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INVESTIGATOR: Prakash, C.; He, G.; Mortley, D.; Bonsi, C.

PERFORMING INSTITUTION:
TUSKEGEE UNIVERSITY
TUSKEGEE, ALABAMA 36088

LINKING DNA MARKERS TO KEY BIOENERGY TRAITS IN MISCANTHUS

NON-TECHNICAL SUMMARY: Molecular markers linked with bioenergy traits in Miscanthus will be identified to help breed fast growing, highly productive and eco-friendly clones of this green energy grass which can serve as a feedstock to produce cellulosic ethanol. The project will enhance science-based knowledge in a multi-disciplinary area of vital importance to national interest, i.e., developing a bioenergy crop to provide cost-efficient and environmentally-sustainable source of biofuel, without impacting our food supply. Implicit in this goal is an effort to build a Center of Excellence in Bioenergy Genomics at Tuskegee University to train future scientists in this cross-cutting discipline that strives to meet green energy needs of the country. High-density, informative polymorphic DNA markers located across the Miscanthus genome will be identified using innovative whole-genome sequencing technique. Genetic loci linked to key bioenergy traits such as yield, yield quality, flowering time, growth and other agronomic traits will be identified on a broad diversity of Miscanthus germplasm using Genome-wide association study. We have already assembled a collection of Miscanthus genotypes at TU and conducted preliminary studies on identification of SSR-DNA markers. Renowned experts from across the country will serve as collaborators. Results from the project will enhance in the rapid development of high yielding cultivars of Miscanthus grass. The project will clearly strengthen our institutional capability in a key mission area of Tuskegee University viz., genomics of bioenergy while enhancing the workforce diversity.

OBJECTIVES: This project seeks to gain molecular insights into the genome of the bioenergy grass Miscanthus by identifying gene-based markers for bioenergy traits, including morphological, agronomical, reproductive, architectural and biochemical traits using Genome-Wide Association Studies (GWAS); and build capacity for bioenergy genomics program at Tuskegee University for training students in this frontier area of science and national priority. The specific research objectives are: 1. to collect, assemble and evaluate a diverse Miscanthus germplasm for agronomic, physiological and biochemical traits of importance to bioenergy; 2. identify and develop gene-based markers for bioenergy traits and discover Single Nucleotide Polymorphism (SNP) markers using RAD sequencing in Miscanthus; and 3. perform Genome-Wide Association Study (GWAS) to associate genomic markers with key bioenergy traits in Miscanthus.

APPROACH: The current collection of Miscanthus germplasm at Tuskegee University will be expanded to represent the global diversity of this species, and will include wild accessions from the USDA National Germplasm Repository in Miami, collections from our collaborators, improved cultivars and hybrids including some that were developed privately. Additional germplasm lines, including ecological races and elite clones, will be procured from other sources including private nurseries, seed companies, and university researchers. Appropriate permits will be obtained from USDA/APHIS to grow wild relatives of Miscanthus considered to be noxious weeds under confined conditions. Genetic and genomic tools developed in sugarcane (evolutionarily the closest crop relative of Miscanthus) will be used to facilitate the rapid and low-cost query of genomic information in Miscanthus. We will especially target the identification of developmentally regulated genes related to morphogenesis to better understand the mechanisms of biomass accumulation in Miscanthus. These functional EST-SSRs, particularly those encoding enzymes for biomass production described above in sugarcane, will be tested for transferability in Miscanthus. The transferable EST-SSRs will then be used along with individual SNPs or SNP haplotypes related to developmental genes for genome-wide association studies in Miscanthus. The data from gene-based SSR and SNP markers, combined with information on morphological, agronomical, and biochemical traits obtained from the evaluation of the Miscanthus germplasm collection, will form the backdrop for genome-wide association studies. We will first use random genomic SSRs from sugarcane to correct for genetic relatedness, and use the software STRUCTURE to determine the population structure within the Miscanthus germplasm collection of this study. Population membership estimates derived using STRUCTURE or principal components analysis (PCA) will be used for structured association analysis by the software TASSEL, developed explicitly for GWAS.

