Progress 10/01/20 to 09/30/21
Outputs
Target Audience:Formal Classroom instruction in Molecular Genetics (GENE 315, synchronous online), Genes and Genomes (GENE 486, synchronous online), University Teaching Experience (AGRO 697, synchronous online), Doctoral Dissertation (AGRO 700, synchronous online) to diverse student population (25 undergraduate and 1 graduate students) including Caucasian, Hispanic, Black, Asian, female and male students during Fall 2020 and Spring 2021. Assisted with coordinating a virtual New Mexico Future Farmers of America Agronomy Career Development Event (April 14) due to COVID19 restrictions. Provided research progress updates to NM Hay Association Board of Directors (9 individuals) via conference calls on May 7 and September 24, 2021. Did NOT assist with organization, coordination, and moderating of 2021 Southwest Hay and Forage Conference which was cancelled due to COVID19. Coordinated online contacts with alfalfa breeding companies to discuss opportunities to employ genomic selection/prediction to improve aflafla drought resilience and conventional phenotyping approaches to improve alfalfa drought tolerance (April 7 and June 3) Served as Chair of the Western Regional Technical Advisory Committee of the USDA-National Plant Germplasm System (NPGS). Collaborated with USDA-ARS personnel to organize/host a virtual annual regional workshop to discuss status of multiple NPGS sites in the western U.S. Audience: USDA-ARS NPGS staff and state Agricultural Experiment Station representatives for the western U.S. (June 15, 2021). Presented two field day presentations on alfalfa genetic technologies and variety development to 80 farmers at Las Cruces and Artesia, NM (August 25 and 26). Presented research results on a genome-wide association study for forage yield and forage quality traits in elite alfalfa germplasm to a professional audience at the Crop Science Society of America Annual Meetings, Nov. 9-12 (Virtual meeting). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One PhD graduate student received training in phenotype data collection for forage biomass, forage quality sampling, seed germination salt tolerance, and disease resistance traits using 220 to 260 elite alfalfa populations. This student received training in conducting genome-wide association analysis (AA) and genomic selection (GS) for these traits in these populations which were genetically fingerprinted with >11,000 DNA markers. Based on AA, significant DNA markers for all traits have been identified in this Ph.D. dissertation research. Data analyses for other traits are in progress. The PhD student has also participated in a high-throughput computing workshops, received training in statistical analysis of data using splines to account for spatial variation in field research studies, and use of statistical software to determine best linear unbiased predictors and estimates (BLUP/BLUE) for multiple alfalfa populations being evaluated for diverse traits. Expertise was also gained in managing and analyzing large datasets (including DNA sequence and phenotype data) relevant to our research program. Extensive training was provided towards evaluating different data analyses approaches utilizing AA and GS to predict alfalfa forage yield potential and nutritional value. In an graduate course, Topics in Agronomy: Genome Data Analysis (AGRO 698), the above Ph.D. candidate served as an instructor to provide advanced training to another Ph.D. candidate from the NMSU onion breeding program. Hands-on training focused on 4 key areas that are affiliated with genome data analysis for marker development and application to plant breeding. These included: (1) management of large raw DNA sequences (genotype-by-sequencing data) from large populations including filtering and quality control evaluation, (2) alignment of sequence data to reference genomes for single nucleotide polymorphism (SNP) marker development, (3) development and filtering of SNP markers, and (4) integration of DNA marker (genotype) data with phenotype data for conducting Genome Wide Association Studies (GWAS) in important crops. One NMSU undergraduate student received extensive "hands-on" training in agronomy, plant breeding and genetic research techniques as a part-time employee with the alfalfa genetics/breeding program.. In a Genetics Internship course (GENE 391), one NMSU undergraduate student generated four experimental alfalfa populations by intermating approximately 20 plants each that were predicted to possess either high or low seedling germination salt tolerance based on their DNA marker profiles. The student conducted a replicated laboratory experiment to evaluate these materials under no salt-stress and high-salt stress conditions during seed germination. Results indicated that predictive use of DNA markers in a genomic selection models were able to improve seedling salt-tolerance by 2 to 10% depending on the quality of the phenotype dataset that was used to train the genomic selection models. In an Agronomy Special Topics course (AGRO 449, Drone Technology in Agriculture), one student received training in different aspects of utilizing Unmanned Aerial Systems (UAS, aka. Drones) in production agriculture and agricultural research. Training topics included an overview of different uses for UAS in agriculture, the basic principles of unmanned aircraft, aircraft operations, and sensor technologies that are utilized for agricultural monitoring and research. Some aspects of imagery processing and imagery analysis were also covered. As a technical assistant and observer trainee, the student also participated in several UAS imagery mapping flights of alfalfa research plots. Instruction was provided on aircraft pre-flight inspection, setup, and operations. Hands-on opportunities included setup of the aircraft and sensor system. All piloting was conducted by a certified UAS pilot. Student was also provided with information on UAS regulations as set by the Federal Aviation Administration (FAA). A brief overview on the current process and requirements for obtaining an FAA Part 107 Pilot Certificate was provided. Mentored 10 undergraduate genetics majors and 4 undergraduate agronomy majors. Provided recommendations on behalf of multiple undergraduate advisees to help them secure professional positions or pursue graduate studies. How have the results been disseminated to communities of interest?Annual reports of our research progress have been provided to a key alfalfa industry collaborator who financially supports our research and is very interested in utilizing DNA marker assisted selection (MAS) to improve alfalfa drought resilience and forage nutritional quality. Two virtual meetings (April 7 and June 3) were conducted with alfalfa breeders and research leaders in private industry to discuss (1) recent advancements and research opportunities to employ genomic selection/prediction to improve alfalfa drought resilience and forage quality, and (2) field-based phenotyping approaches to improve alfalfa drought resilience. Research progress updates were also provided to NM Hay Association Board of Directors (9 individuals) via two virtual board meetings in 2021. A graduate student presented results of their PhD research at the November 2020 Crop Science Society of America Annual Meeting (Virtual, Nov. 2020). Each year the program helps publish an Experiment Station Research Report that describes alfalfa variety performance results across the state. This report is made available online, as well as, in hard copy at the Southwest Hay and Forage Conference to provide hay growers in NM, AZ, and TX with information to help them identify the most suitable varieties for their particular