Source: WASHINGTON STATE UNIVERSITY submitted to
DETERMINING GENETIC FACTORS THAT INFLUENCE PROTEIN QUALITY AND YIELD IN ALFALFA
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1020900
Grant No.
2019-70005-30362
Cumulative Award Amt.
$500,000.00
Proposal No.
2019-02840
Multistate No.
(N/A)
Project Start Date
Sep 1, 2019
Project End Date
Aug 31, 2023
Grant Year
2020
Program Code
[AFRP]- Alfalfa and Forage Program
Project Director
Norberg, S.
Recipient Organization
WASHINGTON STATE UNIVERSITY
240 FRENCH ADMINISTRATION BLDG
PULLMAN,WA 99164-0001
Performing Department
Extension - ANR
Non Technical Summary
This integrated project will use a diverse collection of 200 alfalfa varieties to identify molecular markers and other genetic information that affect 4-year yield and protein degradability, and the linkage of these two traits. Our overall goal is to implement the resulting data into breeding programs so that new varieties of alfalfa can be generated that maximize both the total plant yield (benefit to alfalfa growers) and the subsequent availability of alfalfa-derived nutrients in ruminant digestion (benefit to cattle/dairy industry) - this is an innovative departure from the longstanding work of many breeding programs, which are focused primarily on agronomic traits such as winter hardiness/stand longevity, drought/saline tolerance, etc. The move toward incorporating yield and protein quality into breeding programs is expected to improve sustainability across multiple industries and ecosystems and will involve: i) quantifying alfalfa genetic diversity in protein degradability and yield in a diverse germplasm set; ii) quantifying the relationship between alfalfa protein degradability and important agronomic parameters; and iii) identifying genetic information associated with protein degradability and 4-year yield.The Objectives for this proposal will include: 1) Determine protein quality and stage of maturity at first harvest for 200 varieties at three locations (ID, OR, WA). 2) Quantify the genetic diversity that is related to forage protein quality and yield. 3) Identify molecular markers associated with protein degradability (ADICP, NDICP, soluble protein, microbial protein degradation, fractions of protein) and yield. 4) Extend actionable knowledge to positively affect alfalfa breeders, alfalfa seed/hay producers, and other industry stakeholders.
Animal Health Component
20%
Research Effort Categories
Basic
30%
Applied
20%
Developmental
50%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
51216401080100%
Knowledge Area
512 - Quality Maintenance in Storing and Marketing Non-Food Products;

Subject Of Investigation
1640 - Alfalfa;

