Setaria genome sequencing: an overview

The genus Setaria includes two important C₄ Panicoid grass species, namely S. italica (cultivated) and S. viridis (weed; wild ancestor), which together represent an appropriate model system for architectural, physiological, evolutionary, and genomic studies in related grasses. It is a diploid, inbreeder, self-fertile annual cereal grass having short life cycle and minimal growth requirements. There close relatedness to biofuel crops like switch grass and napier grass further signifies their importance. Further, foxtail millet is an important food and fodder grain crop grown in arid and semi-arid regions in many parts of the world. Therefore, an increasing interest in these species has led to a gradual accumulation and development of genomic data and genetic resources. Setaria genome sequencing is an outcome of such endeavors. These sequencing efforts uncovered several distinctive attributes of Setaria genome that may help in understanding its physiology, evolution and adaptation. This will not only aid in comparative genomics studies of Setaria and related crops including bioenergy grasses but also help in rapid advancements of genomics information for developing varieties with superior traits either through marker-assisted selection (MAS) or using transgenic approaches in these crops.     

Population genetics of Setaria viridis,a new model system

An extensive survey of the standing genetic variation in natural populations is among the priority steps in developing a species into a model system. In recent years, green foxtail (Setaria viridis), along with its domesticated form foxtail millet (S. italica), has rapidly become a promising new model system for C₄ grasses and bioenergy crops, due to its rapid life cycle, large amount of seed production and small diploid genome, among other characters. However, remarkably little is known about the genetic diversity in natural populations of this species. In this study, we survey the genetic diversity of a worldwide sample of more than 200 S. viridis accessions, using the genotyping‐by‐sequencing technique. Two distinct genetic groups in S. viridis and a third group resembling S. italica were identified, with considerable admixture among the three groups. We find the genetic variation of North American S. viridis correlates with both geography and climate and is representative of the total genetic diversity in this species. This pattern may reflect several introduction/dispersal events of S. viridis into North America. We also modelled demographic history and show signal of recent population decline in one subgroup. Finally, we show linkage disequilibrium decay is rapid (<45 kb) in our total sample and slow in genetic subgroups. These results together provide an in‐depth understanding of the pattern of genetic diversity of this new model species on a broad geographic scale. They also provide key guidelines for on‐going and future work including germplasm preservation, local adaptation, crossing designs and genomewide association studies.     

Methods for Performing Crosses in Setaria viridis,a New Model System for the Grasses

Setaria viridis is an emerging model system for C₄ grasses. It is closely related to the bioenergy feed stock switchgrass and the grain crop foxtail millet. Recently, the 510 Mb genome of foxtail millet, S. italica, has been sequenced ^1,2 and a 25x coverage genome sequence of the weedy relative S. viridis is in progress. S. viridis has a number of characteristics that make it a potentially excellent model genetic system including a rapid generation time, small stature, simple growth requirements, prolific seed production ³ and developed systems for both transient and stable transformation ⁴. However, the genetics of S. viridis is largely unexplored, in part, due to the lack of detailed methods for performing crosses. To date, no standard protocol has been adopted that will permit rapid production of seeds from controlled crosses.The protocol presented here is optimized for performing genetic crosses in S. viridis, accession A10.1. We have employed a simple heat treatment with warm water for emasculation after pruning the panicle to retain 20-30 florets and labeling of flowers to eliminate seeds resulting from newly developed flowers after emasculation. After testing a series of heat treatments at permissive temperatures and varying the duration of dipping, we have established an optimum temperature and time range of 48 °C for 3-6 min. By using this method, a minimum of 15 crosses can be performed by a single worker per day and an average of 3-5 outcross progeny per panicle can be recovered. Therefore, an average of 45-75 outcross progeny can be produced by one person in a single day. Broad implementation of this technique will facilitate the development of recombinant inbred line populations of S. viridis X S. viridis or S. viridis X S. italica, mapping mutations through bulk segregant analysis and creating higher order mutants for genetic analysis.     