growing region. One peer-reviewed manuscript discussing "Strategies to increase prediction accuracy in genomic selection of complex traits in alfalfa" was published to discuss molecular approaches to benefit development of superior autopolyploid crops. Presented two field day presentations on alfalfa genetic technologies and variety development to 80 farmers at Las Cruces and Artesia, NM (August 25 and 26). What do you plan to do during the next reporting period to accomplish the goals?Objective 1: We will continue collecting yield data for industry/public alfalfa populations evaluated under normal and deficit irrigation management at Las Cruces, NM as a participant in the NMSU state-wide alfalfa variety testing program. We will also analyze all alfalfa variety performance data for multiple locations across the state as a contribution to the state-wide alfalfa testing program. Multi-spectral aerial imagery of the above alfalfa variety trials will also be conducted with weekly imaging flights throughout the 2022 growing season. Aerial imagery and agronomic data will be integrated to determine correlations between aerial imagery spectral indexes and forage yield and quality. Seed increases of 10 new germplasms derived from the NMGS population will be conducted under cage isolation to produce seed for evaluating these materials in statewide and regional yield trials. Objective 2: Extensive analysis and modeling of DNA marker, forage yield, forage quality data collected in 2018-2020 from the NMGS alfalfa population will continue to be conducted to identify superior populations based on phenotypic selection parameters, association analysis marker selection, and genomic prediction/selection. Vigorous plants from these superior populations (evaluated under summer irrigation termination management for 3-years) were dug from field plots in 2021. These field dug plants, and also greenhouse mother plants from the NMGS alfalfa population, are currently being mated in various combinations in the greenhouse in winter/spring 2022 to develop new drought-resilient germplasm for future evaluation and potentially commercialization. Results of our extensive data collection will be submitted for publication in scientific journals and also as a PhD dissertation. Objective 3: Diploid populations will continue to be evaluated under summer irrigation termination management before imposing phenotypic selection for vigorous plants after 3 years of evaluation.
Impacts
What was accomplished under these goals?
New Mexico State University (NMSU) plant breeding research uses conventional and molecular approaches to improve alfalfa drought tolerance so that farmers can conserve irrigation water resources while meeting the feed demands of the U.S. livestock industry. Since 2016, the NMSU drought-resilient alfalfa variety, NuMex Bill Melton, has been grown in NM, generating an estimated $500,000 annually in hay sales. Nine new alfalfa populations have been developed and are being evaluated in statewide yield trials under well-watered and drought-stressed field conditions to identify those with superior performance in both environments. The program also helps conduct statewide forage yield evaluations of 89 industry and public varieties. Results of these trials are published annually to help NM, AZ, and TX farmers identify the most suitable varieties for their particular growing region. State-of-the-art molecular breeding strategies are being applied to >200 experimental populations to evaluate their potential to accelerate development of drought-resilient alfalfa varieties with high nutritive value and disease resistance. Since irrigated soils tend to have high salt levels, these molecular tools are being assessed for improvement of seed germination salt tolerance. DNA markers significantly associated with each of the above traits have been identified. Statistical models using a recently developed data analysis approach for integrated molecular and agronomic data suggest that DNA-marker based selection approaches may potentially be able to double the rate at which new alfalfa cultivars can be developed with improved nutritive value and resilience to climate change. Opportunities to utilize unique diploid relatives of alfalfa to improve productivity of commercial tetraploid varieties are being investigated. Undergraduate and graduate teaching and research activities helped train future generations of agronomists, plant breeders, and geneticists to benefit agricultural sustainability and human health. Objective 1: Evaluating and enhancing alfalfa germplasm productivity by traditional breeding strategies. The NMSU drought-resilient alfalfa variety, NuMex Bill Melton, continues to perform very well in statewide variety trials. It is being commercially grown in NM and generates an estimated $500,000 annually in hay sales. Industry and public alfalfa varieties were evaluated in forage yield trials at 5 NM locations. Our program conducted all statistical analyses for the NM state-wide alfalfa variety trials (89 populations and 5 locations) and provided summary reports to develop the NM Alfalfa Variety Test Report for 2020. Trial results were published to help NM, TX, and AZ hay growers identify varieties best suited for their farms. In summer 2021, seed was increased for 9 new elite alfalfa germplasms derived from thousands of seedlings and hundreds of mature plants that were previously subjected to selection for drought resilience, resistance to phytophthora root rot and anthracnose diseases, and differing fall dormancies. Several of these new lines are performing well at 3 NM locations under well-watered conditions at Farmington, Las Cruces and Los Lunas, NM, and under summer irrigation termination at Las Cruces, NM. Objective 2: Application of traditional and advanced DNA marker assisted selection (MAS) approaches within elite germplasm to improve alfalfa productivity in drought prone environments. Twenty-seven elite NMSU alfalfa germplasms possessing 75% genetic background from 3 cultivars were developed in 2016 using DNA MAS over two generations for markers that were previously demonstrated to impact biomass production during drought stress. Forage yields of these new materials were evaluated in 2018-2020 under a summer irrigation termination regime, where no water was applied during July through early September. Results suggest that MAS impacted productivity in the elite germplasms. This outcome likely reflected changes in allele frequencies that occurred at targeted genome regions involving both the introgressed new biomass alleles and alternate alleles of similar size that were already present within the three cultivars evaluated. In a second experiment, association analyses (AA) for various traits were conducted on 220 to 260 elite NMGS alfalfa families that were genotyped with 12,884 DNA markers. AA identified 11 significant markers affiliated with seed germination salt tolerance in NMGS over two years. AA for forage yield over 3 years in NM and CA detected four significant markers in NMGS under summer irrigation termination. AA also identified 27 NMGS markers associated with 10 forage quality traits at NM. Several of these significant markers reside in genome regions previously reported to impact alfalfa yield, quality, and salt-stress response traits. These outcomes illustrate the usefulness of AA to detect some QTL with reasonably large effects for improvement of quantitative traits in alfalfa. We also used the AA approach to determine the influence for each of 12,884 DNA markers on forage yield and nutritional value under deficit irrigation management over three years in CA, NM, and WA. Results from a recently-developed data analysis approach