PROGRESS: 2012/09 TO 2017/08
Target Audience:Target audience includes students, scholars and scientists in agriculture and bioenergyand related areas; general public Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We trained two graduate students who conducted their dissertation research on the goals of this project. We also trained several undergraduate students to provide them research experience and training How have the results been disseminated to communities of interest?Yes, we have presented papers in the local Professional AgriculturalWorkers Conference, University-wide conference on student research and at national scientific meetings. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

IMPACT: 2012/09 TO 2017/08
What was accomplished under these goals? To accelerate the development of hybrids and high-performance varieties in the of Miscanthus, we performed GWAS and genome-wide prediction studies by analyzing the associated SNPs (Single Nucleotide Polymorphisms) (~3000 SNPs genotype non missing value) with eight phenotype parameters related to biomass of M. sinensis grown at Tuskegee University, Alabama, USA since 2013. These eight traits include survival (2015 fall and 2017 fall), Culm Length (2014), Height (2015), Biomass Yield fresh weight (2015), sample Yield fresh weight (2015), Sub-sample Yield dry weight (2015), Moisture content (2015), and Biomass Yield dry weight (2015). We could not find an association of any of these traits with the SNPs tested. To increase the probability to identifying specific SNPs associated with critical traits related to biomass, we have continued to collect the data on flower heading time, flowering time, half heading time, and half flowering time until the end of October of this year. Another 7 traits: Culm Length, Height, Biomass Yield fresh weight, sample Yield fresh weight, Sub-sample Yield dry weight, Moisture content, and Biomass Yield dry weight will also be collected. These traits data along with those already collected earlier in 2015 would help in the estimating BLUP (best linear unbiased prediction) of the phenotypic traits. Another way is to increase the number of SNPs to improve the GWAS analysis. Generation of specific SNPs markers associated with key bioenergy traits will molecular breeding in improving the yield of Miscanthus. Miscanthus is a fast-growing bioenergy grass, tolerant of adverse conditions and has high biomass production, and is increasingly attractive as a potential source of cellulosic ethanol. A better understanding of the genetic variation in Miscanthus naturalized populations would help to efficiently select parents for breeding and adapt into changing environments in the US, which further improve the quality of them as a bioenergy grass. Due to the limited molecular markers developed in Miscanthus, sugarcane and corn DNA markers were employed to explore genetic diversity within and among naturalized populations. As sugarcane and corn are closely related to Miscanthus , (53) sugarcane SSR marker and ()[S1] corn SSR markers were initially used in our study to test their transferability in the Miscanthus genome . All (53) pairs of sugarcane markers showed a high transferability. Ten SSR markers (Actin, GA30x, PF00931, PF03856, SMC226CG, SMC248CG, SMC319CG, SMC1039CG, EST-SSR29, and EST-SSR38-2) produced clear and unambiguous polymorphic bands at most loci. Six markers were monomorphic. The sequence of one monomorphic marker, EST-SSR30-1, was obtained and applied in the identification of the genetic variation of the populations. The data from the polymorphic markers were analyzed using Power-Marker 3.25, Structure 2.3.4 and GenALEx6.5 software. The result shows that the highest genetic distance 0.3738 was found in the genotypes of North Carolina (NC) which was sampled from Biltmore deer park (latitude N 35°33.010', longitude W 082°34.034' and elevation 571.5m) and Biltmore, Madison co, Cody rd. latitude N 35°32.032', longitude W 82°32.834' and elevation 698m). The lowest genetic distance 0.2776 showed in Virginia (VA) genotypes sampled from latitude N 37.23, longitude W 80.21 and elevation 655.0152 m. In the cluster and structure analysis, 228 genotypes were divided into six groups and most of the genotypes from different states were mixed in groups. The sequences of EST-SSR markers 30-1 were performed using MEGA6.06 software. The results further confirmed the result from the SSR marker data and the phylogenetic relationship is similar to the dendrograms from the polymorphic markers. Newer genomic tools to dissect complex traits such as the genome-wide association studies (GWASs) and genome-wide phenotype prediction (genomic selection) are very relevant in developing an association between phenotype and genotype data, and thus would be invaluable in crop breeding. Phenotypic traits such as flowering is crucial determinant of the agronomic value of a bioenergy crop. Flowering data and heading data indicates the termination of growth of leaves at the stem apex. Due to short growing seasons and radiation time for plants, it reduces potential biomass accumulation.