A role for differential Rubisco activase isoform expression in C4 bioenergy grasses at high temperature

Rubisco activase (Rca) facilitates the release of sugar‐phosphate inhibitors at Rubisco catalytic sites during CO₂ fixation. Most plant species express two Rca isoforms, the larger Rca‐α and the shorter Rca‐β, either by alternative splicing from a single gene or expression from separate genes. The mechanism of Rubisco activation by Rca isoforms has been intensively studied in C₃ plants. However, the functional role of Rca in C₄ plants where Rubisco and Rca are located in a much higher [CO₂] compartment is less clear. In this study, we selected four C₄ bioenergy grasses and the model C₄ grass setaria (Setaria viridis) to investigate the role of Rca in C₄ photosynthesis. All five C₄ grass species contained two Rca genes, one encoding Rca‐α and the other Rca‐β, which were positioned closely together in the genomes. A variety of abiotic stress‐related motifs were identified in the Rca‐α promoter of each grass, and while the Rca‐β gene was constantly highly expressed at ambient temperature, Rca‐α isoforms were expressed only at high temperature but never surpassed 30% of Rca‐β content. The pattern of Rca‐α induction on transition to high temperature and reduction on return to ambient temperature was the same in all five C₄ grasses. In sorghum (Sorghum bicolor), sugarcane (Saccharum officinarum), and setaria, the induction rate of Rca‐α was similar to the recovery rate of photosynthesis and Rubisco activation at high temperature. This association between Rca‐α isoform expression and maintenance of Rubisco activation at high temperature suggests that Rca‐α has a functional thermo‐protective role in carbon fixation in C₄ grasses by sustaining Rubisco activation at high temperature.     

Evolution of the C4 photosynthetic pathway: events at the cellular and molecular levels

The biochemistry and leaf anatomy of plants using C₄ photosynthesis promote the concentration of atmospheric CO₂ in leaf tissue that leads to improvements in growth and yield of C₄ plants over C₃ species in hot, dry, high light, and/or saline environments. C₄ plants like maize and sugarcane are significant food, fodder, and bioenergy crops. The C₄ photosynthetic pathway is an excellent example of convergent evolution, having evolved in multiple independent lineages of land plants from ancestors employing C₃ photosynthesis. In addition to C₃ and C₄ species, some plant lineages contain closely related C₃–C₄ intermediate species that demonstrate leaf anatomical, biochemical, and physiological characteristics between those of C₃ plants and species using C₄ photosynthesis. These groups of plants have been extremely useful in dissecting the modifications to leaf anatomy and molecular biology, which led to the evolution of C₄ photosynthesis. It is now clear that great variation exists in C₄ leaf anatomy, and diverse molecular mechanisms underlie C₄ biochemistry and physiology. However, all these different paths have led to the same destination—the expression of a C₄ CO₂ concentrating mechanism. Further identification of C₄ leaf anatomical traits and molecular biological components, and understanding how they are controlled and assembled will not only allow for additional insights into evolutionary convergence, but also contribute to sustainable food and bioenergy production strategies.     

Getting the most out of natural variation in C4 photosynthesis

C₄ photosynthesis is a complex trait that has a high degree of natural variation, involving anatomical and biochemical changes relative to the ancestral C₃ state. It has evolved at least 66 times across a variety of lineages and the evolutionary route from C₃ to C₄ is likely conserved but not necessarily genetically identical. As such, a variety of C₄ species are needed to identify what is fundamental to the C₄ evolutionary process in a global context. In order to identify the genetic components of C₄ form and function, a number of species are used as genetic models. These include Zea mays (maize), Sorghum bicolor (sorghum), Setaria viridis (Setaria), Flaveria bidentis, and Cleome gynandra. Each of these species has different benefits and challenges associated with its use as a model organism. Here, we propose that RNA profiling of a large sampling of C₄, C₃–C₄, and C₃ species, from as many lineages as possible, will allow identification of candidate genes necessary and sufficient to confer C₄ anatomy and/or biochemistry. Furthermore, C₄ model species will play a critical role in the functional characterization of these candidate genes and identification of their regulatory elements, by providing a platform for transformation and through the use of gene expression profiles in mesophyll and bundle sheath cells and along the leaf developmental gradient. Efforts should be made to sequence the genomes of F. bidentis and C. gynandra and to develop congeneric C₃ species as genetic models for comparative studies. In combination, such resources would facilitate discovery of common and unique C₄ regulatory mechanisms across genera.     

