. 4. Develop a tool to guide seed companies in the emerging cover crop seed market. Seed companies selling cover crops, including two grant advisory board members, were invited to the 2019 annual Cover Crop Breeding meeting in Raleigh, NC. The meeting included a tour of a hairy vetch breeding nursery. Team members gathered feedback from companies on breeding objectives and market needs. PD Riday hosted a legume cover crop seed company to view cover crop breeding work in Prairie du Sac, WI. Collaborator Kissing Kucek discussed cover crop breeding progress to farmers, seed companies, and researchers during the Upper Midwest Seed Summit in Madison, WI, October, 2019. Collaborator Kissing Kucek participated in a discussion with farmers, researchers, and seed companies during the Midwest Cover Crop Council Annual Meetings, Kansas City, MO, February 2020. Collaborator Kissing Kucek facilitated a roundtable with farmers, researchers, and seed companies during the Midwest Organic Sustainable Education Service Annual Conference, La Crosse, WI, February 2020.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Ali, S. et al. Transcript profiling of hairy vetch (Vicia villosa). Proceedings from the Plant and Animal Genome XXVIII, San Diego, CA. January 2020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Monteros, M. et al. Translational genomics of forage legumes to advance molecular breeding of seed traits in cover crop legumes. Proceedings from the Plant and Animal Genome XXVIII, San Diego, CA. January 2020.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Kissing Kucek, L. H. Riday, et al. 2020. Pod dehiscence in hairy vetch (Vicia villosa Roth). Frontiers in Plant Science.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Kissing Kucek, L. et al. Efforts to domesticate hairy vetch (Vicia villosa Roth): eliminating hard seed and pod shatter. Proceedings from the Plant and Animal Genome XXVIII, San Diego, CA. January 2020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Hernandez, T. et al. Legeraging legume genomic resources to identify genes associated with non-seed shattering in hairy vetch (Vicia villosa Roth). Proceedings from the Plant and Animal Genome XXVIII, San Diego, CA. January 2020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Riday, H; L. Kissing Kucek; et al. Progress in hairy vetch cover crop breeding. IFTBC Meetings. Orlando, FL. February 2019.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Riday, H. L. Kissing Kucek; et al. Breeding for soft seeded non shattering hairy vetch (Vicia villosa Roth) NAPB. Pine Mountain, GA. August 2019
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Monteros et al. Leveraging Legume Genomic Resources to Identify Genes Associated with Non-Seed Shattering in Hairy Vetch (Vicia villosa Roth). ASA-CSSA-SSSA. San Antonio, TX. November 2019.


Progress 02/15/18 to 02/14/19

Outputs
Target Audience:The target audience includes researchers, farmers, and seed companies. Changes/Problems: The lines had poor germination and emergence at Corvallis PMC because of dry conditions in the fall and predation on the few lines that had germinated and emerged following planting. Due to the lapse in Federal funding, the hard seed evaluations were not completed in time to inform selections for the 2019 breeding nurseries. During the lapse in government funding, flow cells purchased for the intended use of sequencing hairy vetch expired. What opportunities for training and professional development has the project provided? Collaborator Kissing Kucek trained one staff member and threeundergraduate students in pod shatter evaluation and one post-baccalaureate and four undergradues in hard seed testing. Collaborator Casey from the Pullman, WA PMC developed a comprehensive guide to Field Book software for field evaluations. Collaborator Casey trained PMC staff at the Corvallis, OR and Knox City, TX PMCs. How have the results been disseminated to communities of interest? Internal results are shared using a Google Drive folder specific to the grant. Results are still preliminary.We are preparing field days and manuscripts to disseminate results to the communities of interest once results are finalized. What do you plan to do during the next reporting period to accomplish the goals? Collaborator Kissing Kucek and PD Riday are analyzing 2018 pod shatter evaluations and preparing a manuscript for submission to Frontiers in Plant Science. Collaborator Kissing Kucek will generate breeding values for the eight hairy vetch nurseries across the country to select the best ~5% for crossing and advance in the spring and summer of 2019. Nanopore sequencing of the MN V1 genotype will begin in May of 2019. Corvallis, OR PMC reestablished the test set in April 2019 by growing out the plants in the greenhouse and transplanting seedlings into the evaluation nursery.The goal is to proceed with data collection once the plants are large enough to evaluate for the criteria established in the protocol.The ARS-FSCRU is prepared to assist the Corvallis PMC with tissue collection. In spring 2019, Knox City and Corvallis PMCs will continue with evaluations of the lines, which includes a spring vigor rating, collecting tissue samples for DNA analysis, harvesting seed pods for shatter resistance estimates, and harvesting the biomass.

