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INVESTIGATOR: Kresovich, S.

PERFORMING INSTITUTION:
CLEMSON UNIVERSITY
CLEMSON, SOUTH CAROLINA 29634

GENOMIC AND BREEDING FOUNDATIONS FOR BIOENERGY SORGHUM HYBRIDS

NON-TECHNICAL SUMMARY: The overall goal of this effort is to build the germplasm, breeding, genetic, and genomic foundations necessary to rapidly and specifically advance cellulosic sorghum as a bioenergy and chemical feedstock. Sorghum is one of the few crops that can fit all of the proposed approaches for renewable fuel production. i.e. from starch, sugar, and/or cellulose. It is productive, highly adaptable to a range of environments and production systems,highly efficient in using water and nutrient inputs, and includes a range of forms able to be grown as either annuals or perennials. We define cellulosic sorghum as an annual or perennial form that is bredand selected to maximize carbon (energy) accumulation per unit time, land area, and/or production input (water, nutrients, pesticides, etc.). Phenotypically, it likely will be extremely tall, heavy tillering,large-barreled, dry stemmed, photoperiod-sensitive material that aggressively ratoons (regrows following harvest) in environments with milder winters (the southeastern and southcentral U.S.).

OBJECTIVES: The overall objective of this proposed research is to build the germplasm, breeding, genetics, and genomics foundations necessary to rapidly and specifically advance cellulosic sorghum as a bioenergy and chemical feedstock. Sorghum is one of the few agricultural crop candidates that can fit all of the proposed approaches for renewable fuel production from starch, sugar, and/or cellulose. It is highly adaptable to a range of environments and production systems, highly efficient in using water and nutrient inputs, and represents a range of forms (starch, sweet, and cellulosic types) able to be grown as either an annual or perennial. For this proposed investigation, we define cellulosic sorghum as an annual or perennial form of sorghum that is bred and selected to maximize carbon accumulation per unit time, land area, and/or production input (including water, nutrients, pesticides, etc.). Phenotypically, it likely will be an extremely tall, heavy tillering, large-barreled dry stems, photoperiod-sensitive material that exhibits the potential to aggressively ratoon (regrow following harvest) in environments with milder winters (the southeastern and southcentral U.S.). The essential, specific objectives of the proposed research are: (1)to develop ten nested association mapping populations (NAMs) and a diversity panel necessary to dissect the genetic bases of carbon accumulation, partitioning, and metabolic pathways of cellulosic sorghum; (2)to phenotype these NAMs and diversity panel for patterns of carbon accumulation, partitioning, and associated metabolic pathways and to correlate these traits with DNA sequence and genic variation that will underlie future breeding and genetic studies of cellulosic sorghum; (3)to lay the genomic foundation (based on DNA sequence generation and analysis, and pipeline and database development) for integrating genomic selection and other genomics-based strategies into cellulosic sorghum breeding programs for energy and chemicals; and (4)to identify and create five cellulosic male-sterile (A lines), maintainer (B lines), and restorer (R lines) germplasm necessary to exploit heterosis specifically targeted at energy and chemical feedstocks.

APPROACH: We will test the hypothesis that energy yield per unit time, land area, and/or input of water/nutrients can be increased by the development of cellulosic sorghum ideotypes that are tall, tiller extensively with dry and large-barreled stalks (maximizing sink strength), tolerate high density planting, and are photoperiod sensitive. This ideotype differs substantially from all current grain or sweet sorghum genepools. We also will test the hypothesis that parallel evolution of/selection for high-biomass sorghum in different botanical races may have involved convergent mutation or ancient introgression of a small number of key mutations, with the result of a high level of correspondence of QTL locations in crosses involving different botanical races. This implies that the genetic control of the traits fundamental to a sorghum biomass ideotype may be relatively simple, and quickly identified to expedite marker-based selection for a high basal level of productivity upon which ongoing selection can further improve. Finally, we will implement a systems biology approach toward tests of multiple hypotheses that specific molecular pathways are underlying QTLs sets by fitting de novo generated sorghum gene-gene coexpression interactions (and their predicted function) to QTLs on a trait-by-trait basis. In this way, we will identify groups of candidate genes with genetic evidence that makes them priorities for functional validation through multiple approaches. This project will complement and supplement a NAM set being developed for grain sorghum, with 10 additional populations that address traits of singular importance to cellulosic sorghum, using one common parent (Grassl) and ten other parental lines exhibiting wide variation in genetic background, geographic origin, biomass composition, and agronomic phenotypes. These new populations will be developed at several key southeastern U.S. regions to ensure adapted cellulosic sorghum germplasm in the future. In complement to this proposal, a DOE Joint Genome Institute Community Sequencing Project (JGICSP) in progress (www.jgi.doe.gov/sequencing/cspseqplans2010.html) and a NSF-BREAD project will provide the raw material, pipelines, and associated databases for us to identify the required SNPs. We also will implement a systems biology approach toward tests of multiple hypotheses that specific molecular pathways are underlying QTL sets by fitting generated sorghum gene-gene coexpression interactions (and their predicted function) to QTLs on a trait-by-trait basis. In this way, we will identify groups of candidate genes with genetic evidence that makes them priorities for functional validation through association genetics, reverse genetics, or transgenic approaches. Lastly, products relevant for breeding of cellulosic hybrids (A, B, and R lines) will be developed and distributed to the user community. Heterosis in cellulosic sorghum is known to be strongly beneficial, impacting yield and composition in manners for which we expect to provide diagnostic DNA markers, while simultaneously advancing the crop for distribution by common practices of the U.S. seed industry.