Efficient genome editing and gene knockout in Setaria viridis with CRISPR/Cas9 directed gene editing by the non-homologous end-joining pathway

The CRISPR/Cas9 system has been used for genome editing in several organisms, including higher plants. This system induces site-specific mutations in the genome based on the nucleotide sequence of engineered guide RNAs. The complex genomes of C4 grasses makes genome editing a challenge in key grass crops like maize (Zea mays), sorghum (Sorghum bicolor), Brachiaria spp., switchgrass (Panicum virgatum), and sugarcane (Saccharum spp.). Setaria viridis is a diploid C4 grass widely used as a model for these C4 crop plants. Here, an optimized CRISPR/Cas9 binary vector that exploits the non-homologous end joining (NHEJ) system was used to knockout a green fluorescent protein (gfp) transgene in S. viridis accession A10.1. Transformation of embryogenic callus by A. tumefaciens generated ten glufosinate-ammonium resistant transgenic events. In the T0 generation, 60% of the events were biallelic mutants in the gfp transgene with no detectable accumulation of GFP protein and without insertions or deletions in predicted off-target sites. The gfp mutations generated by CRISPR/Cas9 were stable and displayed Mendelian segregation in the T1 generation. Altogether, the system described here is a highly efficient genome editing system for S. viridis, an important model plant for functional genomics studies in C4 grasses. Also, this system is a potential tool for improvement of agronomic traits in C4 crop plants with complex genomes.     

Foxtail millet: a model crop for genetic and genomic studies in bioenergy grasses

Foxtail millet is one of the oldest domesticated diploid C₄ Panicoid crops having a comparatively small genome size of approximately 515?Mb, short life cycle, and inbreeding nature. Its two species, Setaria italica (domesticated) and Setaria viridis (wild progenitor), have characteristics that classify them as excellent model systems to examine several aspects of architectural, evolutionary, and physiological importance in Panicoid grasses especially the biofuel crops such as switchgrass and napiergrass. Foxtail millet is a staple crop used extensively for food and fodder in parts of Asia and Africa. In its long history of cultivation, it has been adapted to arid and semi-arid areas of Asia, North Africa, South and North America. Foxtail millet has one of the largest collections of cultivated as well as wild-type germplasm rich with phenotypic variations and hence provides prospects for association mapping and allele-mining of elite and novel variants to be incorporated in crop improvement programs. Most of the foxtail millet accessions can be primarily abiotic stress tolerant particularly to drought and salinity, and therefore exploiting these agronomic traits can enhance its efficacy in marker-aided breeding as well as in genetic engineering for abiotic stress tolerance. In addition, the release of draft genome sequence of foxtail millet would be useful to the researchers worldwide in not only discerning the molecular basis of biomass production in biofuel crops and the methods to improve it, but also for the introgression of beneficial agronomically important characteristics in foxtail millet as well as in related Panicoid bioenergy grasses.     

Faster induction of photosynthesis increases biomass and grain yield in glasshouse-grown transgenic Sorghum bicolor overexpressing Rieske FeS

Sorghum is one of the most important crops providing food and feed in many of the world's harsher environments. Sorghum utilizes the C₄ pathway of photosynthesis in which a biochemical carbon-concentrating mechanism results in high CO₂ assimilation rates. Overexpressing the Rieske FeS subunit of the Cytochrome b₆f complex was previously shown to increase the rate of photosynthetic electron transport and stimulate CO₂ assimilation in the model C₄ plant Setaria viridis. To test whether productivity of C₄ crops could be improved by Rieske overexpression, we created transgenic Sorghum bicolor Tx430 plants with increased Rieske content. The transgenic plants showed no marked changes in abundances of other photosynthetic proteins or chlorophyll content. The steady-state rates of electron transport and CO₂ assimilation did not differ between the plants with increased Rieske abundance and control plants, suggesting that Cytochrome b₆f is not the only factor limiting electron transport in sorghum at high light and high CO₂. However, faster responses of non-photochemical quenching as well as an elevated quantum yield of Photosystem II and an increased CO₂ assimilation rate were observed from the plants overexpressing Rieske during the photosynthetic induction, a process of activation of photosynthesis upon the dark–light transition. As a consequence, sorghum with increased Rieske content produced more biomass and grain when grown in glasshouse conditions. Our results indicate that increasing Rieske content has potential to boost productivity of sorghum and other C₄ crops by improving the efficiency of light utilization and conversion to biomass through the faster induction of photosynthesis.     