PUBLICATIONS (not previously reported): 2012/09 TO 2017/08
Type: Journal Articles Status: Published Year Published: 2016 Citation: Zhao, Y, S Basak, C E. Fleener, M Egnin, Erik J. Sacks, C S. Prakash, and G He. 2016 Genetic diversity of Miscanthus sinensis in US naturalized populations. Global Change Biology-Bioenergy GCB Bioenergy, doi: 10.1111/gcbb.12404 http://onlinelibrary.wiley.com/doi/10.1111/gcbb.12404/full

PROGRESS: 2015/09/01 TO 2016/08/31
Target Audience:Our target audience has primarily been scientists working in the area of bionenergy crops, and faculty and students at Tuskegee University. Once the project is complete, we will atempt to reach out to broader audience including growers Changes/Problems:Beyond the DNA quality issues from Miscanthus clones grown at Tuskegee that has delayed the completion of the project, we have not encountered any major problem so far but we do need more time to complete our genomics research, analyze the results and publish these as scientific papers in good journals. To accomplish these, we plan to request a No-Cost-Extension to the project for antoher year beyond August 31, 2016, What opportunities for training and professional development has the project provided?We trained many undergraduates and one graduate student in genomics, plant breeding, bioinformatics and crop improvement research in this project. How have the results been disseminated to communities of interest?We have dissseminated a short report on the results from our proejct through a research publication from the Tuskegee University Office of Research. What do you plan to do during the next reporting period to accomplish the goals?We have already extracted genomic DNA from 169 cultivars used in the phenotypic study. We had some quality issues with the isolated DNA due high protein contamination of the samples, and thus had to calibrate the process to ensure higher quality sample.We will now conductgenomic studies to uncoverSingle Nucleotide Polymorphism (SNP) markers using a RAD genomic sequencing approach. We will thenperform Genome-Wide Association Study (GWAS) to estimate the level of association between thesegenomic markers with key bioenergy traits in MIscanthus.We willwrite up the results into a scholarly publication in a peer-reviewed journal. Thus, we would be requesting a no-cost-extension of the project for an additonal year to complete the SNP analysis through RAD-seq technique, complete the statistical genetic analysis and develop a scholarly manuscript.

IMPACT: 2015/09/01 TO 2016/08/31
What was accomplished under these goals? Miscanthus is increasingly gaining popularity as a bioenergy grass because of its extremely high biomass productivity.Many clones of this grass were introduced into USA over the past century from East Asia where it originated, and planted for ornamental and landscaping purposes. An understanding of the genetic diversity among these naturalized populations may help in the efficient selection of potential parents in the Miscanthus breeding program. Here we report our study analyzing the genetic diversity of selected natural populations of Miscanthus from six locations across the Eastern United States. We employed DNA markers from other plant species as heterologous probes to look for polymorphic markers in the Miscanthus genome because of the paucity of molecular markers in Miscanthus. We tested 36 sugarcane SSR markers and 38 gene markers for their transferability to Miscanthus. Results showed that 58% SSR and 52% of gene-based markers could amplify Miscanthus DNA samples, respectively. Ten DNA markers (Actin, GA30x, PF00931, PF03856, SMC226CG, SMC248CG, SMC319CG, SMC1039CG, EST-SSR29, EST-SSR38-2) produced clear and unambiguous polymorphic bands, which were subsequently used in a diversity study. The PCR products of one monomorphic marker, EST-SSR30, in 228 Miscanthus DNA samples were sequenced and employed for the identification of the genetic variation of the populations. Genotyping data by the polymorphic markers were analyzed using Power-Marker 3.25, Structure 2.3.4., and GenALEx6.5 software. The highest genetic diversity (0.3738) was found among the North Carolina genotypes taken from Biltmore Deer Park and Biltmore, Madison County, Cody Rd. The lowest genetic diversity (0.2776) was observed among Virginia genotypes. By the cluster and structure analysis, 228 genotypes were formed two major groups, further divided into six sub-groups with DNA sequences. Miscanthus (Miscanthus x giganteus) is a large warm-season Asian grass, and as of recent a new leading biomass crop in the United States. Experience in Europe suggests giant Miscanthus will be productive over a wide geographic range in temperate regions, including marginal land, but is not appropriate for arid regions. Although widely accepted in Europe as a biomass crop very little research in the United States has been developed to asses if this crop would be favorable for growers in the southern region of the country. The objective of this study is document physiological response and growth of Miscanthus Giganteus under natural Macon county conditions. For this study 169 accessions were stduied for one season at the George Washington Agricultural experiment station in Tuskegee Alabama. The experiment was conducted as a complete randomized design (CRD) with 4 replications. Height and yield data, as well as biomass were collected and analyzed. Over a Third of the cultivars did not survive in these conditions. •About 24% of the plants did not survive across all reps and weIdentified nine genotypes of Miscanthus that grew well and thus may prove to bepotential parents for cultivar development Including PMS 138, M. ×giganteus 'Illinois', and Miscanthus sinensis 'PMS-226' that performed well under Alabama conditions.The significant growth documented in the study suggests that the environment of Alabama is favorable for Miscanthus of specific families. Further studies are recommended to select for specific cultivars for Alabama.