Impacts
What was accomplished under these goals? 1. Identify markers associated with seed dormancy and seed shatter, Our team progressed toward sequencing the genome of hairy vetch.Conference calls among collaborators identified one southern hairy vetch type ('AU Merrit') and one northern hairy vetch type (MN V1) for sequencing.During the lapse in government funding, flow cells purchased for the intended use of sequencing hairy vetch expired.New funding to purchase additional flow cells has been acquired by ARS-DFRC advisory board member Derek Bickhart and Noble Research Institute PIs.Staff at the ARS-DFRC tested methods for obtaining high quality high molecular weight DNA needed for the nanopore sequencing. PI Bhamidimarri and Collaborator Ali conducted time sulfuric acid assays to assess the degree of hard seededness in diverse hairy vetch lines.They made crosses between hard and soft seeded lines to determine inheritance of the trait.They also assessed correlations between hard seed and seed size. PI Bhamidimarri and Collaborator Wiering (Univ of MN) developed clonal propagation methods for hairy vetch. Collaborator Monters (NRI) developed a tissue culture method for hairy vetch.Using this protocol, Collaborator Monters is increasing 'AU Merrit' genotypes for sequencing. PD Riday and Collaborator Kissing Kucek coordinated the evaluation of 477 hairy vetch lines for pod shatter and hard seed.Collaborator Kissing Kucek analyzed the data and identified the most extreme 80 lines for hard seed and pod shatter.This set of 80 maternal lines formed the test set for GWAS.10 seed from each of the 80 lines was hand scarified at the ARS-DFRC and sent to Corvallis and Knox City PMCs. The ARS-DFRC sent tissue to Collaborator Monteros of divergent lines for pod shatter.Collaborator Monteros assessed divergent lines for candidate genes for pod shatter in related species. Collaborator Monteros is developing a gene expression atlas for roots, shoots, leaves, flowers, and seeds. PD Riday and Collaborator Kissing Kucek hosted the 2018 Annual Team Meeting in Madison, WI to outline grant priorities, create action plans for completing work, and develop protocols.Collaborator Kissing Kucek presented a review of grant goals and hard seed in vetch research.PI Bhamidimarri presented an update of hairy vetch breeding goals and molecular research from the NRI.Collaborators Englert and Douglas facilitated a conversation to define field protocols at the PMCs.Advisory board member Renzi presented on seed dormancy in hairy vetch and agronomic seed production research on hairy vetch in Argentina. Collaborator Kissing Kucek, PD Riday, and the PMCs developed standardized protocols to install, manage, and evaluate the test set.Periodic conference calls introduced the protocols, checked progress, and problem solved concerns for the field sites. Plant Materials Centers (PMC) at Knox City, TX and Corvallis, OR prepared the study site (weed control, tillage, seedbed) in September/October 2018 according to the protocol and field map.A total of 800 plots (80 maternal lines from the LCCB program (40 lines with high hard seed and 40 lines with low hard seed x 10 blocks), were hand seeded in late September at Corvallis and mid-October at Knox City, following a randomized complete block design.Weed control fabric was installed at Corvallis prior to planting.An electric fence was installed around the plots at Knox City to prevent wildlife damage.Plots were monitored weekly for emergence and weeds were control with tillage and hand rouging.Of the 800 plots seeded at both locations, 625 plots had emerged at Knox City and 60 plots at Corvallis by early December.A fall vigor rating was taken in early December 2018 at Knox City.Due to poor emergence and predation at Corvallis no evaluations were taken prior to February 2019 (plots were re-established in April 2019). 2. Breed improved soft-seeded and low shatter varieties of hairy vetch, Collaborator Kissing Kucek and PD Riday coordinated the evaluation of pod shatter and hard seed on 477 breeding lines from 2017 hairy vetch nurseries. PI Bhamidimarri and Collaborator Ali conducted time sulfuric acid assays to assess the degree of hard seededness in diverse hairy vetch lines.PI Bhamidimarri and Collaborator Ali made crosses between hard and soft seeded lines to develop divergent breeding populations. Collaborator Kissing Kucek conducted a literature review on pod shatter, then developed a method for quantifying pod shatter using an Insight2 push force machine. Collaborator Kissing Kucek and PD Riday coordinated the evaluation of pod shatter on 615 breeding lines from 2018 hairy vetch nurseries. PI Bhamidimarri and Collaborator Ali conducted time sulfuric acid assays to assess the degree of hard seededness in diverse hairy vetch lines.PI Bhamidimarri and Collaborator Ali made crosses between hard and soft seeded lines to develop divergent breeding populations. In the late summer and early fall of 2018, collaborators established eight nurseries for hairy vetch breeding in Oklahoma (NRI), North Carolina (NC State Univ), Maryland (ARS-BARC), New York (Cornell Univ), Wisconsin (ARS-DFRC), and Minnesota (Univ of MN).Each site hand-planted 1200 to 2400 vetch breeding lines.A cold winter throughout the country provided good selection for winter hardiness.Evaluations included emergence, fall vigor, spring survival, and spring vigor.The best ~5% of plants will be selected for crossing and advance. 3. Evaluate improved lines of hairy vetch with farmers, Evaluations of hard seed were shared with a farmer collaborator in Wilton, WI, who used the information to select soft-seeded lines from his participatory breeding hairy vetch nursery. At the 2018 Legume Cover Crop Breeding Annual Meeting in Madison, WI Collaborator Kissing Kucek facilitated a focus group to decide how Participatory Variety Trialing field days would be conducted at 13 nationwide sites to gather feedback on advanced lines from farmers and seed companies. 4. Develop a tool to guide seed companies in the emerging cover crop seed market. Seed companies selling cover crops, including two grant advisory board members, were invited to the 2018 annual Legume Cover Crop Breeding Annual Meeting in Madison, WI and the 2019 annual Cover Crop Breeding meeting in Raleigh, NC.The meeting included a tour of a hairy vetch breeding nursery.Team members gathered feedback from companies on breeding objectives and market needs.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Riday, H. et al. 2019. Progress in hairy vetch cover crop breeding. International Forage and Turf Breeding Conference, March 24-27, Lake Buena Vista, FL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Renzi, J. 2019. Vicia villosa seed dormancy and germination. LCCB Annual Meeting, June 20, Madison, WI. http://connectpro70289319.adobeconnect.com/pukx7oluh46t/?OWASP_CSRFTOKEN=37e8ba032804d8cf17cf5417c792af2263ba6d4f6572d612fffe55ac046a041a