Differences in drought sensitivities and photosynthetic limitations between co-occurring C3 and C4 (NADP-ME) Panicoid grasses

Background and Aims

The success of C₄ plants lies in their ability to attain greater efficiencies of light, water and nitrogen use under high temperature, providing an advantage in arid, hot environments. However, C₄ grasses are not necessarily less sensitive to drought than C₃ grasses and are proposed to respond with greater metabolic limitations, while the C₃ response is predominantly stomatal. The aims of this study were to compare the drought and recovery responses of co-occurring C₃ and C₄ NADP-ME grasses from the subfamily Panicoideae and to determine stomatal and metabolic contributions to the observed response.

Methods

Six species of locally co-occurring grasses, C₃ species Alloteropsis semialata subsp. eckloniana, Panicum aequinerve and Panicum ecklonii, and C₄ (NADP-ME) species Heteropogon contortus, Themeda triandra and Tristachya leucothrix, were established in pots then subjected to a controlled drought followed by re-watering. Water potentials, leaf gas exchange and the response of photosynthetic rate to internal CO₂ concentrations were determined on selected occasions during the drought and re-watering treatments and compared between species and photosynthetic types.

Key Results

Leaves of C₄ species of grasses maintained their photosynthetic advantage until water deficits became severe, but lost their water-use advantage even under conditions of mild drought. Declining C₄ photosynthesis with water deficit was mainly a consequence of metabolic limitations to CO₂ assimilation, whereas, in the C₃ species, stomatal limitations had a prevailing role in the drought-induced decrease in photosynthesis. The drought-sensitive metabolism of the C₄ plants could explain the observed slower recovery of photosynthesis on re-watering, in comparison with C₃ plants which recovered a greater proportion of photosynthesis through increased stomatal conductance.

Conclusions

Within the Panicoid grasses, C₄ (NADP-ME) species are metabolically more sensitive to drought than C₃ species and recover more slowly from drought.     

Bioenergy grass feedstock: current options and prospects for trait improvement using emerging genetic, genomic, and systems biology toolkits

For lignocellulosic bioenergy to become a viable alternative to traditional energy production methods, rapid increases in conversion efficiency and biomass yield must be achieved. Increased productivity in bioenergy production can be achieved through concomitant gains in processing efficiency as well as genetic improvement of feedstock that have the potential for bioenergy production at an industrial scale. The purpose of this review is to explore the genetic and genomic resource landscape for the improvement of a specific bioenergy feedstock group, the C4 bioenergy grasses. First, bioenergy grass feedstock traits relevant to biochemical conversion are examined. Then we outline genetic resources available bioenergy grasses for mapping bioenergy traits to DNA markers and genes. This is followed by a discussion of genomic tools and how they can be applied to understanding bioenergy grass feedstock trait genetic mechanisms leading to further improvement opportunities.     

The photosynthetic response of C3 and C4 bioenergy grass species to fluctuating light

Bioenergy grass species are a renewable energy source, but their productivity has not been fully realized. Improving photosynthetic efficiency has been proposed as a mechanism to increase the productivity of bioenergy grass species. Fluctuating light, experienced by all field grown crops, is known to reduce photosynthetic efficiency. This experiment aimed to evaluate the photosynthetic performance of both C₃ and C₄ bioenergy grass species under steady state and fluctuating light conditions by examining leaf gas exchange. The fluctuating light regime used here decreased carbon assimilation across all species when compared to expected steady state values. Overall, C₄ species assimilated more carbon than C₃ species during the fluctuating light regime, with both photosynthetic types assimilating about 16% less carbon than expected based on steady state measurements. Little diversity was observed in response to fluctuating light among C₃ species, and photorespiration partially contributed to the rapid decreases in net photosynthetic rates during high to low light transitions. In C₄ species, differences among the four NADP-ME species were apparent. Diversity observed among C₄ species in this experiment provides evidence that photosynthetic efficiency in response to fluctuating light may be targeted to increase C₄ bioenergy grass productivity.     

