DEVELOPMENT OF WAXY SORGHUM BREEDING LINES FOR DIVERSE FOOD, FEED, AND FERMENTATION APPLICATIONS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1018534
• Grant No.: 2019-67014-29174
• Cumulative Award Amt.: $500,000.00
• Proposal No.: 2018-06349
• Project Start Date: Mar 1, 2019
• Project End Date: Feb 28, 2023
• Grant Year: 2019
• Program Code: [A1141]- Plant Health and Production and Plant Products: Plant Breeding for Agricultural Production

Recipient Organization
UNIVERSITY OF NEVADA
(N/A)
RENO,NV 89557

Performing Department
AG, NUTR & VET SCI

Non Technical Summary
The purpose of the proposed FASE research is to substantially improve waxy grain sorghum [Sorghum bicolor (L.) Moench] germplasm based upon extensive pilot studies and feedback from commercial end users of waxy inbred lines and hybrid parents publicly released by PI Yerka. The waxy trait is a high-value endosperm trait associated with high digestibility by animals, rapid fermentation into ethanol, the production of alcoholic beverages, and improved baking properties due to high-amylopectin (low amylose) starch content. While the waxy trait had been identified previously, these hybrids are the only publicly-available ones with no yield drag or poor agronomic performance compared to near-isogenic or closely-related wild-type hybrids. Nevertheless, our industry collaborators have identified additional target traits (larger seed size, improved protein content and digestibility, improved germination, sugarcane aphid resistance, and earlier maturity) that would improve their commercial value, expand production into additional regions, and help open underexplored, high-value domestic markets.For example, high protein digestibility (HPD) combined with waxy endosperms results in sorghum flour (which is gluten-free) that performs similarly to wheat flour in some applications. Higher endosperm free amino acid (FAN) profiles associated with some waxy genotypes improve fermentability in ethanol production and craft distilling. Stacked waxy/ HPD lines and high-FAN waxy lines exist, but the waxy genotypes used retain some yield drag that has been overcome in our materials. In addition, published studies using waxy, HPD, stacked waxy/HPD, or high-FAN traits use diverse (not related by descent) genotypes or breeding lines existing within a single program (not publicly available) so that it is difficult to assess the effect of background genetics on endosperm yield and quality. No waxy germplasm has been publicly released having all of the traits targeted by our collaborators. Therefore, significant efforts are needed to improve waxy sorghum germplasm. We will use a set biparental populations derived from common, publicly-available hybrid parent lines (a training population) to develop high-dimensional markers and genomic prediction (GP) models. Simultaneously, we will finish development of a new UNR mapping population that is genetically similar to the training population and incorporates all target traits to validate GP models over multiple generations, integrating marker information for each trait in the process. This will enable us to develop new publicly-available breeding lines having improved endosperm starch yield, fermentability, digestibility, and protein content. This approach has not previously been followed for improving specialty sorghum endosperms, but it will address existing and emerging market opportunities.The most effective way to minimize time-to-market in a breeding program focused on end-of-season (post-flowering/post-selection) traits like grain yield and endosperm quality is to utilize GP strategies. Traditional GP models predict the genotypic breeding value of plants based on marker information obtained when they are young, before flowering, providing a more reliable and inexpensive way to identify potential parents each generation than phenotypic or pedigree-based selection methods alone. Several recent papers have demonstrated the utility of GP methods for sorghum breeding programs in Japan, Brazil, and Australia. These studies emphasized the utility of GP for achieving genetic gains, but none of them phenotyped for endosperm traits. The proposed efforts will use this new tool to substantially improve waxy grain sorghum germplasm while incorporating the target traits listed above.Effective GP model development requires genomic marker-trait associations. Two recent genomics papers identified SNPs associated with grain quality (including waxy, fat, and protein traits) using the grain sorghum association panel (SAP) or QTLs in waxy/HPD mapping populations. While these papers published sequence data, a consistent problem with using it is that it tends to lack sufficient read depth to guide marker development (and therefore GP model development) by other researchers, even when working with similar pedigrees. Our efforts will address this by conducting whole-genome resequencing of parent lines (selected recurrent parents and endosperm trait donor parents) and the two best BC1F5 lines, and releasing the models and data; thus paving the way for others to locally adapt our germplasm using GP.Our team has expertise in plant breeding, bioinformatics, fermentation science, and genomic prediction. We have letters of support from Richardson Seeds (the largest sorghum classical breeding company in the U.S.), NuLife Market (currently commercially producing PI Yerka's waxy sorghum hybrids), the International Crops Research Institute for the Semiarid Tropics (ICRISAT, a major international agricultural R&D organization focused on nutritionally-enhanced dual-purpose sorghum and biofuels, headquartered in India), Benson Hill Biosystems (developer of Breed® software specifically designed to assist collaborators in using GP), the USDA-ARS grain quality lab in Manhattan, KS (Dr. Scott Bean), a large local brewery/distillery (The Depot Craft Brewery and Distillery; Mr. Brandon Wright), and a local farmer (Baker Ranch and Brewery Beef; Mr. Karl Baker) who uses distiller's grains and sorghum forage in local beef production. Each has agreed to partner with us on the proposed work for little or no cost, and to provide feedback during the line development process. Thus, we are in an ideal position to lead internationally-impactful improvement of waxy sorghum for diverse food, feed, and fermentation applications while building local markets in the northern Nevada community. Funding is requested for GP modeling and breeding line development. Future funding will be sought to finish and test breeding lines and hybrids resulting from these efforts.Our long-term goal is to utilize genomics-assisted plant breeding methodologies to develop high-value sorghum germplasm that will grow well in northern Nevada's high-elevation environments and contribute to innovative collaboration and agricultural sustainability around the world. The specific objectives of this research are to:Genotype and phenotype a training population, developed from publicly-available materials, provided by Richardson Seeds through an MTRA (Year 1).Develop markers and GP models for breeding line development (Years 1-3).Use breeding lines derived from the new UNR mapping population (whose parents are closely related to the training population) to validate the GP models (Years 3-4).Conduct collaborative research with industry, government, and international partners to identify and publicly release the most promising breeding lines from the Yerka Lab (Year 4).This work will result in the public release of sorghum breeding lines from UNR having different combinations of high-value traits that may be further selected upon, domestically and internationally, to generate locally-adapted, high-yielding hybrids. The sequence information at improved read depths, markers, and genomic prediction models optimized on the new mapping population having the high-value traits desired by our collaborators will be made publicly available to promote wider adoption of GP in global sorghum breeding programs. The breeding lines will be immediately used by our academic, government, and industry collaborators across the U.S. and in India, wherever highly-digestible sorghum grain and biomass are needed. It will have large implications for markets in Africa, South America, China and Australia through ICRISAT.