indicate that this trait-specific marker effect information can be integrated into genomic selection models to predict alfalfa yield and nutritional quality with 60 to 90% accuracy. Future research is required to confirm these outcomes. If validated, we estimate that DNA-marker based selection approaches can approximately double the rate at which new alfalfa cultivars can be developed with improved resilience to climate change. Such new varieties will help U.S. farmers conserve water and meet the livestock industry's feed demands. In future work, identification of "superior" plants for developing new breeding lines will be based on conventional phenotypic selection, AA, or genomic selection approaches. Performance of populations developed by these different breeding strategies will be comparatively evaluated to determine their effectiveness towards improving alfalfa, yield, quality and abiotic stress tolerance traits. Objective 3: Evaluate the potential of inbred diploid (2x; 2n=2x=16) alfalfa germplasm to enhance productivity of commercial tetraploid (4x; 2n=4x=32) populations. We hypothesize that natural 2x populations of alfalfa may provide an enriched source of favorable alleles to benefit productivity of commercial 4x cultivars. Furthermore, opportunities to enhance favorable allele frequencies may be greater in diploids where inbreeding can purge deleterious alleles much faster than in tetraploids. Once superior noninbred or inbred 2x plants are identified, regular occurrence of unreduced gamete production (i.e., 2n gametes) in diploids can be used to transfer their desirable alleles to commercial 4x populations. In this study, 194 noninbred 2x alfalfa populations were phenotypically evaluated in the greenhouse in 2018. They were also self-pollinated for one generation to develop 2x inbred lines. Seventy-nine vigorous noninbred lines were identified and 62 of these also produced sufficient self-pollinated seed. The 79 noninbred populations and 62 of their corresponding inbred lines were planted in the field in fall 2019. They were phenotypically evaluated multiple times during 2020 and 2021 under summer irrigation termination management. This evaluation will continue in 2022 with future phenotypic selection based on vigor and persistence. Selected diploid plants from phenotypically superior lines will be advanced for additional inbreeding. They will also be mated with elite tetraploid cultivars. Their 2x-4x interploidy hybrid progeny will then evaluated for yield and other traits under well-watered and deficit irrigation management regimes to determine if their performance exceeds that of conventional commercial tetraploid populations.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Medina, C., Kaur*, H., Ray, I., Yu, L.-X. L. (2021). Strategies to Increase Prediction Accuracy in Genomic Selection of Complex Traits in Alfalfa (Medicago sativa L.). Cells. https://doi.org/10.3390/cells10123372
- Type:
Other
Status:
Published
Year Published:
2020
Citation:
Lauriault, L., Ray, I., Pierce, C., Djaman, K., Flynn, R., Marsalis, M., Allen, S., Havlik, C., Martinez, G., and West, M. (2020). The 2020 New Mexico Alfalfa Variety Test Report. Las Cruces, NM: Agricultural Experiment Station and Cooperative Extension Service, New Mexico State University.
https://aces.nmsu.edu/pubs/variety_trials/AVT20.pdf
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Kaur, H., Medina-Culma, C., Pierce, C., Singh Gill, A., Reich, J., Yu, L-X., Ray, I. (2020). Association analyses for forage yield and quality under deficit irrigation management in elite alfalfa (Medicago sativa L.) germplasm. Crop Science Society of America Annual Meeting Abstracts. Nov. 9-12. Virtual meeting.
- Type:
Other
Status:
Published
Year Published:
2021
Citation:
Ray, I. (2021). SAES-422 Multistate Research Activity Report for New Mexico in 2020. Pullman, WA: USDA-ARS W-6 Management and Utilization of Plant Genetic Resources Unit.
|
Progress 10/01/19 to 09/30/20
Outputs
Target Audience:Formal Classroom instruction in Molecular Genetics (GENE 315, face-2-face) and Genetics Seminar (GENE 440, mix of face-2-face and online) to diverse student population (30 undergraduate and 1 graduate students) including Caucasian, Hispanic, Native American, Asian, female and male students during Fall 2019 and Spring 2020. Did NOT coordinate New Mexico Future Farmers of America Agronomy Career Development Event competition as this event was cancelled due to COVID19 restrictions. Provided research progress updates to NM Hay Association Board of Directors (9 individuals) on January 31 (face-2-face), and conference calls on May 22, and November 11, 2020. Assisted with organization, coordination, and moderating of Southwest Hay and Forage Conference, Ruidoso, NM Jan. 30-31, 2020, Ruidoso, NM. Approximately 130 Ag. industry and farmer clientele were present including Caucasian, Hispanic, Native American, females and males. Served as Chair of the Western Regional Technical Advisory Committee of the USDA-National Plant Germplasm System (NPGS). Collaborated with USDA-ARS personnel to organize/host a virtual annual regional workshop to discuss status of multiple NPGS sites in the western U.S. Audience: USDA-ARS NPGS staff and state Agricultural Experiment Station representatives for the western U.S. (June 16, 2020). Presented research results on a genome-wide association study for forage yield and forage quality traits in elite alfalfa germplasm to a professional audience at the Crop Science Society of America Annual Meetings (Nov. 9-11, 2020, Virtual meeting). Changes/Problems:Due to COVID19, efforts originally intended for publication development were reallocated to address significant online teaching needs and to adjust operating procedures to safely conduct research. Publication development was also negative impacted by retirement of key USDA-ARS personnel (research leader) at Pullman Washington. Hence, as Chair of the W6 Regional Technical Advisory Committee, this required a significant amount of extra time be directed towards collaborating with other USDA-ARS and Washington State Univ. personnel to develop/submit a proposal to renew funding in 2022-2026 for the W6 Multistate Research Project: "Maximizing Conservation and Utilization of Plant Genetic Resources and their Information". What opportunities for training and professional development has the project provided?One PhD graduate student received training in phenotype data collection for forage biomass, forage quality sampling, and disease resistance traits using 220 to 260 elite alfalfa populations. This student received training in conducting genome-wide association analysis (AA) and genomic selection (GS) and prediction modeling for these traits in these populations which were genetically fingerprinted with >11,000 DNA markers. Based on AA, significant DNA markers for all traits have been identified in this Ph.D. dissertation research. Data analyses for other traits are in progress. The PhD student also participated in a high-throughput computing workshops, received training in statistical analysis of data using splines to account for spatial variation in field research studies, and use of statistical software to determine best linear unbiased predictors and estimates (BLUP/BLUE) for multiple alfalfa populations being evaluated for diverse traits. Expertise was also gained in managing and analyzing large datasets (including DNA sequence and phenotype data) relevant to our research program. Provided recommendations on behalf of 5 undergraduate advisees to help them secure professional positions or pursue advanced studies in the following areas/institutions: Diagnostic Genetics MS Degree Program, Univ. of Texas MD Anderson Cancer