PROGRESS: 2014/06 TO 2016/08
Target Audience:The target audience included the scientific community, sorghum growers, commodity groups, food/feed companies, processing and engineering groups, and renewable energy companies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The overall project provided training and professional development in the areas of plant breeding, genetics, bioinformatics and biomass compositional analysis. These opportunities involved Dr. Liz Cooper, Dr. Geoff Morris (Post-Doctoral Research Associates); Rick Boyles, Zach Brenton, Will Phoelman, Nadia Shakoor (Graduate Students); Matt Myers, Brad Rauh (Research Associates); and Alex Cox (Technician). How have the results been disseminated to communities of interest?The results have been reported to the scientific community via research publications (see "Products" section) and conference presentations (Plant and Animal Genome XXIV, Crop Science Society of America 2015). Given the focus of this project on the development of sorghum resources to understand carbon allocation and partitioning in this crop, the results were of interest to the overall sorghum community. Results of this project were demonstrated in the field to individuals associated with sorghum as a commodity (United Sorghum Checkoff Program), food companies (Murphy Brown, Smithfield Foods), bio-based chemicals/renewables (Chemtex/Beta Renewables) and national funding agencies interested in biofuel feedstock development (Department of Energy ARPA-E). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

IMPACT: 2014/06 TO 2016/08
What was accomplished under these goals? We created an 11-family Nested Association Mapping population of 250 individuals per family. Three of these populations are in the F6 and are currently being genotyped. The other 8 families are in the F5 and will be advanced to the F6 this winter in a southern nursery. Phenotypic data was generated in the F3 populations for yield and composition. We collected over 40 phenotypic measurements including total yield as well as percent cellulose, hemicellulose, lignin and nonstructural sugars among others. The phenotypic data was generated in the F3s to determine phenotypic segregation patterns. This data will be recollected in the F6 in 2017. Genomic markers for the other eight families will be generated in the spring of 2017 for public release. In addition to the Nested Association Mapping population, we created a 400-accession diversity panel, which includes accessions representing sorghums global genetic and geographic diversity. We have generated 232,000 genomic markers using genotyping-by-sequencing. We have phenotyped this panel for three years for over 40 phenotypic traits. Using genome-wide association scans, we successfully identified strong candidate genes for the accumulation of high structural sugars as well increased structural carbon (cellulose and hemicellulose). We tested over 400 cellulosic hybrids for bioenergy production. We released 10 diverse commercial ready hybrids through the South Carolina Crop Improvement Association. Through the SCCIA, we tested the top five performing hybrids in 2013 in the Official Variety Trial. Each produced over 15 dry tons per acre over three locations statewide. These lines were licensed to a bioenergy company and currently being tested by another. Female development takes longer than male development. The female seed has been backcrossed four times and will be backcrossed again this winter nursery.

PUBLICATIONS (not previously reported): 2014/06 TO 2016/08
1. Type: Journal Articles Status: Published Year Published: 2016 Citation: Brenton ZW, Cooper EA, Myers MT, Boyles RE, Shakoor N, Zielinski KJ, Rauh BL, Bridges WC, Morris GP, Kresovich S. 2016. A genomic resource for the development, improvement, and exploitation of sorghum for bioenergy. Genetics 204(1):21-33.
2. Type: Journal Articles Status: Published Year Published: 2016 Citation: Shakoor N, Ziegler G, Dilkes BP, Brenton Z, Boyles R, Connolly EL, Kresovich S, Baxter IR. 2016. Integration of experiments across diverse environments identifies the genetic determinants of variation in Sorghum bicolor seed element composition. Plant Physiol 170(4):1989-1998.
3. Type: Journal Articles Status: Published Year Published: 2015 Citation: Fernandez MG, Strand K, Hamblin MT, Westgate M, Heaton E, Kresovich S. 2015. Genetic analysis and phenotypic characterization of leaf photosynthetic capacity in a sorghum (Sorghum spp.) diversity panel. Genet Resour Crop Ev 62(6):939-950.
4. Type: Journal Articles Status: Published Year Published: 2014 Citation: Shakoor N, Nair R, Crasta O, Morris G, Feltus A, Kresovich S. 2014. A Sorghum bicolor expression atlas reveals dynamic genotype-specific expression profiles for vegetative tissues of grain, sweet and bioenergy sorghums. BMC Plant Biol 14:35.
5. Type: Journal Articles Status: Published Year Published: 2014 Citation: Rhodes DH, Hoffmann Jr L, Rooney WL, Ramu P, Morris GP, Kresovich S. 2014. Genome-wide association study of grain polyphenol concentrations in global sorghum [Sorghum bicolor (L.) Moench] germplasm. J Agric Food Chem. 62(45):10916-10927.