Field Of Science
1080 - Genetics;
Keywords
Goals / Objectives
Goal 1-Quantifying alfalfa genetic diversity in protein degradability and yield in a diverse germplasm set.Goal 2-Quantifying the relationship between alfalfa protein degradability and important agronomic parameters.Goal 3 -Identifying genetic information associated with protein degradability and 4-year yield.
Project Methods
Objective 1. Determine protein quantity and quality and stage of maturity at first harvest of 200 alfalfa varieties at three locations in the PNW. Reduce confounding factors with quality and yield.Task 1.1 - Seed selection and agronomy. The diverse set of germplasm comes from active breeding programs in academia, industry, and government. Collectively, S&W Seed Co., Alforex Seed Co., Legacy Seed Co, and Blue River Hybrid Seed Co. contributed 150 alfalfa genotypes, which were matched with an additional 150 alfalfa genotypes and accessions from the USDA- ARS National Plant Germplasm System (NGPS). Since the quantity for many of the selections/varieties is very limited we used an augmented treatment design with eleven blocks and two controls, Vernal and HiGest 360, with a plot size of 1 meter by 4.6 meters. To eliminate fall dormancy effects, we included the standard fall dormancy checks 1-6.Task 1.2 - Wet Chemistry. With the samples from Task 1.1, co-PD Zanton will oversee efforts to determine alfalfa forage protein quality including work with the Near Infrared Spectroscopy Consortium (see Letter of Cooperation) to develop calibrations to decrease the amount of wet chemistry required using the process described by Laskowski Morren, McIntosh, and Anderson (Progressive Forage Magazine, 12/28/2016). To accomplish this task, a subset of alfalfa varieties will be analyzed in Year-1 for chemical fractions associated with protein degradability, which are required for use in the CNCPS (Higgs et al., 2015) by methods approved by AOAC International (2005), where available. All blanks, standards, and alfalfa samples will be evaluated in duplicate in each run and each in vitro run will be replicated three times for a total of 6 replicates per sample. Calculation of the Fraction A and Fraction B, and the rate of Fraction B protein degradation (kd) will be calculated as described previously (Broderick et al., 2004b).Task 1.2 - Data Analysis. The chemical analyses and in vitro protein degradability results will be used to develop NIRS calibration equations. Year-2 will include 260 alfalfa samples to be analyzed using these chemical- and in vitro methods to evaluate the performance of the NIRS predictions. NIRS predictions for protein degradability and chemical fractions will be used to quantify genetic diversity and molecular markers for alfalfa.Objective 2. Quantify the genetic diversity of alfalfa that is related to forage protein quality and yield to breed new alfalfa varieties for higher forage quality and yield.Data Analysis. Alfalfa forage protein quality data generated from Objective 1 and field agronomic data including fall dormancy and yield will be analyzed across three locations with regression and correlation procedures. Correlations and regression between protein quality data and fiber quality data which generated from our last proposal will be also analyzed. This will provide multiple forage quality parameters that relate to each other and to forage yield. Objective 3. Identify molecular markers associated with protein degradability (ADICP, NDICP, soluble protein, microbial protein degradation, fractions of protein) and yield traits in alfalfa to aid alfalfa breeders.Task 3.1 - Gene identification. A diverse panel of alfalfa populations will be used for GWAS mapping to identify molecular markers associated with protein quality traits. A genotyping-by- sequencing (GBS) strategy will be used for developing thousands of markers throughout the genome. Single nucleotide polymorphisms (SNP) will be mapped using a HapMap; after which, it will undergo GWAS analysis in combination with phenotypic data of protein quality traits. Principal component analysis (PCA) will also be performed on the marker data for individuals within each population as well as across populations for protein quality. The analysis will use SAS PROC PRINCOMP (SAS Institute Inc. 2011, SAS OnlineDoc 9.3, Cary, NC, USA). A covariance matrix will be obtained and used for association analysis. Linkage disequilibrium (LD) between markers will be assessed by calculation of r2 between markers, LD statistics will be calculated and LD decay will be evaluated by an exponential probability density function using PROC NLIN in SAS software (Yu et al., 2011).Task 3.1 - Data Analysis. Significant alleles associated with each trait will be identified. We will use the GBS FreeBayes pipeline to assign the genotypes. The algorithm is based on the fitting of several mixture models with five components, one for each of the five possible genotypes (Yu et al., 2017).Task 3.2 - High-throughput platform. Markers that are found to be tightly linked to the quality loci will be used for developing diagnostic high-throughput markers for MAS.Task 3.2 - Data Analysis. Multiplex primers will be used for evaluating the resistance locus or candidate gene. All markers will be scored in a given genotype. Single markers with two- character states will be tested for significant phenotypic differences between genotype groups by the t-test for each trait, and Mann-Whitney U test for marker quality. Marker combinations will undergo analysis of variance (ANOVA) for each trait, and Kruskal-Wallis test for chip quality. Spurious associations can be reduced by taking population structure into account in a mixed linear model (MLM) analysis.Objective 4. Extend the knowledge gained from the project to positively impact alfalfa breeders, alfalfa seed and hay producers, and others in the alfalfa industryInformation Dissemination Plan. Results from field trials will be shared with Extension Forage Specialists and County Agents, Agribusiness crop agronomists, and producers from ID, OR, and WA during winter meetings following the first and second years of the project. At the end of the project, a special PNW Extension/AES report will also be published with descriptions of the alfalfa cultivars, including fall dormancy, yield, and protein content. Program Impact Evaluation. Formative evaluations will categorize priorities and/or develop new programs. Information collected during all phases of the programming and evaluation, will serve as Process Evaluations to determine if alterations in materials, methodology, or techniques are needed. In total, data collected during these evaluations (such as adoption of practices integrated with systematic economic analysis) will be utilized to determine the overall long-term impact of the programming (i.e., economic, social, and conditional change).Data Management Plan. The results from this research will be published in the peer-reviewed scientific journals. DNA sequence data will be submitted to The National Center for Biotechnology Information (NCBI). The phenotypic data will be collected in the second year of the project and continuing for the third and fourth years. These data will be submitted to database centers such as Dryad (http://datadryad.org/) or Alfalfa Breeder's toolbox (https://www.noble.org/research/clusters/breeders-toolbox/). For phenotypic data of germplasm, we will upload the evaluation data into the USDA Germplasm Resources Information Network (GRIN-Global) Database.