Animal Health Component

15%

Research Effort Categories

Basic

5%

Applied

15%

Developmental

80%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
202	1520	1081	100%

Knowledge Area
202 - Plant Genetic Resources;

Subject Of Investigation
1520 - Grain sorghum;

Field Of Science
1081 - Breeding;

Keywords

genomic prediction

genomics

marker assisted breeding

sorghum

whole genome resequencing

Goals / Objectives
Our long-term goal is to utilize genomics-assisted plant breeding methodologies to develop high-value sorghum germplasm that will grow well in northern Nevada's high-elevation environments and contribute to innovative collaboration and agricultural sustainability around the world. The specific objectives of this research are to:Genotype and phenotype a training population, developed from publicly-available materials, provided by Richardson Seeds through an MTRA (Year 1).Develop markers and GP models for breeding line development (Years 1-3).Use breeding lines derived from the new UNR mapping population (whose parents are closely related to the training population) to validate the GP models (Years 3-4).Conduct collaborative research with industry, government, and international partners to identify and publicly release the most promising breeding lines from the Yerka Lab (Year 4).

Project Methods
Objective 1: Genotype and phenotype a training population derived from publicly-available materials (Year 1).Training population development. Richardson Seeds will provide, plant, maintain, collect general field observations for, and harvest grain from four biparental cross populations from which sufficient seeds for both parents and their recombinant inbred lines (RILs) are available to the BC1F5 generation. The parents are in A/B (seed parent) and R (pollen parent) heterotic groups. Traits include white grain, tan plant color, bmr12 alleles, early or medium maturity, drought tolerance and high grain yield, and grain quality.Phenotyping. Parents and each generation of progeny will be grown in Winter 2018. The experimental design will be an augmented design with two check genotypes randomly interspersed to total 20% of the rows in each field (environment). Standard plant population densities and agronomic practices will be used for growth and production. General field observations and grain quality analyses (phenotypic data) will be collected. FAN and amino acid content (needed to reduce fermentation times), grain composition, physical kernel properties, and protein digestibility will be determined.Genotyping. Four replicates will be planted and phenotyped, but only one replicate will be genotyped.The samples (100 plants evenly representing no more than 10 families × 4 biparental populations × 4 generations between BC1F1 and BC1F5; 1600 plants total) will be subjected to DNA extraction. Whole genome resequencing (WGR) of the parent lines will be completed in Spring 2019. Sequencing will be done using 50x coverage to reconstruct reference genomes using NovaSeq6000. WGR will provide needed reference data for imputation and to clarify complexities that may hinder SNP discovery (repeats, tandem duplications, structural variation). GBS and GWA will be conducted on ~1,600 BC1F3 to BC1F5 progeny covering the diversity of target traits in the training population. The current version of the sorghum genome will serve as the reference. The data will enable robust marker-trait associations suitable for GP model development.Objective 2: Develop markers and GP models (Years 1-3).Sub-objective 2A: Develop a validation population for GP model optimization.UNR mapping population development. A BC1 mapping population derived from various combinations of trait donors will be finished and GBS will be conducted to identify parental alleles known (or suspected) to be associated with target trait phenotypes. Backcrosses are being made to waxy versions of B Wheatland (to enrich for the B heterotic group), R Tx430 (to enrich for the R heterotic group), and B Tx623 (ms8) to diversify the germplasm and incorporate nuclear male-sterility alleles. The BC1 population of 12 backcrosses will be finished by Fall 2019. Molecular markers will be used to enrich for target alleles and desirable plants will be selfed. The BC1F1 will be advanced one generation using single-seed descent, selecting 50 seeds per original BC1 to form 12 recombinant inbred line populations (RILs). Head rows will be planted in the BC1F2 generation to ensure adequate BC1F3 and BC1F4 seed for growth, genotyping, and phenotyping in four environments each season.Phenotyping. BC1F3 and BC1F4 will