Center;Research technician, Dept. of Pediatrics/ Oncology, Univ. of Texas Southwestern in Dallas; Research assistant, Human Genome Sequencing Center, Baylor College of Medicine; 5th grade STEM instructor at Las Cruces Public Schools; Medical Doctor training, School of Medicine, University of New Mexico. One NMSU undergraduate student received extensive "hands-on" training in agronomy, plant breeding and genetic research techniques. Student participation in this training area was limited due to COVID19 restrictions on research activities. How have the results been disseminated to communities of interest?Annual reports of our research progress have been provided to a key alfalfa industry collaborator who is very interested in utilizing DNA marker assisted selection (MAS) to improve alfalfa drought resilience. Research progress updates were also provided to NM Hay Association Board of Directors (9 individuals) in January 2020. A graduate student presented results of their PhD research at the 2020 Crop Science Society of America Annual Meeting (Virtual, Nov. 2020). Each year the program helps publish an Experiment Station Research Report that describes alfalfa variety performance results across the state. This report is made available online, as well as, in hard copy at the Southwest Hay and Forage Conference to provide hay growers in NM, AZ, and TX with information to help them identify the most suitable varieties for their particular growing region. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: We will continue collecting data from a fall dormancy trial to characterize newly developed elite NMSU alfalfa populations for natural plant height in late fall. We will continue collecting yield data for industry/public alfalfa populations evaluated under normal and deficit irrigation management at Las Cruces, NM as a participant in the NMSU state-wide alfalfa variety testing program. We will also analyze all alfalfa variety performance data for multiple locations across the state as a contribution to the state-wide alfalfa testing program. Seed increases of 9 new populations will be conducted under cage isolation to produce seed for evaluating these materials in statewide and regional yield trials. Objective 2: Extensive analysis and modeling of DNA marker, forage yield, and nutritional quality data collected in 2018-2020 will be conducted to identify superior populations based on phenotypic selection parameters, association analysis marker selection, and genomic prediction/selection. Vigorous plants from these superior populations (evaluated under summer irrigation termination management for 3-years) will be dug from field plots and mated in various combinations to develop new drought-resilient germplasm for future evaluation and potentially commercialization. Results of our extensive data collection will be submitted for publication in scientific journals and also as a PhD dissertation. Objective 3: Diploid populations will continue to be evaluated under summer irrigation termination management before imposing phenotypic selection for vigorous plants after 2-3 years of evaluation.
Impacts
What was accomplished under these goals?
New Mexico State University (NMSU) plant breeding research uses conventional and molecular approaches to improve alfalfa drought tolerance so that farmers can conserve irrigation water resources while meeting the feed demands of the U.S. livestock industry. Since 2016, the NMSU drought-resilient alfalfa variety, NuMex Bill Melton, has been grown in NM, generating an estimated $500,000 annually in hay sales. Nine new alfalfa populations have been developed and are being evaluated in statewide field trials under well-watered and drought-stressed field conditions to identify those with superior performance in both environments. The program also helps conduct statewide forage yield evaluations of approximately 50 to 90 industry and public varieties. Results of these trials are published annually to help NM, AZ, and TX farmers identify the most suitable varieties for their particular growing region. State-of-the-art molecular breeding strategies are being applied to >200 experimental populations to evaluate their potential to accelerate development of drought-resilient alfalfa varieties with high nutritive value and disease resistance. Since irrigated soils tend to have high salt levels, these molecular tools are being assessed for improvement of seed germination salt tolerance. DNA markers significantly associated with each of the above traits have been identified. Opportunities to utilize unique diploid relatives of alfalfa to improve productivity of commercial tetraploid varieties are being investigated. Undergraduate and graduate teaching and research activities helped train future generations of agronomists, plant breeders, and geneticists to benefit agricultural sustainability and human health. Objective 1: Evaluating and enhancing alfalfa germplasm productivity by traditional breeding strategies. The NMSU drought-resilient alfalfa variety, NuMex Bill Melton, continues to perform very well in statewide variety trials. It is being commercially grown in NM and generates an estimated $500,000 annually in hay sales. Sixty industry and public alfalfa varieties were evaluated in forage yield trials at 5 NM locations. Our program conducted all statistical analyses for the NM state-wide alfalfa variety trials (60 populations at 5 locations) and provided summary reports to develop the NM Alfalfa Variety Test Report for 2020. Trial results are published annually to help NM, TX, and AZ hay growers identify varieties best suited for their farms. In summer 2020, seed was increased for 9 new elite alfalfa germplasms derived from thousands of seedlings and hundreds of mature plants that were previously subjected to selection for drought resilience, resistance to phytophthora root rot and anthracnose diseases, and differing fall dormancies. Several of these new lines are performing well at 2 NM locations under well-watered conditions at Las Cruces and Los Lunas, NM, and under summer irrigation termination at Las Cruces, NM. Objective 2: Application of traditional and advanced DNA marker assisted selection (MAS) approaches within elite germplasm to improve alfalfa productivity in drought prone environments. Twenty-seven elite NMSU alfalfa germplasms possessing 75% genetic background from 3 cultivars were developed in 2016 using DNA MAS over two generations for markers that were previously demonstrated to impact biomass production during drought stress. Forage yields of these new materials were evaluated in 2018-2020 under a summer irrigation termination regime, where no water was applied during July through early September. Results suggest that MAS impacted productivity in the elite germplasms. This outcome likely reflected changes in allele frequencies that occurred at targeted genome regions involving both the introgressed new biomass alleles and alternate alleles of similar size that were already present within the three cultivars evaluated. In a second experiment, association analyses (AA) for various traits were conducted on 220 to 260 elite NMGS alfalfa families that were genotyped with 11,000 DNA markers. AA identified 11 significant markers affiliated with seed germination salt tolerance in NMGS over two years. AA for forage yield over 3 years in NM and CA detected four significant markers in NMGS under summer irrigation termination. AA also identified 27 NMGS markers associated with 10 forage quality traits at NM. Several of these significant markers reside in genome regions previously reported to impact alfalfa yield, quality, and salt-stress response traits. These outcomes illustrate the usefulness