Kinetic variation in grass phosphoenolpyruvate carboxylases provides opportunity to enhance C4 photosynthetic efficiency

The high rates of photosynthesis and the carbon-concentrating mechanism (CCM) in C₄ plants are initiated by the enzyme phosphoenolpyruvate (PEP) carboxylase (PEPC). The flow of inorganic carbon into the CCM of C₄ plants is driven by PEPC’s affinity for bicarbonate (K_HCO3), which can be rate limiting when atmospheric CO₂ availability is restricted due to low stomatal conductance. We hypothesize that natural variation in K_HCO3 across C₄ plants is driven by specific amino acid substitutions to impact rates of C₄ photosynthesis under environments such as drought that restrict stomatal conductance. To test this hypothesis, we measured K_HCO3 from 20 C₄ grasses to compare kinetic properties with specific amino acid substitutions. There was nearly a twofold range in K_HCO3 across these C₄ grasses (24.3 ± 1.5 to 46.3 ± 2.4 μm ), which significantly impacts modeled rates of C₄ photosynthesis. Additionally, molecular engineering of a low-HCO₃⁻ affinity PEPC identified key domains that confer variation in K_HCO3. This study advances our understanding of PEPC kinetics and builds the foundation for engineering increased-HCO₃⁻ affinity and C₄ photosynthetic efficiency in important C₄ crops.     

Potential of Plants from the Genus Agave as Bioenergy Crops

Agave is a succulent genus within the monocot family Agavaceae. The plants have a large rosette of thick fleshy leaves, each ending generally in a sharp point, and are native to arid and semi-arid regions from the southern USA to northern South America. The most important commercial species is Agave tequilana grown for production of tequila. Several cultivated species of Agave such as Agave sislana and Agave salmiana can perform well in areas where rainfall is insufficient for the cultivation of many C₃ and C₄ crops. The key feature of the crassulacean acid metabolism photosynthetic pathway used by agaves is the stomata opening and CO₂ uptake during the night, thus allowing less water to be lost by transpiration. Alcoholic beverages, sweeteners, fibers, and some specialty chemicals are currently the main products coming from agave plants. The recovered information related to productivity, biofuel processability, by-products, etc. suggests that some Agave species have a real potential to compete economically with other bioenergy crops. But more than compete, it could complement the list of bioenergy crops due to its capacity to grow with very little rainfall and/or inputs and still reach good amount of biomass, so unused semi-arid land could be productive. Although Agave has great potential to be developed as a bioenergy crop, more laboratory and field research are needed.     

Can perennial C4 grasses attain high efficiencies of radiant energy conversion in cool climates?

Although C₄ plants are considered to have higher conversion efficiency and productivity than C₃, plants, this advantage may not be realized under sub-optimal conditions. Two perennial C₄ rhizomatous grasses of cool temperate origin, Miscanthus × giganteus and Spartina cyno-suroides, have been suggested as potential fuel crops for north-western Europe. The conversion efficiencies of these species were examined for 2 years in fertilized, irrigated, replicated plots in south-eastern England. In the second year, the energy conversion efficiencies for shoot and total biomass production were 0.040 and 0.051, respectively, for S. cynosuroides, with significantly higher values of 0.060 and 0.078, respectively, for M. × giganteus. The M. × giganteus crop attained shoot productivity of 2.87 kg m^?2 between April and September, exceeding the highest values typically obtained with intensively managed C₃, crops. Canopy development was early, and high interception and conversion efficiencies were maintained over most of the growing season. This study provides the evidence that the superior potential light conversion efficiencies associated with C₄ photosynthesis can be realized under cool temperate conditions and that such climatic conditions do not Inherently impair the C₄ process.     

Functional group and fertilization affect the composition and bioenergy yields of prairie plants

Prairies used for bioenergy production have potential to generate marketable products while enhancing environmental quality, but little is known about how prairie species composition and nutrient management affect the suitability of prairie biomass for bioenergy production. We determined how functional‐group identity and nitrogen fertilization affected feedstock characteristics and estimated bioenergy yields of prairie plants, and compared those prairie characteristics to that of corn stover. We tested our objectives with a field experiment that was set up as a 5 × 2 incomplete factorial design with C₃ grasses, C₄ grasses, legumes, and multi‐functional‐group mixtures grown with and without nitrogen fertilizer; a fertilized corn treatment was also included. We determined cell wall, hemicellulose, cellulose, and ash concentrations; ethanol conversion ratios; gross caloric ratios; aboveground biomass production; ethanol yields; and energy yields for all treatments. Prairie functional‐group identity affected the biomass feedstock characteristics, whereas nitrogen fertilization did not. Functional group and fertilization had a strong effect on aboveground biomass production, which was the major predictor of ethanol and energy yields. C₄ grasses, especially when fertilized, had among the most favorable bioenergy characteristics with high estimated ethanol conversion ratios and nongrain biomass production, relatively high gross caloric ratios, and low ash concentrations. The bioenergy characteristics of corn stover, from an annual C₄ grass, were similar to those of the biomass of perennial C₄ grasses. Both functional‐group composition and nitrogen fertility management were found to be important in optimizing bioenergy production from prairies.     