be grown as described in Objective 1. The BC1F4 generation will be harvested for extra seed (the BC1F5) for distribution to NuLife Market in Kansas and ICRISAT in India ahead of public release; see Objective 3. Phenotyping for grain quality in the BC1F1 (for original marker development) and BC1F5 breeding lines (for public release), will be conducted as in Objective 1.Genotyping. Four replicates (environments) will be planted each season and phenotyped, but only one replicate will be genotyped. The mapping population parents will be subjected to WGR and progeny (100 plants representing 10 randomly-selected RILs from each of the 12 BC1F1 plants; 1200 plants total) will be subjected to GBS.Genome reconstruction and marker development. We will assemble the genomes of six parents common among the training and mapping populations (Year 1) and two elite breeding lines (Year 4) using the 10X Genomics platform. Genomes will be annotated. WGR data from the parent lines, and GBS data from the BC1F1 progeny, will be used in GWA, and to improve GP models by integrating marker information for target traits not in the training population.Sub-objective 2B: Model development for predicting phenotypic traits using the training population where molecular marker and environmental information are used as covariates.The training population will be used to build the prediction models that will be validated on the mapping population lines (BC1F3 to BC1F5). Published GP models for sorghum will be expanded upon using marker information as it becomes available during the breeding process. WGR and GBS data from later generations will be included, once available, for GP model validation (by assessing the ability of the integrated GP models to predict phenotypes in later generations based on the genotypes of earlier ones). We anticipate that the models we develop will perform at least as well or better than currently published sorghum GP models in terms of prediction accuracy, because previous studies incorporated marker information only as main effect, and did not include GxE in the GP models.Objective 3: Use breeding lines derived from the UNR mapping population to validate the GP models (Years 3-4).GP models will be tested using four cross-validation schemes: (i) predict observed lines in observed environments [some lines tested in some environments but not in others]; (ii) predict unobserved lines in observed environments [new developed lines that have not been observed in any trial]; (iii) predict observed lines in unobserved environments [new locations]; and (iv) predict unobserved lines in unobserved locations [new lines that have never been tested would be tested in new environments]. Cases (i) and (ii) are less complicated to confront and similar scenarios can be found elsewhere in the literature. However, if we consider the usefulness of GP methods by restricting their application to only those cases where predictions are performed in already observed environments, this may not be useful for predicting in the future. Thus a realistic application of GP methods should consider those cases where the aim is to predict the performance of the lines (already observed in other environments or not) in unobserved environments [i.e., cases (i) and (ii)]. The predictive ability will be measured on a trial basis (i.e., no matter how the training set is integrated at the end, predicted and observed values for a single trial will be contrasted) using Pearson's product-moment correlation coefficient. These efforts will be coordinated among Co-PIs through Breed® software (Benson Hill Biosystems).Objective 4: Identify and publicly release the most promising breeding lines (Year 4). We will conduct a final assessment of the germplasm by all of our collaborators in Year 4. The BC1F5 will be grown in the following four environments: Reno, NV (managed by PI Yerka); Telangana, India (managed by Dr. Ashok Kumar Are); Scott City, Kansas (managed by NuLife Market staff and collaborators); and Vega, TX (managed by Richardson Seeds). A subset of the 10 best RILs descended from each BC1F1 plant will be provided to each collaborator (~ 50 g seed/RIL/generation). Tissue samples will be collected for the most promising lines targeted by our collaborators as meriting future development. The two best-performing lines will be subjected to WGR to make detailed comparisons among them and their original parents. Each collaborator will be able to keep the materials provided to them for their own use. PI Yerka will curate all data and feedback regarding germplasm performance and sequence data.