PUBLICATIONS: 2015/09/01 TO 2016/08/31
1. Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Amole, O., C.S. Prakash, Dr. Guohao He, Dr. D. Mortley. 2016 Growth and Biomass Production of (169) Miscanthus Giganteus Grown under natural conditions in Macon County Alabama. Joint Annual Research Symposium, Tuskegee University. March 17-18, 2016
2. Type: Journal Articles Status: Submitted Year Published: 2016 Citation: Genetic diversity of Miscanthus sinensis in US naturalized populations Zhao, Y, S Basak, C E. Fleener, M Egnin, Erik J. Sacks, C S. Prakash, and G He. 2016 Genetic diversity of Miscanthus sinensis in US naturalized populations. Global Change Biology-Bioenergy (in review)
3. Type: Book Chapters Status: Published Year Published: 2016 Citation: Zhao Y, R Williams , C. S. Prakash, Guohao He. 2016 Identification and Characterization of gene-based SSR markers in date palm (Phoenix dactylifera L.) Book Chapter in Press SPRINGER PROTOCOL SERIES: METHODS IN MOLECULAR BIOLOGY - Date Palm Biotechnology Protocols , Volume 2: Germplasm Conservation and Molecular Breeding Eds. Jameel M. Al-Khayri, S. Mohan Jain, and Dennis V. Johnson
4. Type: Journal Articles Status: Published Year Published: 2015 Citation: Abdalla N, CS Prakash & A McHughen. 2015. Genome editing for crop improvement: Challenges and opportunities. GM Crops and Food. 7 (1):183-205. DOI:10.1080/21645698.2015.1129937 http://www.tandfonline.com/action/showAxaArticles?journalCode=kgmc20
5. Type: Book Chapters Status: Published Year Published: 2016 Citation: Prakash, CS. 2016. Lifebox. In Plant Biotechnology and Genetics. 2nd Edition. C. Neal Stewart (Ed). P378-381. John Wiley & Sons, New York

PROGRESS: 2014/09/01 TO 2015/08/31
Target Audience:Scientific community, academic community, Tuskegee University students Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One graduate student complered her masters thesis under this project, and a second graduate student is half way through his thesis research on his project. Wehave trained several undergraduate students in research methodology also in this project. How have the results been disseminated to communities of interest?So far our results have been disseminated to student and faculty at Tuskegee University and Iowa State University through poster presentations at scientific meetings on these two campuses. We are publishing a research paper in a peer-reviewed scholarly journal to share our research results with the global scientific community. We plan to share results with larger audience through social media networks once the research results are published. What do you plan to do during the next reporting period to accomplish the goals?We now plan to identifymeaningful genomic markers associated with productivity traits in MIscanthus. Specific objectives are a) to record observations on key phenotypic traits critical to biofuel production in the established trial of Miscanthus (DOE population study) at Tuskegee University farm, b) Isolate genomic DNA from the 168 genotypes of Miscanthus from the DOE population study, c) Conduct SNP analysis of the DOE population using markers obtained from University of Illinois, d) Perform Genome-wide association study to test for relation between SNP markers and biofuel traits in the Miscanthus (DOE population). Molecular markers linked with bioenergy traits in Miscanthus compared to phenotypic observations will help to target and identify desired breeds, to increase further biofuel production in the US.

