Economic and Sustainability Impacts of Yield and Composition Variation in Bioenergy Crops: Switchgrass (Panicum virgatum L.).

Economically viable production of biobased products and fuels requires high-yielding, high-quality, sustainable process-advantaged crops, developed using bioengineering or advanced breeding approaches. Understanding which crop phenotypic traits have the largest impact on biofuel economics and sustainability outcomes is important for the targeted feedstock crop development. Here, we evaluated biomass yield and cell-wall composition traits across a large natural variant population of switchgrass (Panicum virgatum L.) grown across three common garden sites. Samples from 331 switchgrass genotypes were collected and analyzed for carbohydrate and lignin components. Considering plant survival and biomass after multiple years of growth, we found that 84 of the genotypes analyzed may be suited for commercial production in the southeastern U.S. These genotypes show a range of growth and compositional traits across the population that are apparently independent of each other. We used these data to conduct techno-economic analyses and life cycle assessments evaluating the performance of each switchgrass genotype under a standard cellulosic ethanol process model with pretreatment, added enzymes, and fermentation. We find that switchgrass yield per area is the largest economic driver of the minimum fuel selling price (MSFP), ethanol yield per hectare, global warming potential (GWP), and cumulative energy demand (CED). At any yield, the carbohydrate content is significant but of secondary importance. Water use follows similar trends but has more variability due to an increased dependence on the biorefinery model. Analyses presented here highlight the primary importance of plant yield and the secondary importance of carbohydrate content when selecting a feedstock that is both economical and sustainable.

A single amino acid change led to structural and functional differentiation of PvHd1 to control flowering in switchgrass.

Switchgrass, a forage and bioenergy crop, occurs as two main ecotypes with different but overlapping ranges of adaptation. The two ecotypes differ in a range of characteristics, including flowering time. Flowering time determines the duration of vegetative development and therefore biomass accumulation, a key trait in bioenergy crops. No causal variants for flowering time differences between switchgrass ecotypes have, as yet, been identified. In this study, we mapped a robust flowering time quantitative trait locus (QTL) on chromosome 4K in a biparental F2 population and characterized the flowering-associated transcription factor gene PvHd1, an ortholog of CONSTANS in Arabidopsis and Heading date 1 in rice, as the underlying causal gene. Protein modeling predicted that a serine to glycine substitution at position 35 (p.S35G) in B-Box domain 1 greatly altered the global structure of the PvHd1 protein. The predicted variation in protein compactness was supported in vitro by a 4 °C shift in denaturation temperature. Overexpressing the PvHd1-p.35S allele in a late-flowering CONSTANS-null Arabidopsis mutant rescued earlier flowering, whereas PvHd1-p.35G had a reduced ability to promote flowering, demonstrating that the structural variation led to functional divergence. Our findings provide us with a tool to manipulate the timing of floral transition in switchgrass cultivars and, potentially, expand their cultivation range.

Genome analyses reveal population structure and a purple stigma color gene candidate in finger millet.

Finger millet is a key food security crop widely grown in eastern Africa, India and Nepal. Long considered a 'poor man's crop', finger millet has regained attention over the past decade for its climate resilience and the nutritional qualities of its grain. To bring finger millet breeding into the 21st century, here we present the assembly and annotation of a chromosome-scale reference genome. We show that this ~1.3 million years old allotetraploid has a high level of homoeologous gene retention and lacks subgenome dominance. Population structure is mainly driven by the differential presence of large wild segments in the pericentromeric regions of several chromosomes. Trait mapping, followed by variant analysis of gene candidates, reveals that loss of purple coloration of anthers and stigma is associated with loss-of-function mutations in the finger millet orthologs of the maize R1/B1 and Arabidopsis GL3/EGL3 anthocyanin regulatory genes. Proanthocyanidin production in seed is not affected by these gene knockouts.

Identification of microRNAs responsive to arbuscular mycorrhizal fungi in Panicum virgatum (switchgrass).