Progress 03/01/19 to 02/28/23

Outputs
Target Audience:Six target audiences were reached by this project: Nevada farmers, community members, UNR students, the national sorghum research community, an industrypartner, and industry breeders. The Nevada farmers and community members were reached by our team's efforts putting together a fourth annual field day in August 2021 wherein the public was invited to tour our field sites in Reno, NV (the Valley Road Experiment Station) and an online workshop in April 2021 (Walia, M., M. Yerka, R. Washington-Allen, and M. Andrade-Rodriguez. Nevada Sorghum Workshop. University of Nevada Cooperative Extension. 29 April 2021. https://www.youtube.com/watch?v=6-L0G8AeXuM).The field day was an integrated (research + extension) activity because it was hosted by Dr. Maninder Walia, University of Nevada Cooperative Extension (UNCE) Field CropsSpecialist, and included UNR faculty and students, who described their research. Students (third target audience)were reached by the team's efforts to include undergraduates and graduate students inresearch and teaching activities. Three undergraduate studentsparticipated in theresearch throughout the year, under the direction of the graduate student assigned to this project. All students receivedtraining and oversight by the PI on this project. In addition, PI Yerka used the field site in afield trip for her AGSC 460/660 "Intro to Plant Breeding" course, which included eight undergraduates, to teach them about breeding for new environments and how to incorporate molecular markers intobreeding programs to make efficient selections among superior plants. The national sorghum research community (fourth target audience)was reached when PI Yerka was invited to give a zoom seminar by the Center for Sorghum Improvement (CSI) on February 8, 2022 (M. Yerka.News from Nevada: The first four years of a new sorghum breeding program in the Mountain West. 8 February 2022. Center for Sorghum Improvement.Invited Presentation.) Our industry collaborator (fifth target audience), Mr. Larry Richardson,President of Richardson Seeds, the biggest sorghum breeding company in the U.S., provided diverse varieties cost-freewhich we grew and harvested to develop and test molecular markers for breeding for fermentation quality. He also providedcost-freeaccess to nursery and phenotyping sites routinely used by his head breeder in Texas and Argentina. The plant breeders working for Richardson Seeds (sixth target audience)were fully integrated throughoutthe year by virtue of providing winter nursery sites in three different locations across northern Argentina. PI Yerka, a graduate student, and atechnician visited the various locations in CA andTXto collaborate with their breeders on this research. This project is theirfirst opportunity to integrate molecular breeding into the program and to learn about how to develop and use genomicprediction, so the training conducted by our team for their company is highly significant and impactful in the long term onfarmers who rely on their company for seed. PI Yerka grew 400 breeding lines both in Parlier, CA and Lubbock, TX in2021. Another 230 lines were grown at Richardson Seeds' global headquarters in Vega, TX. The goal is to develop new varieties of sorghum that are adapted to Northern Nevada having desirable fermentationand animal feed traits. These breeding lines comprise the validation population for this work and are scheduled to be finishednext winter (2023; a two-year delay due to complications with the COVID pandemic). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? One graduate student was able to pursue, and successfully defended, a Ph.D. in biochemistry. He was trained in bioinformatics and worked closely with farmers, community members, brewers, and Richardson Seeds through this project. One graduate student has been able to learn how to develop genomic prediction models. Ten undergraduate students and three technicians in the Yerka Lab have been able to learn about applied agriculture, plant breeding, and basic laboratory techniques such as DNA extraction, PCR, grain color phenotyping, and nucleotide sequencing. How have the results been disseminated to communities of interest? Results were disseminated to the public and local farmers through annual field days in August 2019-2021and in online workshops in April 2020-21. One poster was presented at a graduate student conference at UNR regarding the bioinformatics of this work that has now been published. PI Yerka presented an invited seminar to the Center for Sorghum Improvement to a national audience across sorghum research sectors in February 2022. Two manuscripts, one book chapter, two presented posters. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Genotype and phenotype a training population, developed from publicly-available materials, provided by Richardson Seeds through an MTRA (Year 1). Completed. Develop markers and GP models for breeding line development (Years 1-3).Completed. Use breeding lines derived from the new UNR mapping population (whose parents are closely related to the training population) to validate the GP models (Years 3-4).Completed. Conduct collaborative research with industry, government, and international partners to identify and publicly release the most promising breeding lines from the Yerka Lab (Year 4). Substantially Completed; we are delayed with the public release due to the COVID-19 pandemic having hampered lab productivity during the majority of the grant, delaying the multi-environment analyses needed for publication. The breeding lines and mapping populations have been developed but require about 2 more years to fully characterize for public release.

Publications

• Type: Journal Articles Status: Submitted Year Published: 2023 Citation: Yerka, M.K., Z. Liu, S. Bean, D. Nigam, C. Hayes, D. Druetto, G. Krishnamoorthy, S. Fangman, G. Cucit, J. Atim, J. Pedraza, and Y. Jiao. 2023. An enhanced genome assembly and functional, high-throughput molecular markers enable genomics-assisted breeding of waxy sorghum [Sorghum bicolor (L.) Moench]. Molecular Breeding. Submitted.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Yerka, M.K.*, Z. Liu, Y. Jiao. 2022. Development of KASP markers for waxy alleles and identification of a Copia retrotransposon as being causal of the wxa allele in low-amylose grain sorghum. National Association of Plant Breeders Annual Conference. Poster.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Peiris, S.K.H, X. Wu, S.R. Bean, M. Perez-Fajardo, C. Hayes, M.K. Yerka, K.S.V. Jagadish, T. Ostmeyer, F.M. Aramouni*, T. Tesso, R. Perumal, W.L. Rooney, M. Kent, B. Bean. 2021. Near infrared spectroscopic evaluation of starch properties of diverse sorghum populations. Processes. 9:1942. https://doi.org/10.3390/pr9111942.
• Type: Book Chapters Status: Submitted Year Published: 2023 Citation: Boyles, R. E., Rhodes, D. H., Yerka, M. K., Hayes, C. M., Brenton, Z. W., Behnke, M.-F., Emendack, Y. & Sanchez, J. 2022. Progress in, Targets of, and Resources for Mining Alleles in Sorghum Candidate Genes in Allele Mining in Crops Plants Vol. 1 (ed Chittaranjan Kole) Ch. 12, (Springer Nature, Submitted). Invited co-authorship.