Progress 09/01/19 to 08/31/23

Outputs
Target Audience:The target audience includes:alfalfa plant breeders, alfalfa researchers, alfalfa producers and agribusiness related to alfalfa. Changes/Problems:Covid significantly slowed the process of this grant since the ARS laboratory was shut down for almost 2 years. We accomplished what we could and got two extensions on the grant and made significant progress on the grant once the ARS laboratory was opened back up. This was accomplished in July 2023 and we have been making good progress finishing things up. What opportunities for training and professional development has the project provided?Steve Norberg attended the Western Crop Science Society meeting in June of 2023 and presented on the impact of fall dormancy of alfalfa on forage quality. How have the results been disseminated to communities of interest?A meeting of the alfalfa breeders in North America occurred over zoom to share the results of this grant. Also research on fall dormancy impact on forage quality was given to the Western Crop Science Society of America. A scientific journal was published to help scientists and learn from this research. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? To accomplish these goals, we have developed four Objectives: Determine protein quality and stage of maturity at first harvest of 200 alfalfa varieties at three locations in the PNW. Reduce confounding factors with quality and yield; Accomplished. Quantify the genetic diversity of alfalfa that is related to forage protein quality and yield to breed new alfalfa varieties for higher forage quality and yield; Accomplished. Identify molecular markers associated with protein degradability (ADICP, NDICP, soluble protein, microbial protein degradation, fractions of protein) and yield traits in alfalfa; Will be accomplished in August 2023. Extend the knowledge gained from the project to positively impact alfalfa breeders, alfalfa seed and hay producers, and others in the alfalfa industry. Partially accomplished, molecular marker work on proteins still needs to be accomplished.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Medina, S. Lin, G. Wang, D. Combs, G. Shewmaker, S. Fransen, D. Llewellyn, S. Norberg, L. Yu. 2022. Identification of Genetic Loci Associated with Five Agronomic Traits in Alfalfa Using Multi-Environment Trials. Theoretical and Applied Genetics. Published April 13, 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Norberg, S., C. Culma, Z. Xu, D. Llewellyn, L. Yu, G. Wang, S. Fransen, D. Combs, G. Shewmaker, S. Lin. 2023. Influence of Fall Dormancy on Alfalfa Quality. Western Crop Science Society of America.


Progress 09/01/21 to 08/31/22

Outputs
Target Audience:Our audience is alfalfa breeders, alfalfa seed/hay producers, and other alfalfa industry stakeholders. Changes/Problems:Covid shutdown of lab has altered the timeline of protein degradability findings. We are doing out best to make progress as fast as we can this year we had a private lab do some quality testing to accelerateus up in some areas. What opportunities for training and professional development has the project provided?Attendance at North American Alfalfa Improvement Conference How have the results been disseminated to communities of interest?Results have been shared at conferences for alfalfa advancement as well as animal nutrition conferences and in the form of book chapter on genomics of alfalfa which are listed below. Lin, S., O.S. Norberg, D. Combs. 2021. Genomics of Forage Quality in Alfalfa, Chapter 7 In The Alfalfa Genome. Springer Nature L. Yu and C. Kole editors. Lin, S., C. Medina-Culma, G. Zanton, G. Wang, G. Shewmaker, S. Fransen, D. Llewellyn, L. Yu., S. Norberg. 2022. Determining Genetic Factors That Influence Protein Quality & Yield in Alfalfa. 2022 North American Alfalfa Improvement Conference. Invited International Proceedings Norberg, S., S. Lin, C. Medina-Culma, G. Zanton, G. Wang, G. Shewmaker, S. Fransen, D. Llewellyn, L. Yu. 2022. Identifying Molecular Markers Associated With Quality & Quantifying Their Potential to Increase Alfalfa Value. 2022 North American Alfalfa Improvement Conference. Dreger, S., D.A. Llewellyn, O.S. Norberg, S. Fransen, G. Wang, D. Combs, G. Shewmaker, E. van Santen, L.X. Yu. 2022. Evaluation of Forage Quality of Alfalfa from 200 Varieties Produced in the Pacific Northwest. Proceedings of the 56th Annual Pacific Northwest Animal Nutrition Conference, Boise, ID, January 17-18, 2022. What do you plan to do during the next reporting period to accomplish the goals?Manuscript on yield,fall dormancy, plant height, and mean stage of development bycutting and molecular markers influencing this will be published. Other manuscripts on forage quality, alfalfa yield will also be published. Final results on protein degradability will be accomplished and NIRS calibration for protein degradability will be developed. Finally a paper on molecular markers influencing protein degradability will be researched and published.