of AA to detect some markers/genome regions with reasonably large effects for improvement of quantitative traits in alfalfa. The NMGS DNA marker and phenotypic trait data are being further evaluated for their ability to accurately predict each trait using different genomic selection models. In future work, identification of "superior" plants for developing new breeding lines will be based on conventional phenotypic selection, AA, or genomic selection approaches. Performance of populations developed by these different breeding strategies will be comparatively evaluated to determine their effectiveness towards improving alfalfa, yield, quality and abiotic stress tolerance traits. Objective 3: Evaluate the potential of inbred diploid (2x; 2n=2x=16) alfalfa germplasm to enhance productivity of commercial tetraploid (4x; 2n=4x=32) populations. We hypothesize that natural 2x populations of alfalfa may provide an enriched source of favorable alleles to benefit productivity of commercial 4x cultivars. Furthermore, opportunities to enhance favorable allele frequencies may be greater in diploids where inbreeding can purge deleterious alleles much faster than in tetraploids. Once superior noninbred or inbred 2x plants are identified, regular occurrence of unreduced gamete production (i.e., 2n gametes) in diploids can be used to transfer their desirable alleles to commercial 4x populations. In this study, 194 noninbred 2x alfalfa populations were phenotypically evaluated in the greenhouse in 2018. They were also self-pollinated for one generation to develop 2x inbred lines. Seventy-nine vigorous noninbred lines were identified and 62 of these also produced sufficient self-pollinated seed. The 79 noninbred populations and 62 of their corresponding inbred lines were planted in the field in fall 2019. They were phenotypically evaluated multiple times during 2020 under summer irrigation termination management. This evaluation will continue in 2021 with future phenotypic selection based on vigor and persistence. Selected diploid plants from phenotypically superior lines will be advanced for additional inbreeding. They will also be mated with elite tetraploid cultivars. Their 2x-4x interploidy hybrid progeny will then evaluated for yield and other traits under well-watered and deficit irrigation management regimes to determine if their performance exceeds that of conventional commercial tetraploid populations.
Publications
- Type:
Other
Status:
Published
Year Published:
2020
Citation:
Lauriault, L., Ray, I., Pierce, C., Djaman, K., Flynn, R., Marsalis, M., Allen, S., Havlik, C., Martinez, G., and West, M. (2020). The 2019 New Mexico Alfalfa Variety Test Report. Las Cruces, NM: Agricultural Experiment Station and Cooperative Extension Service, New Mexico State University. https://aces.nmsu.edu/pubs/variety_trials/AVT19.pdf
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Kaur, H., Medina-Culma, C., Pierce, C., Singh Gill, A., Reich, J., Yu, L-X., Ray, I. (2020). Association analyses for forage yield and quality under deficit irrigation management in elite alfalfa (Medicago sativa L.) germplasm. Crop Science Society of America Annual Meeting Abstracts. Nov. 9-12. Virtual meeting.
|
Progress 10/01/18 to 09/30/19
Outputs
Target Audience:Formal Classroom instruction in Molecular Genetics (GENE 315), Genetics Seminar (GENE 440), and Genes and Genomes (GENE 486) to diverse student population (31 undergraduate and 2 graduate students) including Caucasian, Hispanic, Native American, Asian, female and male students during Fall 2018 and Spring 2019. Hosted a field/greenhouse tour of research projects and alfalfa breeding methods to 3 NMSU undergraduate students (AGRO 462, Plant Breeding Lab) on Nov. 8, 2018. Coordinated Future Farmers of America Agronomy Career Development Event competition with 43 participants on April 5, 2019 including Caucasian, Hispanic, female and male students. Provided research progress updates to NM Hay Association Board of Directors (9 individuals) on January 25, May 17, August, 29 and September 29, 2019. Assisted with organization, coordination, and moderating of Southwest Hay and Forage Conference, Ruidoso, NM Jan. 24-25, 2019, Ruidoso, NM. Approximately 100 Ag. industry and farmer clientele were present including Caucasian, Hispanic, Native American, females and males. Presented research results on a genome-wide association study for salt tolerance at seed germination in elite alfalfa germplasm to approximately multiple alfalfa scientists at the Western Crop Science Society Annual Meetings (June 25-26, 2019, Pasco, WA). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One MS graduate student and one PhD graduate student received training in phenotype data collection for forage biomass, forage quality sampling, seed germination salt tolerance, and disease resistance traits using 220 to 260 elite alfalfa populations. Both graduate students received training in conducting association analysis for these traits in these populations which were genetically fingerprinted with 7,281 DNA markers. Significant DNA markers for seed germination salt tolerance and phytophthora root rot resistance have been identified in their M.S. thesis and Ph.D. dissertation research. Data analyses for other traits are in progress. The MS student completed his thesis in summer 2019 and is now pursuing a PhD at another institution. The PhD student participated in a high-throughput computing workshop offered in summer 2019 at the Univ. of Wisconsin-Madison. Expertise was gained in managing and analyzing large datasets (including DNA marker and phenotype data) relevant to our research program. Three NMSU undergraduate students received extensive "hands-on" training in agronomy, plant breeding and genetic research techniques. How have the results been disseminated to communities of interest?Annual reports of our research progress have been provided to a key alfalfa industry collaborator who is very interested in utilizing DNA marker assisted selection (MAS) to improve alfalfa drought resilience. This venture may provide opportunities to commerically market superior products developed by our research program to benefit forage production in the western US. Research progress updates were also provided to NM Hay Association Board of Directors (9 individuals) in January, May, August and September 2019. An MS student presented results of their thesis research at the 2019 Western Society of Crop Science Annual Meetings in Pasco, WA. They also presented a poster of their research project at a well-attended community open-house hosted by the NMSU College of Agriculture, Consumer, and Environmental Sciences in April 2019. Each year our program helps publish an Experiment Station Research Report that describes alfalfa variety performance results across the state. This report is made available online, as well as, in hard copy at the Southwest Hay and Forage Conference to provide hay growers in NM, AZ, and TX with information to help them identify the most suitable varieties for their particular growing region. One peer-reviewed article was published in the journal, Crop Science. This article described genome regions influencing water-use efficiency and carbon and nitrogen metabolism in drought-stressed alfalfa. Potential candidate genes influencing these traits were identified. Those results suggest that genes involved in trehalose biosynthesis may play a key role in alfalfa carbon and nitrogen metabolism during drought. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