Progress 03/01/21 to 02/28/22

Outputs
Target Audience:Six target audiences were reached by this project: Nevada farmers, community members, UNR students, the national sorghum research community, an industrypartner, and industry breeders. The Nevada farmers and community members were reached by our team's efforts putting together a fourth annual field day in August 2021 wherein the public was invited to tour our field sites in Reno, NV (the Valley Road Experiment Station) and an online workshop in March 2021 (Walia, M., M. Yerka, R. Washington-Allen, and M. Andrade-Rodriguez. Nevada Sorghum Workshop. University of Nevada Cooperative Extension. 29 April 2021. https://www.youtube.com/watch?v=6-L0G8AeXuM).The field day was an integrated (research + extension) activity because it was hosted by Dr. Maninder Walia, University of Nevada Cooperative Extension (UNCE) Field CropsSpecialist, and included UNR faculty and students, who described their research. Students (third target audience)were reached by the team's efforts to include undergraduates and graduate students inresearch and teaching activities. Three undergraduate studentsparticipated in theresearch throughout the year, under the direction of the graduate student assigned to this project. All students receivedtraining and oversight by the PI on this project. In addition, PI Yerka used the field site in afield trip for her AGSC 460/660 "Intro to Plant Breeding" course, which included eight undergraduates, to teach them about breeding for new environments and how to incorporate molecular markers intobreeding programs to make efficient selections among superior plants. The national sorghum research community (fourth target audience)was reached when PI Yerka was invited to give a zoom seminar by the Center for Sorghum Improvement (CSI) on February 8, 2022 (M. Yerka.News from Nevada: The first four years of a new sorghum breeding program in the Mountain West. 8 February 2022. Center for Sorghum Improvement.Invited Presentation.) Our industry collaborator (fifth target audience), Mr. Larry Richardson,President of Richardson Seeds, the biggest sorghum breeding company in the U.S., provided diverse varieties cost-freewhich we grew and harvested to develop and test molecular markers for breeding for fermentation quality. He also providedcost-freeaccess to nursery and phenotyping sites routinely used by his head breeder in Texas and Argentina. The plant breeders working for Richardson Seeds (sixth target audience)were fully integrated throughoutthe year by virtue of providing winter nursery sites in three different locations across northern Argentina. PI Yerka, a graduate student, and atechnician visited the various locations in CA andTXto collaborate with their breeders on this research. This project is theirfirst opportunity to integrate molecular breeding into the program and to learn about how to develop and use genomicprediction, so the training conducted by our team for their company is highly significant and impactful in the long term onfarmers who rely on their company for seed. PI Yerka grew 400 breeding lines both in Parlier, CA and Lubbock, TX in2021. Another 230 lines were grown at Richardson Seeds' global headquarters in Vega, TX. The goal is to develop new varieties of sorghum that are adapted to Northern Nevada having desirable fermentationand animal feed traits. These breeding lines comprise the validation population for this work and are scheduled to be finishednext winter (2023; a two-year delay due to complications with the COVID pandemic). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? One graduate student was able to pursue, and successfully defended, a Ph.D. in biochemistry. He was trained in bioinformatics and worked closely with farmers, community members, brewers, and Richardson Seeds through this project. One graduate student has been able to learn how to develop genomic prediction models. Three undergraduate students and a technician in the Yerka Lab have been able to learn about applied agriculture, plant breeding, and basic laboratory techniques such as DNA extraction, PCR, grain color phenotyping, and nucleotide sequencing. How have the results been disseminated to communities of interest? Results and ongoing progress were disseminated to the public and local farmers through a field day in August 2021 and an online workshop in April 2021. One poster was presented at a graduate student conference at UNR regarding the bioinformatics of this work that has now been published. PI Yerka presented an invited seminar to the Center for Sorghum Improvement to a national audience across sorghum research sectors in February 2022. Published manuscripts. What do you plan to do during the next reporting period to accomplish the goals? Breeding lines in the training and validation populations are being advanced two more generations in 2022-2023 by Richardson Seeds and PI Yerka. Additional genotypic and phenotypic data will be collected. The initial GP model development based off of publicly available sequence was finished in 2020 and will be updated using genotypic and phenotypic data. Molecular markers for sorghum traits of interest to this project, and to sorghum breeders generally, will be integrated into the GP models. PI Yerka's annual field day and annual online sorghum workshop, co-hosted with UNCE and collaborating UNR researchers, will again be held to integrate community members and local stakeholders in the project. PI Yerka will continue to use the breeding lines in this project to teach her Intro to Plant Breeding course in Fall 2022.