BACKGROUND: MicroRNAs (miRNAs) are important post-transcriptional regulators involved in the control of a range of processes, including symbiotic interactions in plants. MiRNA involvement in arbuscular mycorrhizae (AM) symbiosis has been mainly studied in model species, and our study is the first to analyze global miRNA expression in the roots of AM colonized switchgrass (Panicum virgatum), an emerging biofuel feedstock. AM symbiosis helps plants gain mineral nutrition from the soil and may enhance switchgrass biomass production on marginal lands. Our goals were to identify miRNAs and their corresponding target genes that are controlling AM symbiosis in switchgrass. RESULTS: Through genome-wide analysis of next-generation miRNA sequencing reads generated from switchgrass roots, we identified 122 mature miRNAs, including 28 novel miRNAs. By comparing miRNA expression profiles of AM-inoculated and control switchgrass roots, we identified 15 AM-responsive miRNAs across lowland accession "Alamo", upland accession "Dacotah", and two upland/lowland F1 hybrids. We used degradome sequencing to identify target genes of the AM-responsive miRNAs revealing targets of miRNAs residing on both K and N subgenomes. Notably, genes involved in copper ion binding were targeted by downregulated miRNAs, while upregulated miRNAs mainly targeted GRAS family transcription factors. CONCLUSION: Through miRNA analysis and degradome sequencing, we revealed that both upland and lowland switchgrass genotypes as well as upland-lowland hybrids respond to AM by altering miRNA expression. We demonstrated complex GRAS transcription factor regulation by the miR171 family, with some miR171 family members being AM responsive while others remained static. Copper miRNA downregulation was common amongst the genotypes tested and we identified superoxide dismutases and laccases as targets, suggesting that these Cu-miRNAs are likely involved in ROS detoxification and lignin deposition, respectively. Other prominent targets of the Cu miRNAs were blue copper proteins. Overall, the potential effect of AM colonization on lignin deposition pathways in this biofuel crop highlights the importance of considering AM and miRNA in future biofuel crop development strategies.

A high-density linkage map of finger millet provides QTL for blast resistance and other agronomic traits.

Finger millet [Eleusine coracana (L.) Gaertn.] is a critical subsistence crop in eastern Africa and southern Asia but has few genomic resources and modern breeding programs. To aid in the understanding of finger millet genomic organization and genes underlying disease resistance and agronomically important traits, we generated a F2:3 population from a cross between E. coracana (L.) Gaertn. subsp. coracana accession ACC 100007 and E. coracana (L.) Gaertn. subsp. africana , accession GBK 030647. Phenotypic data on morphology, yield, and blast (Magnaporthe oryzae) resistance traits were taken on a subset of the F2:3 population in a Kenyan field trial. The F2:3 population was genotyped via genotyping-by-sequencing (GBS) and the UGbS-Flex pipeline was used for sequence alignment, nucleotide polymorphism calling, and genetic map construction. An 18-linkage-group genetic map consisting of 5,422 markers was generated that enabled comparative genomic analyses with rice (Oryza sativa L.), foxtail millet [Setaria italica (L.) P. Beauv.], and sorghum [Sorghum bicolor (L.) Moench]. Notably, we identified conserved acrocentric homoeologous chromosomes (4A and 4B in finger millet) across all species. Significant quantitative trait loci (QTL) were discovered for flowering date, plant height, panicle number, and blast incidence and severity. Sixteen putative candidate genes that may underlie trait variation were identified. Seven LEUCINE-RICH REPEAT-CONTAINING PROTEIN genes, with homology to nucleotide-binding site leucine-rich repeat (NBS-LRR) disease resistance proteins, were found on three chromosomes under blast resistance QTL. This high-marker-density genetic map provides an important tool for plant breeding programs and identifies genomic regions and genes of critical interest for agronomic traits and blast resistance.

Quantitative trait locus mapping combined with variant and transcriptome analyses identifies a cluster of gene candidates underlying the variation in leaf wax between upland and lowland switchgrass ecotypes.