IMPACT: 2014/09/01 TO 2015/08/31
What was accomplished under these goals? Miscanthus is a bioenergy grass known for its fast growth, tolerant to adverse conditions and high biomass yielding capacity, thus increasingly attractive as a potential source of cellulosic ethanol. There are several clusters of naturalized populations of Miscanthus across the Eastern and South-Eastern US. An understanding of genetic variation in such naturalized populations of this grass may help in selecting appropriate parents for breeding superior cultivars that are adapted across wide-ranging and changing environments. As sugarcane is closely related Miscanthus, we employed its DNA markers as probes to explore genetic diversity within and among naturalized populations of the bioenergy grass. When 53 sugarcane specific gene markers and EST-SSR markers were initially tested for their transferability in the Miscanthus genome, all of them showed a high transferability, and thus were homologous to each other. Of thirteen sugarcane EST-SSR markers tested, seven such markers (SSR29, SSR30-2, SSR31-2, SSR33, SSR35, SSR37 and SSR38-2) readily amplified the Miscanthus DNA, producing clear and unambiguous polymorphic bands at most loci. Six markers (SSR30-1, SSR31-1, SSR32, SSR34, SSR36 and SSR38-1) were monomorphic. Polymorphic marker data were used for cluster analysis using NTSYSpc software. The genetic similarity among 228 genotypes was 0.0044. Eleven clusters in the dendrogram were formed and genotypes collected from North Carolina were spread into ten clusters suggesting that North Carolina naturalized population had large genetic variation. Plants arising from Virginia (VA) formed only three clusters indicating low genetic variation. Some EST-SSRs were monomorphic, one (SSR30-1) of such SSRs was used for sequencing to exam genetic variation at DNA sequence level due to its association with cellulose synthase gene. Genetic distance of among genotypes was calculated using the DNA sequence data of Miscanthus collected from each state using MEGA software. The highest mean of genetic distance 0.012 was found in the genotypes of North Carolina (NC) which was sampled from latitude N 35°33.010', longitude W 082°34.034' and elevation 571.5 m. The lowest mean of genetic distance 0.004 was observed in the Virginia (VA) population, sampled from latitude N 37.23, longitude W 80.21 and elevation 655.0152 m. On the basis of DNA sequence data, phylogenetic relationship was constructed among all genotypes using MEGA software. Miscanthus genotypes from North Carolina (NC) and Pennsylvania (PA) showed large variation spread across four groups, while genotypes from Virginia (VA) had the smallest variation and concentrated in one group. Both polymorphic marker data and sequence data showed large genetic variation in North Carolina, implying that genotypes might be collected from several different sources, while Virginia genotypes with small genetic variation may have come from single source.