Impacts
What was accomplished under these goals? Goal 1. Manuscript is in late revisions after markers determining yield, fall dormancy, plant height, and mean stage of development at cutting molecular markers have been identified. Goal 2. Due to covid we are still doing wet chemistry on protein degradability but have made significant progress. Goal 3. Again, yield work is almost done but protein degradability is still being determined.

Publications

  • Type: Book Chapters Status: Published Year Published: 2021 Citation: Lin S., Norberg S., Combs D. Genomics of Forage Quality in Alfalfa. In L-X Yu and C. Kole Edits The Alfalfa Genome, Pages 129-143 Springer Nature Publisher. 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Norberg, S., S. Lin, C. Medina-Culma, G. Zanton, G. Wang, G. Shewmaker, S. Fransen, D. Llewellyn, L. Yu. 2022. Identifying Molecular Markers Associated With Quality & Quantifying Their Potential to Increase Alfalfa Value. 2022 North American Alfalfa Improvement Conference.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Dreger, S., D.A. Llewellyn, O.S. Norberg, S. Fransen, G. Wang, D. Combs, G. Shewmaker, E. van Santen, L.X. Yu. 2022. Evaluation of Forage Quality of Alfalfa from 200 Varieties Produced in the Pacific Northwest. Proceedings of the 56th Annual Pacific Northwest Animal Nutrition Conference, Boise, ID, January 17-18, 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Lin, S., C. Medina-Culma, G. Zanton, G. Wang, G. Shewmaker, S. Fransen, D. Llewellyn, L. Yu., S. Norberg. 2022. Determining Genetic Factors That Influence Protein Quality & Yield in Alfalfa. 2022 North American Alfalfa Improvement Conference.


Progress 09/01/20 to 08/31/21

Outputs
Target Audience:Our audience is alfalfa breeders, alfalfa seed/hay producers, and other alfalfa industry stakeholders. Changes/Problems:Covid shut down the ARS laboratory that was to do protein degradability work for over a year. This has slowed all the results of protein work significantly. All samples are currently at the laboratory getting analysis. Covid also made the field data collection more difficult. What opportunities for training and professional development has the project provided?Sen Lin and Steve Norberg attended the American Society of Agronomy Meetings virtually in 2020. How have the results been disseminated to communities of interest?Results have been diseminated at the American Society of Agronomy Meetings virtually in 2020. At producer meetings results have been shared at Alfalfa U., sponsored by High Plains Journal virtually with participants from three countries. The results was also shared with 7th Arizona Alfalfa and Forage Crops Workshop. Results was also shared with alfalfa breeders from the four major programs in the North America and they expressed interest in trying the molecular markers found. What do you plan to do during the next reporting period to accomplish the goals?Yield of this last year of production will continue to be measured and quality analysis will continue especially with protein degradation analysis. Near infrared spectroscopy calibration will be accomplished for multiple protein parameters and molecular markers identified for yield and protein quality. Results will be shared in many forms including scientific journal articles, presentations with many audiences.

Impacts
What was accomplished under these goals? Goal 1. Forage yield has been determined as has protein quality for 2020 crop as has first cutting of alfalfa in 2021. Goal 2. Is still in progress as covid has slowed lab work down in the ARS laboratory. Goal 3. Also still in progress.