NMSU plant breeding research uses conventional and molecular approaches to improve alfalfa drought tolerance so that farmers can conserve irrigation water resources while meeting the feed demands of the U.S. livestock industry. Since 2016, the NMSU drought-resilient alfalfa variety, NuMex Bill Melton, has been grown in NM, generating an estimated $500,000 annually in hay sales. Nine new alfalfa populations have been developed and are being evaluated in statewide field trials under well-watered and drought-stressed field conditions to identify those with superior performance in both environments. The program also helps conduct statewide forage yield evaluations of approximately 75 industry and public varieties. Results of these trials are published annually to help NM, AZ, and TX farmers identify the most suitable varieties for their particular growing region. State-of-the-art molecular breeding strategies are being applied to >200 experimental populations to evaluate their potential to accelerate development of drought-resilient alfalfa varieties with high nutritive value and disease resistance. Since irrigated soils tend to have high salt levels, these molecular tools are being assessed for improvement of seed germination salt tolerance. Opportunities to utilize unique relatives of alfalfa to improve productivity of commercial varieties are being investigated. Undergraduate and graduate teaching and research activities helped train future generations of agronomists and plant breeders to benefit agricultural sustainability. Objective 1: Evaluating and enhancing alfalfa germplasm productivity by traditional breeding strategies. The NMSU drought-resilient alfalfa variety, NuMex Bill Melton, continues to perform very well in statewide variety trials. It is being commercially grown in NM and generates an estimated $500,000 annually in hay sales. Ninety-five industry and public alfalfa varieties were evaluated in forage yield trials at 5 NM locations. Trial results were published to help NM, TX, and AZ hay growers identify varieties best suited for their farms. In summer 2019, seed was increased for 9 new elite alfalfa germplasms derived from thousands of seedlings and hundreds of mature plants that were previously subjected to selection for drought resilience, resistance to phytophthora root rot and anthracnose diseases, and differing fall dormancies. In fall 2019, these new lines were planted in statewide variety trials for yield evaluation during 2020-2022 under well-watered conditions at Las Cruces and Los Lunas, NM, as well as, a summer irrigation termination at Las Cruces, NM. Objective 2: Application of traditional and advanced DNA marker assisted selection (MAS) approaches within elite germplasm to improve alfalfa productivity in drought prone environments. Twenty-seven elite NMSU alfalfa germplasms were previously developed in 2016 using DNA MAS over two generations for markers that were previously demonstrated to impact biomass production during drought stress. Forage yields of these new materials were evaluated in 2018/2019 under a summer irrigation termination regime, where no water was applied during July through early September. Results suggest that MAS was largely ineffective beyond the first generation of introgression. This outcome likely occurred, because the elite genetically broad-based cultivars into which the markers were being transferred possessed many alleles of similar size to the original/donor marker alleles which hindered MAS progress. In a second experiment, association analyses (AA) for various traits were conducted on 220 to 260 elite NMGS alfalfa families that were genotyped with 7,281 DNA markers. AA for the phytophthora root rot trait identified a single significant marker (R2=0.17) located at 48.3 Mb on M. truncatula chromosome 4. This region has been reported to influence forage yield in different alfalfa populations and may reflect that resistance to key pathogens benefits forage productivity over time. AA also identified 8 significant markers (R2=0.11 to 0.33) affiliated with seed germination salt tolerance in NMGS over two years. Five markers were validated by other researchers who reported similar genome regions associated with this trait in diverse alfalfa populations. AA for forage yield over four harvests in NM detected no significant markers in NMGS under summer irrigation termination. These outcomes may reflect that previous generations of NMGS experienced multiple cycles of selection for biomass productivity. For important genes influencing this trait, such selection could reduce the magnitude of their phenotypic effects preventing a declaration of statistical significance. Collectively, these outcomes illustrate the usefulness of AA to detect some QTL with reasonably large effects for improvement of quantitative traits in alfalfa (e.g. seedling salt tolerance). However, they also demonstrate the limitation of AA, which ignores genes with smaller effects that underpin other quantitative traits (e.g. yield) in elite germplasm. A more detailed examination of the top 30 markers (i.e., those with the lowest p-values) based on AA for forage yield in each harvest was conducted. The approximate positions of those biomass markers in the M. truncatula genome indicated that they frequently colocalized in specific chromosome intervals potentially identifying genome regions impacting biomass productivity over time. Many of these regions have been reported to impact forage yield in different Medicago populations. Although these markers were not declared to be significant based on AA, they may provide valuable resources for genomic selection breeding strategies to improve forage yield in alfalfa. Objective 3: Evaluate the potential of inbred diploid alfalfa germplasm enhance productivity of commercial tetraploid populations. Natural diploid (2n=2x=16) populations of alfalfa may provide an enriched source of favorable alleles to benefit productivity of commercial tetraploid (2n=4x=32) alfalfa culitvars. Evidence indicates that temperature stress during flower development can disrupt meiotic processes in plants, and increase 2n gamete frequencies to facilitate allele transfer from diploids to tetraploids. During hot summer months of 2018 and cool winter months of 2019, hundreds of flowers from yellow-flowered diploid alfalfa were mated with elite purple-flowered tetraploids in the greenhouse. Hundreds of progeny from these crosses were phenotyped in 2019 and evaluation of flower color indicated that most were derived by self-pollination. But, fifteen 2x-4x and 4x-2x hybrids possessing variegated flowers were recovered, primarily from crosses made during hot summer months. A second experiment is also underway to test the hypothesis that, opportunities to enhance favorable allele frequencies may be much greater in diploids where inbreeding can rapidly purge deleterious alleles. In this study, 172 diploid alfalfa populations were self-pollinated for one generation in 2018 with a goal to develop highly inbred diploid lines from these materials. One hundred vigorous lines were retained and self-pollinated and cross-pollinated seed from each line was planted in the field in fall 2019 for future phenotypic selection for vigor and persistence. Selected diploid plants from this material will be advanced for additional inbreeding. They will also be crossed to elite tetraploid cultivars under high temperature stress and their interploidy progeny evaluated for yield and other traits under well-watered and deficit irrigation management regimes to determine if their performance exceeds that of conventional commercial tetraploid populations.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Santantonio, N., C.A. Pierce, R.L. Steiner, and I.M. Ray. (2019). Genetic mapping of water-use efficiency and carbon and nitrogen metabolism in drought-stressed alfalfa. Crop Science 59:92-106.