PUBLICATIONS: 2014/09/01 TO 2015/08/31
1. Type: Conference Papers and Presentations Status: Accepted Year Published: 2015 Citation: Suma Basak, C.S. Prakash, Guohao He, Marceline Egnin and Erik Sacks* 2015 INSIGHT INTO THE GENETIC DIVERSITY AMONG NATURALIZED POPULATIONS OF MISCANTHUS, A BIOENERGY GRASS, USING GENOMIC TOOLS. Paper presented at Tuskegee University Joint Annual Research Symposium (JARS) to beheld on February 17-28, 2015
2. Type: Conference Papers and Presentations Status: Accepted Year Published: 2014 Citation: Suma Basak, C.S. Prakash, Guohao He, Marceline Egnin and Erik Sacks. 2014 GENETIC DIVERSITY AMONG BIOENERGY GRASS MISCANTHUS IN THE NATURALIZED POPULATIONS OF USA USING MOLECULAR MARKERS. Professional Agricultural Workers Conference, December 9, 2014. Tuskegee University
3. Type: Theses/Dissertations Status: Awaiting Publication Year Published: 2015 Citation: Basak, Suma. 2015. Genetic Diversity of Miscanthus in the US Naturalized Populations Based on Molecular Markers. Masters Thesis submitted to Tuskegee University
4. Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2014 Citation: Basak, S, C Prakash, G He, M Egnin & E Sacks, 2014. Genetic Diversity Among Bioenergy Grass Miscanthus In The Natural Populations of US Using Molecular Markers. George Washington Carver Legacy Symposium. April 23, 2014, Iowa State University, Ames, IA.
5. Type: Journal Articles Status: Published Year Published: 2015 Citation: Wang, Y., Zhang, X.G., Zhao, Y.L., Prakash, C.S., He, G.H., and Yin, D.M. (2015) A Large Diverse ?12-FADs Gene Family in Peanut: Insights into the novel genes of the FAD2 enzyme involved in high-oleate fluxes. Genome doi: 10.1139/gen-2015-0008 ePaper at http://www.nrcresearchpress.com/doi/abs/10.1139/gen-2015-0008?src=recsys#.VdN5E xNVhBd
6. Type: Journal Articles Status: Published Year Published: 2015 Citation: Ncube-Kanyika BTC Lungu D, Mweetwa AM, Kaimoyo E, Njung?e VM, Monyo ES, Siambi M, He G, Prakash CS, Zhao Y, De Villiers SM, 2015. Identification of groundnut SSR markers suitable for multiple resistance traits QTL mapping in African germplasm. Electronic Journal of Biotechnology 18(2): 61-67. http://dx.doi.org/10.1016/j.ejbt.2014.10.004

PROGRESS: 2013/09/01 TO 2014/08/31
Target Audience: Target audience include the scientific community, growers especially limited resource famrers in Black Belt region, graduate students, undergraduate students, high school students, along with scientists and technicians at Tuskegee University. This is primarily a research project but we involve students (both high school and college) in the program to train them in various research methodologies as outreach and teaching form an important component of our activitity Changes/Problems: Only major change in the project was the addition of a genetic diversity study of naturalized populations of Miscanthus across the Eastern US that we conducted in collaboration with University of Illinois. As ouMiscnathus population established in Tuskegee University was not yet ready for analysis in 2013 and 2014, we went to University of Illinois to collect the leaf samples from an experiment already established on their Urbana campus, and conducted a DNA marker study using sugarcane SSR markers. This provided unique insights into the nature of genetic diversity among various introduced lines of this ornamental plant that may help further in breeding of this grass for bioenergy. What opportunities for training and professional development has the project provided? We trained a graduate student and two undergraduate students in this project who learnt much about the field testing, experimental plot lay outs, field management, data observartion and analysis, DNA isolation, PCR analysis, scoring of polymorphic bands, and the use of computational biology software How have the results been disseminated to communities of interest? A conference paper was presented at the Professional Agricultural Workers Conference in Tuskegee, AL during December 2013 and also another paper was presented at the George Washington Carver Symposium in Ames, Iowa during April 2014. A masters thesis was developed from a graduate student's research from this project, and will be on public domain with open access. What do you plan to do during the next reporting period to accomplish the goals? We have continued to gather agronimic and other field data on the Miscanthus population established at Tuskegee from the five trials. We will also isoalte genomic DNA from the leaf samples of these lines, and pursue DNA marker studies to associate agronimic and bioenergy traits with select markers