Impacts
What was accomplished under these goals? Richardson Seeds advanced two more generations in the training population attached to this project, thus decreasing the time to commercialization of specialty sorghums available on a large scale to U.S. farmers. PI Yerka advanced two generations of the validation population attached to this project. These efforts have expanded the genetic diversity of drought-tolerant crops available to farmers for forage, high-value specialty market (beer), and industrial uses in western states where short growing seasons and dependence on decreasing irrigation water derived from snowmelt limit the suitability of other crops. The development, genotyping, and phenotyping of the training and validation populations is about one year behind due to COVID-19, but it is ongoing. Co-PI Reka Howard's efforts at the University of Nebraska-Lincoln developing the GP models for sorghum are ongoing. Due to very long accounting staff shortages at UNR during the COVID-19 pandemic, we were not able to pay for or access the DNA sequences of the training and validation populations for three generations and we are trying to make up for it now.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Walia, M., M. Yerka, R. Washington-Allen, M. Andrade-Rodriguez, and G. McCuin. 2021, Sorghum Production in Nevada. University of Nevada, Reno FS-21-09.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Peiris, S.K.H, X. Wu, S.R. Bean, M. Perez-Fajardo, C. Hayes, M.K. Yerka, K.S.V. Jagadish, T. Ostmeyer, F.M. Aramouni*, T. Tesso, R. Perumal, W.L. Rooney, M. Kent, B. Bean. 2021. Near infrared spectroscopic evaluation of starch properties of diverse sorghum populations. Processes. 9:1942. https://doi.org/10.3390/pr9111942.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Baggett JP, R.L. Tillett, E.A. Cooper, and M.K. Yerka. 2021. De novo identification and targeted resequencing of SSRs efficiently fingerprints Sorghum bicolor sub-population identity. PLoS ONE 16(3): e0248213. https://doi.org/10.1371/journal.pone.0248213.
• Type: Theses/Dissertations Status: Published Year Published: 2021 Citation: Baggett, J.P. 2021. Improvements in semi-arid agriculture: Sorghum bicolor sub-population molecular marker development and mild salinity stress in Vitis. Ph.D. Thesis. University of Nevada, Reno. Reno.

Progress 03/01/20 to 02/28/21

Outputs
Target Audience:Four target audiences were reached by this project: Nevada farmers, community members, UNR students, and an industry partner. The Nevada farmers and community members were reached by our team's efforts putting together a field day on August 12, 2020wherein the public was invited to one of our field sites in Fallon, NV (the Newlands Experiment Station). Due to COVID-18, only 8 people attended, including three farmers, as determined by sign-in sheets. The field day was an integrated (research + extension) activity because it was hosted by Dr. Maninder Walia, UNCE Statewide Extension Specialist, and included UNR faculty and students, who described their research. Students, the third target audience, were reached by the team's efforts to include undergraduates and graduate students in research and teaching activities. Three undergraduate students (funded from a separate account) participated in the research throughout the year, under the direction of the graduate student assigned to this project. All students received training and oversight by the PI on this project. In addition, PI Yerka used the Valley Road Experiment Station field site in two field trips for her AGSC 460/660 "Intro to Plant Breeding" course, which included one graduate studentand three undergraduates, to teach them about breeding for new environments and how to incorporate molecular markers into breeding programs to make efficient selections among superior plants. Our industry collaborator, Mr. Larry Richardson, President of Richardson Seeds, the biggest sorghum breeding company in the U.S., provided diverse varieties cost-free which we grew and harvested to develop and test molecular markers for breeding for fermentation quality. He also provided cost-free access to nursery and phenotyping sites routinely used by his head breeder in Texas and Argentina. PI Yerka grew 200 breeding lines associated withthis work in Fallon, NV; and another 400 breeding lines in Parlier, CAin 2020. The goal is to develop new varieties of sorghum that are adapted to Northern Nevada having desirable fermentation and animal feed traits. These breeding lines comprise the validation population for this work and are scheduled to be finished next winter (2022). This is a year later than previously expected due to the reduced opportunities for travel and lab work due to COVID-19. The plant breeders working for Richardson Seeds, the fourth group reached by this project, were fully integrated throughout the year by virtue of providing winter nursery sites in three different locations across northern Argentina. PI Yerka and a technician visited the various locations in Argentina to collaborate with their breeders on this research. This project is their first opportunity to integrate molecular breeding into the program and to learn about how to develop and use genomic prediction, so the training conducted by our team for their company is highly significant and impactful in the long term on farmers who rely on their company for seed. Changes/Problems:The project was affected by COVID-19 in two ways. First, DNA sequencing information for the training population was delayed when UNR lost the staff member in charge of managing professors' accounts, as she was a National Guard member and was called to active duty. It took many months to find a suitable replacement. Due to the size of the bill, payment had to be made by a staff member, and due to staffing shortages throughout the university, the remaining staff were scrambling to keep up with everyone's purchases. We have received the DNA sequences now and are currently going through about 18 months of backlogged bioinformatics and modeling work. We should be able to complete it by the project's end date. If not, we will apply for a no-cost extension. Second, the project was affected when lab operations had to be scaled back to a skeleton crew. PI Yerka lost most of her undergraduate students, so the remaining ones had to work across many more projects. This necessarily reduced the number of entries that could be evaluated. Richardson Seeds has very graciously offered PI Yerka 1200 rows in three Argentine environments in Winter 2021-2022 to make up for these losses. The worst case scenario is that breeding lines will be 1-2 generations less "finished" by the time to project ends and there may be a delay in publicly releasing them. If so, we will apply for a no-cost extension. What opportunities for training and professional development has the project provided?One graduate student has been able to pursue a Ph.D. in biochemistry, learn bioinformatics, and work closely with farmers, community members, brewers, and Richardson Seeds through this project. One graduate student has been able to learn how to develop genomic prediction models. Three undergraduate students in the Yerka Lab have been able to learn about applied agriculture, plant breeding, and basic laboratory techniques such as DNA extraction, PCR, grain color phenotyping, and nucleotide sequencing. How have the results been disseminated to communities of interest?Results and ongoing progress were disseminated to the public and local farmers through a field day in August 2020. Eightfarmers andcommunity members attended. One poster was presented at a graduate student conference at UNR regarding the bioinformatics of this work that has now been published. Due to COVID-19, no posters were presented at national or international conferences. What do you plan to do during the next reporting period to accomplish the goals?Breeding lines in the training and validation populations are being advanced two more generations in 2021-2022 by Richardson Seeds and PI Yerka. Additional genotypic and phenotypic data will be collected. The initial GP model development based off of publicly available sequence was finished in 2020 and will be updated using 2019-2020 genotypic and phenotypic data in 2021-2022 now that we have the sequences back from the provider. Molecular markers for sorghum traits of interest to this project, and to sorghum breeders generally, will be integrated into the GP models. PI Yerka's annual field day will again be held to integrate community members and local stakeholders in the project. PI Yerka will also continue to use the breeding lines in this project to teach her Intro to Plant Breeding course in Fall 2021.