Carbon implications of converting cropland to bioenergy crops or forest for climate mitigation: a global assessment

The potential for climate change mitigation by bioenergy crops and terrestrial carbon sinks has been the object of intensive research in the past decade. There has been much debate about whether energy crops used to offset fossil fuel use, or carbon sequestration in forests, would provide the best climate mitigation benefit. Most current food cropland is unlikely to be used for bioenergy, but in many regions of the world, a proportion of cropland is being abandoned, particularly marginal croplands, and some of this land is now being used for bioenergy. In this study, we assess the consequences of land‐use change on cropland. We first identify areas where cropland is so productive that it may never be converted and assess the potential of the remaining cropland to mitigate climate change by identifying which alternative land use provides the best climate benefit: C₄ grass bioenergy crops, coppiced woody energy crops or allowing forest regrowth to create a carbon sink. We do not present this as a scenario of land‐use change – we simply assess the best option in any given global location should a land‐use change occur. To do this, we use global biomass potential studies based on food crop productivity, forest inventory data and dynamic global vegetation models to provide, for the first time, a global comparison of the climate change implications of either deploying bioenergy crops or allowing forest regeneration on current crop land, over a period of 20 years starting in the nominal year of 2000 ad . Globally, the extent of cropland on which conversion to energy crops or forest would result in a net carbon loss, and therefore likely always to remain as cropland, was estimated to be about 420.1 Mha, or 35.6% of the total cropland in Africa, 40.3% in Asia and Russia Federation, 30.8% in Europe‐25, 48.4% in North America, 13.7% in South America and 58.5% in Oceania. Fast growing C₄ grasses such as Miscanthus and switch‐grass cultivars are the bioenergy feedstock with the highest climate mitigation potential. Fast growing C₄ grasses such as Miscanthus and switch‐grass cultivars provide the best climate mitigation option on ≈485 Mha of cropland worldwide with ~42% of this land characterized by a terrain slope equal or above 20%. If that land‐use change did occur, it would displace ≈58.1 Pg fossil fuel C equivalent (C_eq oil). Woody energy crops such as poplar, willow and Eucalyptus species would be the best option on only 2.4% (≈26.3 Mha) of current cropland, and if this land‐use change occurred, it would displace ≈0.9 Pg C_eq oil. Allowing cropland to revert to forest would be the best climate mitigation option on ≈17% of current cropland (≈184.5 Mha), and if this land‐use change occurred, it would sequester ≈5.8 Pg C in biomass in the 20‐year‐old forest and ≈2.7 Pg C in soil. This study is spatially explicit, so also serves to identify the regional differences in the efficacy of different climate mitigation options, informing policymakers developing regionally or nationally appropriate mitigation actions.     

Grass Invasion into Switchgrass Managed for Biomass Energy

Switchgrass (Panicum virgatum) is a C₄ perennial grass and is the model herbaceous perennial bioenergy feedstock. Although it is indigenous to North American grasslands east of the Rocky Mountains and has been planted for forage and conservation purposes for more than 75 years, there is concern that switchgrass grown as a biofuel crop could become invasive. Our objective is to report on the invasion of C₄ and C₃ grasses into the stands of two switchgrass cultivars following 10 years of management for biomass energy under different N and harvest management regimes in eastern Nebraska. Switchgrass stands were invaded by big bluestem (Andropogon gerardii), smooth bromegrass (Bromus inermis), and other grasses during the 10 years. The greatest invasion by grasses occurred in plots to which 0 N had been applied and with harvests at anthesis. In general, less grass encroachment occurred in plots receiving at least 60 kg of N ha^?1 or in plots harvested after frost. There were differences among cultivars with Cave-in-Rock being more resistant to invasion than Trailblazer. There was no observable evidence of switchgrass from this study invading into border areas or adjacent fields after 10 years of management for biomass energy. Results indicate that switchgrass is more likely to be invaded by other grasses than to encroach into native prairies or perennial grasslands seeded on marginally productive cropland in the western Corn Belt of the USA.