IMPACT: 2013/09/01 TO 2014/08/31
What was accomplished under these goals? We conducted a genetic diversity study of natural, introduced population of this grass using DNA markers. A total of 228 Miscanthus genotypes were collected from six states (Ohio, North Carolina, Washington D.C., Kentucky, Pennsylvania, and Virginia). The genotypes collected from a state were considered as a naturalized population. Thus, six naturalized populations were analyzed to reveal the genetic diversity of Miscanthus among states and within state using sugarcane SSR markers because of lack of SSR markers in Miscanthus and close related species of Miscanthus and sugarcane. To this end, sugarcane DNA markers, including SSR markers and gene markers, were firstly tested for their transferability to Miscanthus. All fifty-three sugarcane DNA markers tested showed that expected size of bands were generated by these markers through PCR amplification, indicating high transferability of sugarcane DNA markers on 228 Miscanthus sinensis genotypes. As ost gene markers showing less polymorphism among Miscanthus genotypes, the dataset derived from polymorphic EST-SSR markers were used for the diversity study. As a result, genetic distance (GD) within each population was calculated to estimate the extent of their divergence using MEGA software. The highest genetic distance was 135.048 found in Pennsylvania (PA) state which was sampled from latitude N 39°57.814', longitude W 075°23.778' and elevation 92.0496 m. However, the lowest genetic distance (0.008) was observed in North Carolina 1.5 (NC1.5) and Kentucky (KY) genotypes. The North Carolina 1.5 (NC1.5) genotypes were sampled from latitude N 35°33.010', longitude W 082°34.034' and elevation 571.5 m and Kentucky (KY) genotypes from latitude N 37.8034, longitude W -83.6628 and elevation 1293 m. The result suggested that Miscanthus genotypes in PA had a large genetic variation while genotypes in NC and KY showed small variation. The average genetic similarity value among 228 genotypes was 0.0044 based on the PCR amplified bands data. Cluster analysis was performed using UPGMA method in NTSYSpc 2.21q software on the basis of genetic similarity matrix. Eleven distinct clusters were formed in the dendrogram and the results indicated that the genotypes (DC416, OH1412, NC1.52210, NC332 and PA1019) were clustered in a unique group whereas; the other genotypes were clustered together. Furthermore, the most genotypes produced the largest genetic similarity group located in X cluster, suggesting that the geological and various environmental conditions contributed to shaping the adaptation in Miscanthus. In non-polymorphic SSR markers, SSR alleles could be similar in length, but might be different in descent. To further understand genetic diversity among these Miscanthus genotypes, PCR products of 228 genotypes amplified by one of non-polymorphic EST-SSR 30-1 maker was sequenced. Sequence data showed a nucleotide variation among these genotypes. Phylogenetic relationships among all genotypes were constructed on the basis of amplified sequences using the MEGA5 software. The results indicated that the genotypes OH1310 and DC433 produced the most distinct groups and most of the genotypes from different states were mixed in other groups. These newly developed genic SSR markers in Miscanthus will be useful for further analyzing population genetics and evolutionary history of Miscanthus or its closely related species. Those markers also could be valuable for germplasm evaluation, genetic studies and molecular breeding of Miscanthus in US and will be helpful in choosing different genes or vigorous parents with respect to traits of interest for efficient breeding, selecting resistant and highly productive varieties for biofuel or ethanol production, tracking the potential genetic materials responsible for adaptation into different environmental situations. Moreover, the reliable information on the genetic diversity of Miscanthus naturalized populations can be generated by studying those markers that may be used as new tools for identifying varieties and will allow us to enhance our understanding of the distribution and extent of genetic variation within and among Miscanthus naturalized populations.

PUBLICATIONS: 2013/09/01 TO 2014/08/31
1. Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Basak, S, C Prakash, G He, M Egnin & E Sacks, 2014. Genetic Diversity Among Bioenergy Grass Miscanthus In The Natural Populations of US Using Molecular Markers. George Washington Carver Legacy Symposium. April 23, 2014, Iowa State University, Ames, IA.
2. Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: J. Davis*, Y. Zhao, C. S. Prakash, G. He,. 2013 Sequence diversity of cellulose synthase genes in Miscanthus. Proceedings of the Prof. Ag Workers Conference. December 2013. Tuskegee, AL
3. Type: Theses/Dissertations Status: Awaiting Publication Year Published: 2014 Citation: Basak, S. 2014. Genetic Diversity of Miscanthus in the US Naturalized Populations Based on Molecular Markers. Masters Thesis, under submission to Tuskegee University