Impacts
What was accomplished under these goals? 1. Richardson Seeds advanced two more generations in the training population attached to this project, thus decreasing the time to commercialization of specialty sorghums available on a large scale to U.S. farmers. PI Yerka advanced two generations of part of the validation population attached to this project (due to COVID-19, only the highest priority lines could be advanced; we hope to make up for this by advancing the remaining lines over winter 2021-2022). These efforts haveexpandedthe genetic diversity of drought-tolerant crops available to farmers for forage, high-value specialty market (beer), and industrial uses in western states where short growing seasons and dependence on decreasing irrigation water derived from snowmelt limit the suitability of other crops. The development, genotyping, and phenotyping of the training and validation populations is about one year behind due to COVID-19, but it is ongoing. 2. Co-PI Reka Howard's efforts at the University of Nebraska-Lincolndeveloping the GP models for sorghum are ongoing. Due to very long accounting staff shortages at UNR during the COVID-19 pandemic, we were not able to pay for oraccess the DNA sequences of the training and validation populations for three generations and we are trying to make up for it now.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Baggett JP, R.L. Tillett, E.A. Cooper, and M.K. Yerka. 2021. De novo identification and targeted resequencing of SSRs efficiently fingerprints Sorghum bicolor sub-population identity PLoS ONE 16(3): e0248213.