- Type:
Other
Status:
Published
Year Published:
2019
Citation:
Lauriault, L. M., Ray, I., Pierce, C., Burney, O., Flynn, R. P., Marsalis, M. A., O'Neill, M. K., Cunningham, A., Havlik, C., West, M. (2019). The 2018 New Mexico Alfalfa Variety Test Report. Las Cruces, NM: Agricultural Experiment Station and Cooperative Extension Service, New Mexico State University.
https://aces.nmsu.edu/pubs/variety_trials/AVT18.pdf
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2019
Citation:
Gill, A.S. 2019. Genome-wide association study for salt tolerance at seed germination in elite alfalfa (Medicago sativa L.) germplasm. M.S. thesis New Mexico State University.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Gill, A. S., Yu, L.-X., Hawkins, C., Reich J., Ray, I. (2019). Genome-Wide Association Study for Salt Tolerance at Seed Germination in Elite Alfalfa (Medicago sativa L.) Germplasm. Madison, WI: Western Society of Crop Science Annual Meeting Abstracts. June 24-25, Pasco, WA.
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Progress 10/01/17 to 09/30/18
Outputs
Target Audience:Formal Classroom instruction in Molecular Genetics (GENE 315), Genetics Seminar (GENE 440), and Advanced Plant Breeding (AGRO 610) to diverse student population (27 undergraduate and 6 graduate students) including Caucasian, Hispanic, Native American, Asian, female and male students during Fall 2017 and Spring 2018. Hosted a field/greenhouse tour of research projects and alfalfa breeding methods to 6 NMSU undergraduate students (AGRO 462, Plant Breeding) on Nov. 14, 2017. Coordinated Future Farmers of America Agronomy Career Development Event competition with 38 participants on April 6, 2018 including Caucasian, Hispanic, female and male students. Provided research progress updates to NM Hay Association Board of Directors (9 individuals) on Jan 18 and Sept 29, 2018. Assisted with organization, coordination, and moderating of Southwest Hay and Forage Conference, Ruidoso, NM Jan. 18-19, 2018, Ruidoso, NM. Approximately 100 Ag. industry and farmer clientele were present including Caucasian, Hispanic, Native American, females and males. Presented research results on DNA marker assisted selection research to improve alfalfa drought resilience to several hundred Ag. Industry personnel and farmers at the Western Alfalfa and Forage Symposium (Nov. 29, 2017, Reno, NV). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One NMSU M.S. graduate student and one Ph.D. graduate student received training in phenotype data collection for forage biomass, forage quality, seed germination salt tolerance, and disease resistance traits using 220 to 260 elite alfalfa populations. Both graduate students also received training in conducting association analysis for these traits in these populations, which were genetically fingerprinted with 7,281 DNA markers. To-date five significant DNA markers for seed germination salt tolerance and one significant DNA marker for phytophthora root rot resistance have been identified. This work will provide an important component of each student's M.S. thesis and Ph.D. dissertation research. Departmental graduate seminars describing research progress were presented by both students in fall 2018. Four NMSU undergraduate students received extensive "hands-on" training in agronomy, plant breeding and genetic research techniques. One undergraduate student completed their degree in 2018 and is currently employed by the Bureau of Land Management. How have the results been disseminated to communities of interest?Annual reports of our research progress have been provided to a key alfalfa industry collaborator who is very interested in utilizing DNA marker assisted selection (MAS) to improve alfalfa drought resilience. Research progress updates were also provided to NM Hay Association Board of Directors (9 individuals) in January and September 2018. In November 2017, a presentation describing the outcome of our DNA marker assisted selection (MAS) research was presented to several hundred Ag. Industry personnel and farmers at the Western Alfalfa and Forage Symposium (Reno, NV). Each year the program publishes an Experiment Station Research Report that describes alfalfa variety performance results across the state. This report is made available online, as well as, in hard copy at the Southwest Hay and Forage Conference to provide hay growers in NM, AZ, and TX with information to help them identify the most suitable varieties for their particular growing region. Two peer-reviewed articles were published in the journal, Crop Science. The first article described outcomes based on the integrated results of five alfalfa forage yield QTL mapping projects. This work identified specific regions of the alfalfa genome that influence forage biomass in diverse environments and populations. Potential candidate genes impacting yield were also identified. Such results will provide useful information for designing future MAS based breeding work to improve alfalfa productivity. The second article described genome regions influencing water-use efficiency and carbon and nitrogen metabolism in drought-stressed alfalfa. Potential candidate genes influeincing these traits were identified. Those results suggest that genes involved in trehalose biosynthesis may play a key role in alfalfa carbon and nitrogen metabolism during drought. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
NMSU plant breeding research uses conventional and molecular approaches to improve alfalfa drought tolerance so that farmers can conserve irrigation water resources while meeting the feed demands of the U.S. livestock industry. Since 2016, the NMSU drought-resilient alfalfa variety, NuMex Bill Melton, has been grown in NM, generating an estimated $500,000 annually in hay sales. Forty-four new alfalfa populations have been developed and are being evaluated under drought-stressed field conditions to identify those with superior performance. The program also helps conduct statewide forage yield evaluations of approximately 100 industry and public varieties. Results of these trials are published annually to help NM, AZ, and TX farmers identify the most suitable varieties for their particular growing region. State-of-the-art molecular breeding strategies are being applied to >200 experimental populations to evaluate their potential to accelerate development of drought-resilient alfalfa varieties with high nutritive value and disease resistance. Since irrigated soils tend to have high salt levels, these molecular tools are being assessed for improvement of seed germination salt tolerance. Opportunities to utilize unique relatives of alfalfa to improve productivity of commercial varieties are being investigated. Undergraduate and graduate teaching and research activities helped train future generations of agronomists and plant breeders to benefit agricultural sustainability. Objective 1: Evaluating and enhancing alfalfa germplasm productivity by traditional breeding strategies. The NMSU drought-resilient alfalfa variety, NuMex Bill