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Lin, S., C.A. Medina, S. Norberg, D. Combs, G.Wang, G. Shewmaker, S. Fransen, D.Llewellyn, Long-Xi Yu. 2021. Genome-Wide Association Studies Identifying Multiple Loci Associated with Alfalfa Forage Quality. Frontiers in Plant Science. https://doi.org/10.3389/fpls.2021.648192
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Lin, S., C. Medina-Culma, S. Norberg, D. Combs, G. Wang, G. Shewmaker, S. Fransen, D. Llewellyn, E. van Santen and L. Yu. 2020. Genome-Wide Association Study Identified Multiple Loci Related to Different Traits on Alfalfa Quality. Virtual 2020 American Society of Agronomy Annual Meeting.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Norberg, S., L. Yu, D.K. Combs, G.E. Shewmaker, G.J. Wang, D. A. Llewellyn, S.C. Fransen and E. van Santen. 2020. Quantifying the Potential Increase In Alfalfa Value and Identifying Molecular Markers That Influence Quality. National Invited. Virtual NACAA Annual Meeting
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Norberg, O.S., L. Yu, D.K. Combs, G.E. Shewmaker, G.J. Wang, D. A. Llewellyn, S.C. Fransen and E. van Santen. 2019. Determining Genetic Factors that Influence Forage Quality in Alfalfa. 2019 American Society of Agronomy. San Antonio, TX


Progress 09/01/19 to 08/31/20

Outputs
Target Audience: Nothing Reported Changes/Problems:Due to NIFA move to Kansas City, it delayed the approval for money to be released which occurred on 5/12/2020 so it has given us less than a month to do our project before this report. We have made some progress. The major set back has been to do the wet chemistry required to do protein calibrations. Also now that Covid 19 has occurred it has made laboratory work stop so further delays may occur until laboratories can operate normally. We have moved it on our time line to Summer / Fall for wet chemistry to occur in 2020. 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? Table 1. Summary of work to be completed by research/Extension team. Time point Responsible PI Scope of Work F '19/20 Norberg, Zanton, NIRSC NIRS spectra from 2018 quality samples sent to NIRSC to identify number and which wet chemistry needs to be done. Accomplished 1st Harvest Norberg, Wang, Shewmaker, Zanton, NIRSC Mean stage by count and harvest is accomplished with moisture, yield and sample collection, (Accomplished) drying and grinding to 2 mm. Send Samples to Norberg for final grind. Then all 660 samples are forwarded to NIRSC then scanned and 220 selected for wet chemistry and subsequently used to modify calibration. 2nd Harvest and other harvests Norberg, Wang, Shewmaker Yield, fall dormancy recorded compared to checks Su/F '19/20 Zanton, NIRSC Wet chemistry accomplished and NIRSC scanning and first NIRSC calibrations established on previous years ~440 samples. Moved from W/S to Su/F Su/F '20 Norberg, Zanton Geoff Zanton and NIRSC send results Steve Norberg for review and analysis and results forwarded to Yu for use F/W '20/21 Yu Starts genetic process of developing molecular markers from first year's data W '20/21 Llewellyn, and Norberg Start Extension activities sharing field results and quality data from four locations As Needed Norberg Attend training, submit progress reports 1st Harvest '21 Norberg, Wang, Shewmaker, Zanton, NIRSC Mean stage by count and harvest is accomplished with moisture, yield and sample collection, drying and grinding to 2 mm. Send Samples to Norberg for final grind. Then all 660 samples are forwarded to NIRSC and data is predicted. Table 3 - Continued. Summary of work to be completed by research/Extension team. Time point Responsible PI Scope of Work 2nd Harvest and others Norberg, Wang, Shewmaker Yield, fall dormancy recorded compared to checks F '21 Yu Starts genetic process of developing molecular markers from second year's data F/W '21 Llewellyn and Norberg Continue Extension activities sharing field results and quality data from four locations F '21 Norberg 2 year grant ends submit Final Report

Impacts
What was accomplished under these goals? Due to NIFA move to Kansas City, it delayed the approval for money to be released which occurred on 5/12/2020 so it has given us less than a month to do our project before this report.We have made some progress on Goal 1. Goal 1 - Samples for the first two years spectra from Near Infrared Spectroscopy Consortium (NIRSC) and 444 samples were identified from previous years harvest. These samples were then sent to NIRSC for scanning to develop calibration for protein parameters once wet chemistry is done. Harvest of 2020 first cutting has been accomplished at all three locations and the grinding of samples has begun.

Publications