PROGRESS: 2014/06/01 TO 2015/05/31
Target Audience: The target audience includes growers, commodity groups, processing and engineering groups, scientific community and energy companies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Opportunities for training include Post-doctoral researchers-Liz Cooper (now at Clemson University). Graduate students include: Rick Boyles (Clemson University), Will Poelhm (Clemson University), and Zach Brenton (Clemson University.) Technical personnel include: Matt Myers, Brad Rauh (Clemson University). How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Winter nursery activities will continue in Mexico for 2015-2016 to continue making progress on population development. For each of the five races of sorghum, we have at least two NAM populations advancing to the F5 or finishing the final generation at F6 and increasing seed to provide to USDA's Plant Genetic Resource Conservation Unit in Griffin, GA. Seven F3 populations will be phenotyped for a significant number of agronomic and compositional traits in replicated trials in South Carolina and Georgia. These populations then will be genotyped and association studies will be performed for particular bioenergy traits. It is expected that when all populations are completed, all individuals will be genotyped-by-sequencing.

IMPACT: 2014/06/01 TO 2015/05/31
What was accomplished under these goals? The specific objectives of the proposed research are: (1) to develop ten nested association mapping populations (NAMs) and a diversity panel necessary to dissect the genetic bases of carbon accumulation and partitioning of cellulosic sorghum; (2) to phenotype these NAMs and diversity panel for patterns of carbon accumulation and partitioning and to correlate these traits with DNA sequence variation that will underlie future breeding/genetic studies; (3) to lay the foundation for integrating genomic selection and other genomics-based strategies into cellulosic sorghum breeding programs; and (4) to identify and create and characterize cellulosic male-sterile (A lines), maintainer (B lines), and restorer (R lines) germplasm necessary to exploit heterosis specifically targeted at energy production. Following winter nursery activities in Mexico for 2014-2015 we will have made significant progress in population development. For each of the five races of sorghum, we have at least two NAM populations advancing to the F4 or F5 generation. In all, 15 NAM populations have been generated. We are behind schedule in population of development because of the complexities of handling and inducing flowering in photoperiod sensitive lines. We have ceased the summer increase in South Carolina, plants are exceedingly tall (and difficult to pollinate and collect viable seeds) and frequently the seeds do not mature and will not try again in Mexico during summer as in 2014 when the entire planting was wiped out by hurricane. When this project ends, we intend to continue advancing populations through support to be provided by the national sorghum commodity group. Seven F3 populations have been phenotyped for a significant number of agronomic and compositional traits in replicated trials in South Carolina and Georgia. These populations then will be genotyped and association studies will be performed for particular bioenergy traits. It is expected that when all populations are completed, all individuals will be genotyped-by-sequencing.

PUBLICATIONS: 2014/06/01 TO 2015/05/31
1. Type: Journal Articles Status: Published Year Published: 2014 Citation: Rhodes, D.R., et al. 2014. Genome-wide association study of grain phenol concentrations in global sorghum [Sorghum bicolor (L.) Moench] germplasm. J. Ag. Food Chem. 62: 10916-27.
2. Type: Journal Articles Status: Published Year Published: 2014 Citation: Shakoor, N., et al. 2014. A Sorghum bicolor expression atlas reveals dynamic genotype-specific expression profiles for vegetative tissues of grain, sweet, and bioenergy sorghums. BMC Plant Biology 14:35.
3. Type: Journal Articles Status: Awaiting Publication Year Published: 2015 Citation: Lasky, JR., H.D. Upadhyaya, P. Ramu, S. Deshpande, C.T. Hash, J. Bonnette, T.E. Juenger, K. Hyma, C. Acharya, S.E. Mitchell, E.S. Buckler, Z. Brenton, S. Kresovich, and G.P. Morris. In Press. Genome-environment associations in sorghum landraces predict adaptive traits. Science Advances.
4. Type: Journal Articles Status: Awaiting Publication Year Published: 2015 Citation: Salas Fernandez, M.G., K. Strand, M.T. Hamblin, M. Westgate, E. Heaton, and S. Kresovich. In Press. Genetic analysis and phenotypic characterization of leaf photosynthesis capacity in sorghum. Gen. Res. Crop Evol.