Progress 03/01/19 to 02/29/20

Outputs
Target Audience:Four target audiences were reached by this project: Nevada farmers, community members, UNR students, and an industry partner. The Nevada farmers and community members were reached by our team's efforts putting together a field day on August 13, 2019 wherein the public was invited to one of our field sites in Fallon, NV (the Newlands Experiment Station). 15 people attended, including five farmers, as determined by sign-in sheets. The field day was an integrated (research + extension) activity because it was co-hosted by me and Dr. Maninder Walia, UNCE Statewide Extension Specialist. Channel 2 News from Reno attended andinterviewed Dr. Yerkaand several farmers. The interviews were aired on that day's 5:00 PM and 6:00 PM news as the frontline story, "UNR Researchers Study Crops That Could Be Used For Beer." The focus of these interviews was on which crops are being grown and evaluated by UNR and UNCE researchers, and how they can help make Nevada agriculture more sustainable by reducing water use, yet still remain profitable by targeting specialty markets. Students, the third target audience, were reached by the team's efforts to include undergraduates and graduate students in research and teaching activities. Three undergraduate students (funded from a separate account) participated in the research throughout the year, under the direction of the graduate student assigned to this project. All students received training and oversight by the PI on this project. In addition, PI Yerka used the Valley Road Experiment Station field site in two field trips for her AGSC 460/660 "Intro to Plant Breeding" course, which included three graduate students and three undergraduates, to teach them about breeding for new environments and how to incorporate molecular markers into breeding programs to make efficient selections among superior plants. Our industry collaborator, Mr. Larry Richardson, President of Richardson Seeds, the biggest sorghum breeding company in the U.S., provided diverse varieties cost-free which we grew and harvested to develop and test molecular markers for breeding for fermentation quality. He also provided cost-free access to multiple winter nursery and phenotyping sites routinely used by his head breeder in Texas, Mexico, and Argentina. PI Yerka grew 200 breeding lines (parental varieties and their progeny, based on initial breeding work associated with this project in 2018) in Mexico and Texas in 2019. The goal is to develop new varieties of sorghum that are adapted to Northern Nevada having desirable fermentation and animal feed traits. These breeding lines comprise the validation population for this work and are scheduled to be finished next winter (2021). The plant breeders working for Richardson Seeds were fully integrated into this project throughout the year by virtue of providing and developing the training population in Vega, TX and four different locations across northern Argentina. PI Yerka and John Baggett visited the various facilities multiple times to train them on the use and integration of molecular markers in breeding and to plan and conduct research. This project is their first opportunity to integrate molecular breeding into the program and to learn about how to develop and use genomic prediction, so the training conducted by our team for their company is highly significant and impactful in the long term on farmers who rely on their company for seed. Changes/Problems:Because this is the first time Richardson Seeds has attempted to integrate molecular breeding into their workflow, many conversations and some troubleshooting had to take place to keep the project on track and moving forward. One particular challenge was that their team was largely unaware of what a recombinant inbred line (RIL) population was, and although they were fully integrated into discussions during preparation of the grant proposal prior to its submission, they did not alert PI Yerka that her suggestion of using RILs for their training population would not be possible because they do not use them. This is not entirely their fault - they simply did not realize that a RIL is a specific thing - they probably thought it was a general description of a pedigree. PI Yerka worked very closely with the head breeder to re-design the mapping populations once the mistake was identified. Therefore, while the training population in the proposal was intended to be multiple generations of pre-existing RILs, the current training population is a set of four bulk populations in the F2- F6 generations that are segregating for each of the traits of interest to the proposal, including waxy endosperm and other starch digestibility properties. The validation population necessarily had to be optimized to match the training population, so instead of a backcrossing method followed by line development, it is a MAGIC population pyramiding all of the traits of interest to the project and comprised in the training population. This new approach to the validation population is a better match to the training population because it enables the analysis of bulk populations segregating for multiple traits of interest, as opposed to highly structured populations such as RILS and BCs. The project remains on time because these alternative materials were already available and under development at Richardson Seeds and in PI Yerka's lab. What opportunities for training and professional development has the project provided?One graduate student has been able to pursue a Ph.D. in biochemistry, learn bioinformatics, and work closely with farmers, community members, brewers, and Richardson Seeds through this project. One graduate student has been able to learn how to develop genomic prediction models. Three undergraduate students in the Yerka Lab have been able to learn about applied agriculture, plant breeding, and basic laboratory techniques such as DNA extraction, PCR, and nucleotide sequencing. How have the results been disseminated to communities of interest?Results and ongoing progress were disseminated to the public and local farmers through a field day on August 13, 2019. Five farmers and ten community members attended, and a local news station (Channel 2) interviewed, filmed, and broadcast the event as the lead story on the 5:00 and 6:00 news. Results were also disseminated to the publicand to other researchers and students at UNRat the CABNR Annual Field Day in Reno. PI Yerka had a table and poster, managed by her and the graduate student attached to this project, which provided information about the project and about plant breeding and sorghum genetics and utilization in general. Two posters were developed and presented at scientific conferences regarding the bioinformatics of this work that are now being prepared in a manuscript. The first was at the 2019 Annual NIFA PI meeting held during the NAPB Annual Meetings in Georgia. The second was at the 2020 International Plant Animal Genome (PAG) meetings in California. What do you plan to do during the next reporting period to accomplish the goals?Breeding lines in the training and validation populations will be advanced two more generations in FY 2020 by Richardson Seeds and PI Yerka. Additional genotypic and phenotypic data will be collected. The initial GP model development is expected to be finished in FY 2020 using FY 2019-2020 genotypic and phenotypic data and publicly available data sets. Molecular markers for sorghum traits of interest to this project, and to sorghum breeders generally, will be integrated into the GP models. PI Yerka's annual field day will again be held to integrate community members and local stakeholders in the project. PI Yerka will also continue to use the breeding lines in this project to teach her Intro to Plant Breeding course in Fall 2020.

Impacts
What was accomplished under these goals? 1. Richardson Seeds advanced two generations in the training population attached to this project, thus decreasing the time to commercialization of specialty sorghums available on a large scale to U.S. farmers. PI Yerka used molecular markers to assist them in achieving homozygosity of the population for waxy alleles. PI Yerka also harvested tissue for the F2-F4/F5 generations for genotyping. PI Yerka advanced twogenerations of the validation population attached to this project, thus expanding the genetic diversity of drought-tolerant crops available to farmers for forage, high-value specialty market (beer), and industrial uses in western states where short growing seasons and dependence on decreasing irrigation water derived from snowmelt limit the suitability of other crops.The development, genotyping, and phenotyping of the training and validation populations is on time and ongoing. 2. Co-PI Reka Howard (University of Nebraska-Lincoln) trained a graduate student who is developing the GP models for sorghum using publicly-available genomics data sets. PI Yerka provided marker information for known loci of interest to the waxy breeding program associated with various starch digestibility traits. This work is on time and ongoing.

Publications