PROGRESS: 2012/09/01 TO 2013/08/31
Target Audience: Target audience include graduate students, undergraduate students, high school students, scientists and technicians at Tuskegee University. This is primarily a research project but we involved students (both high school and college) in the program to train them in various research methodologies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? We are training one masters student who is conducting research under this project as a part of her masters dissertation. We have also involved many undergraduate and also high school students during the planting of this crop and thus enabling them to be trained in various areas such as field plot design, land preparation, and weed management in the bionernergy plantations. 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? We will soon conduct a transferability test where fifty-three sugarcane Simple Sequence Repeats primers will be used in a Polymerase Chain Reaction amplification ofMiscanthusDNAs. DNA marker polymorphism data and gene sequence data will be used to perform cluster analysis and phylogenetic analysis.

IMPACT: 2012/09/01 TO 2013/08/31
What was accomplished under these goals? In collaboration with Dr. Erik Saks and colleagues at the Institute of Genomic Biology and Energy Biosciences Institute (University of Illinois, Urbana-Champaign), we established a series of repicated trials of Miscanthusand other bionenergy grasses at the experimental farms of the Tuskegee University.In addition to Miscanthus, the test plots also contained studies of energy cane, sugar cane and Miscane. Details of these studies: Mxg Study: To identify potential new cultivars of triploid M. xgiganteus that are high yielding and adapted to different U.S. environments. Seven new genotypes of M. xgiganteus (4 from a cross between selected parents and 3 from seed collected in the wild in Japan) are being compared with the commercial standard clone at five locations, including Urbana, IL, Dixon Springs, IL, Jonesboro, AR, Mills River, NC, and Tuskegee, AL. DOE Study: To quantify genotype, environment and GxE effects for M. sinensis. The 169 genotypes in this planting are also being evaluated in Urbana, IL, Boulder, CO, Canada, Japan, Korea and China. The set grown in the U.S. is a subset of those grown ex-U.S. This study will allow us to conduct association analyses to identify markers associated with key yield and adaptation traits. Ogi Study: Tetraploid M. sacchariflorus, known as Ogi in Japan, are a key parent for making triploid M. xgiganteus. The goal of this study is to identify the best genotypes for use as parents. Height, yield and flowering time will be evaluated among 50 entries at Tuskegee, AL, Dixon Springs, IL and Urbana, IL. Southern Trial: To compare overwintering ability of a set of 9 Miscanthus lines that are adapted to subtropical environments at three locations that vary in severity of winter: Tuskegee, AL, Dixon Springs, IL and Urbana, IL. Cane Trial: To compare growth and overwintering ability of different sugarcane, energycane and Miscane genotypes at Tuskegee, AL and Dixon Springs, IL. Total 15 entries. Table showing the details of five trials of bionenergy grass at Tuskegee University Trial Name Location Width (ft) Tall (ft) # Entries Area (sq ft) Area (acres) # Border plants Cane Gp2 Trial-2013 AL 51 132 15 6732 0.15 92 DOE 2013 Trial AL 74 266 169 19684 0.45 134 Mxg 2013 AL 174 129 14 22446 0.52 180 Ogi Trial 2013 AL 72 132 50 6372 0.15 256 Southern Misc-2013 AL 33 72 9 2376 0.05 92 We also initiated a study aimed at testing the genetic adaptation of Miscanthus to changing environments in the US., to explore the genetic variation within and among nature populations. The specific objectives of this research are to (a) test the transferability of sugarcane DNA markers in the genome of Miscanthus (b) determine genetic diversity within and among natural populations on the basis of sugarcane molecular markers. (c) conduct phylogenetic analysis of Miscanthus natural populations in the US using DNA sequences of cellulose synthase and disease resistance genes. Samples collection: a total of two hundred thirty six leaf samples were collected from six natural populations (OH, NC, DC, KY, PA and VA) of Miscanthus from the University of Illinois at Urbana Champaign. DNA isolation: DNA of Miscanthus was extracted from young leaf samples using the modified CTAB DNA extraction protocol,

PUBLICATIONS: 2012/09/01 TO 2013/08/31
No publications reported this period.