Melton, continues to perform very well in statewide variety trials. This variety is being commercially grown in NM and generates an estimated $500,000 annually in hay sales. Ninety-four industry and public alfalfa varieties were evaluated in forage yield trials at NM locations. Trial results were published to help NM, TX, and AZ hay growers identify varieties best suited for their farms. Seven hundred fifty vigorous plants were dug from field plots of multiple alfalfa populations previously subjected to 3 years of deficit irrigation management. These materials were intermated in various combinations to develop 17 germplasms possessing different fall dormancies. Among these, 9 germplasms were subjected to additional screening for resistance to phythophthora root rot and anthracnose pathogens. Surviving plants were intermated within each population and this seed will be planted in field isolation cages in spring 2019 to generate breeder seed for 9 elite NMSU populations in summer 2019. This seed will be planted in statewide variety trials in fall 2019 for yield evaluation during 2020-2023. Objective 2: Application of traditional and advanced DNA marker assisted selection (MAS) approaches within elite germplasm to improve alfalfa productivity in drought prone environments. Twenty-seven elite NMSU alfalfa germplasms were previously developed in 2016 using DNA MAS for markers that have previously been demonstrated to impact biomass production during drought stress. During 2018, forage yields of these materials were evaluated in replicated field experiments under a summer irrigation termination regime, where no water was applied during July through early September. Results are pending data analyses. In a second experiment, association analyses (AA) for various traits were conducted on 220 to 260 elite NMGS alfalfa half-sib families that were genotyped with 7,281 DNA markers. AA for the phytophthora root rot trait identified a single significant marker (R2=0.17) located at 48.3 Mb on M. truncatula chromosome 4. This region has been reported to influence forage yield in different alfalfa populations and may reflect that resistance to key pathogens provides important contributions to forage productivity over time. AA also identified 5 significant markers (R2=0.15 to 0.26) affiliated with seed germination salt tolerance in NMGS. Two of these markers were validated by other researchers who identified similar genome regions associated with this trait in a panel of 198 diverse alfalfa populations. AA for forage yield over four harvests in NM detected no significant markers in NMGS. These outcomes may reflect that previous generations of NMGS experienced multiple cycles of selection for biomass productivity. For important genes influencing this trait, such selection could reduce the magnitude of their phenotypic effects preventing a declaration of statistical significance. Collectively, these outcomes illustrate the usefulness of AA to detect some QTL with reasonably large effects for improvement of quantitative traits in alfalfa. However, they also demonstrate the limitation of AA, which ignores genes with smaller effects that underpin quantitative traits in elite germplasm. A more detailed examination of the top 30 markers (i.e., those with the lowest p-values) based on AA for forage yield in each harvest was conducted. The approximate positions of those biomass markers in the M. truncatula genome indicated that they frequently colocalized in specific chromosome intervals potentially identifying genome regions impacting biomass productivity over time. Many of these regions have been reported to impact forage yield in different Medicago populations. Although these markes were not declared to be significant based on AA, they may provide valuable resources for genomic selection breeding strategies to improve forage yield in alfalfa. Objective 3: Evaluate the potential of inbred diploid alfalfa germplasm enhance productivity of commercial tetraploid populations. Natural diploid (2n=2x=16) populations of alfalfa and M. truncatula may provide an enriched source of favorable alleles to benefit productivity of commercial tetraploid (2n=4x=32) alfalfa populations. In addition, evidence indicates that temperature stress during flower development can disrupt meiotic processes in plants, and increase 2n gamete frequencies to facilitate allele transfer from diploids to tetraploids. To evaluate the impact of temperature stress to facilitate 2x-4x hybridization we conducted an experiment where, greenhouse climate controls were adjusted ln late fall/winter 2018 so that large temperature fluctuations occurred during flowering. During this time, hundreds of flowers from yellow-flowered diploid alfalfa and M. truncatula plants were mated with elite purple-flowered tetraploids. Seed from these crosses is currently ripening. To determine if interploidy hybridizations occurred, this seed will be planted in spring 2019 for evaluation of flower color and root tip karyotyping. Hybrids are expected to possess variegated flower color and be tetraploid, or perhaps, triploid. DNA marker genotyping of 2x and 4x parents will also be used to confirm interploidy hybridity. A second experiment is also underway to test the hypothesis that, opportunities to enhance favorable allele frequencies may be much greater in diploids where inbreeding can rapidly purge deleterious alleles. In this study, 20 plants from each of 172 diploid alfalfa populations were self-pollinated for one generation in 2018. Our long-term goal is to develop highly inbred diploid lines from these materials. If our first experiment indicates that temperature stress facilitates allele transfer between ploidy levels, then flowering inbred diploid lines will be subjected to temperature stress and then mated with elite tetraploids. If such 2x-4x crosses are successful, interploidy hybrids will be evaluated for forage yield and other traits under well-watered and deficit irrigation management regimes to determine their potential to improve the productivity of commercial tetraploid populations.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Ray, I.M., M.J. Monteros, B. Julier,, M.K. Sledge, E.C. Brummer. (2018). Identification of Consensus Regions Associated with Shoot Biomass Production in the Medicago Genome. Crop Sci. 58:1037-1060.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2019
Citation:
Santantonio, N., C.A. Pierce, R.L. Steiner, and I.M. Ray. (In Press). Genetic mapping of water-use efficiency and carbon and nitrogen metabolism in drought-stressed alfalfa. Crop Science.
- Type:
Other
Status:
Published
Year Published:
2018
Citation:
Lauriault, L. M., Ray, I., Pierce, C., Burney, O., Flynn, R. P., Marsalis, M. A., O'Neill, M. K., Cunningham, A., Havlik, C., West, M. (2018). The 2017 New Mexico Alfalfa Variety Test Report. Las Cruces, NM: Agricultural Experiment Station and Cooperative Extension Service, New Mexico State University. http://aces.nmsu.edu/pubs/variety_trials/AVT17.pdf
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