KEY MESSAGE: Mapping combined with expression and variant analyses in switchgrass, a crop with complex genetics, identified a cluster of candidate genes for leaf wax in a fast-evolving region of chromosome 7K. Switchgrass (Panicum virgatum L.) is a promising warm-season candidate energy crop. It occurs in two ecotypes, upland and lowland, which vary in a number of phenotypic traits, including leaf glaucousness. To initiate trait mapping, two F2 mapping populations were developed by crossing two different F1 sibs derived from a cross between the tetraploid lowland genotype AP13 and the tetraploid upland genotype VS16, and high-density linkage maps were generated. Quantitative trait locus (QTL) analyses of visually scored leaf glaucousness and of hydrophobicity of the abaxial leaf surface measured using a drop shape analyzer identified highly significant colocalizing QTL on chromosome 7K (Chr07K). Using a multipronged approach, we identified a cluster of genes including Pavir.7KG077009, which encodes a Type III polyketide synthase-like protein, and Pavir.7KG013754 and Pavir.7KG030500, two highly similar genes that encode putative acyl-acyl carrier protein (ACP) thioesterases, as strong candidates underlying the QTL. The lack of homoeologs for any of the three genes on Chr07N, the relatively low level of identity with other switchgrass KCS proteins and thioesterases, as well as the organization of the surrounding region suggest that Pavir.7KG077009 and Pavir.7KG013754/Pavir.7KG030500 were duplicated into a fast-evolving chromosome region, which led to their neofunctionalization. Furthermore, sequence analyses showed all three genes to be absent in the two upland compared to the two lowland accessions analyzed. This study provides an example of and practical guide for trait mapping and candidate gene identification in a complex genetic system by combining QTL mapping, transcriptomics and variant analysis.

Genomic mechanisms of climate adaptation in polyploid bioenergy switchgrass.

Long-term climate change and periodic environmental extremes threaten food and fuel security1 and global crop productivity2-4. Although molecular and adaptive breeding strategies can buffer the effects of climatic stress and improve crop resilience5, these approaches require sufficient knowledge of the genes that underlie productivity and adaptation6-knowledge that has been limited to a small number of well-studied model systems. Here we present the assembly and annotation of the large and complex genome of the polyploid bioenergy crop switchgrass (Panicum virgatum). Analysis of biomass and survival among 732 resequenced genotypes, which were grown across 10 common gardens that span 1,800 km of latitude, jointly revealed extensive genomic evidence of climate adaptation. Climate-gene-biomass associations were abundant but varied considerably among deeply diverged gene pools. Furthermore, we found that gene flow accelerated climate adaptation during the postglacial colonization of northern habitats through introgression of alleles from a pre-adapted northern gene pool. The polyploid nature of switchgrass also enhanced adaptive potential through the fractionation of gene function, as there was an increased level of heritable genetic diversity on the nondominant subgenome. In addition to investigating patterns of climate adaptation, the genome resources and gene-trait associations developed here provide breeders with the necessary tools to increase switchgrass yield for the sustainable production of bioenergy.

A rapid thioacidolysis method for biomass lignin composition and tricin analysis.

BACKGROUND: Biomass composition varies from plant to plant and greatly affects biomass utilization. Lignin is a heterogeneous phenolic polymer derived mainly from p-coumaryl, coniferyl, and sinapyl alcohols and makes up to 10-25% of lignocellulosic biomass. Recently, tricin, an O-methylated flavone, was identified as a lignin monomer in many grass species. Tricin may function as a nucleation site for lignification and is advocated as a novel target for lignin engineering to reduce lignin content and improve biomass digestibility in grasses. Thioacidolysis is an analytical method that can be adapted to analyze both lignin monomeric composition and tricin content in the lignin polymer. However, the original thioacidolysis procedure is complex, laborious, and time consuming, making it difficult to be adopted for large-scale screening in biomass research. In this study, a modified, rapid higher throughput thioacidolysis method was developed. RESULTS: In combination with gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS), the modified thioacidolysis method can be used to simultaneously characterize the lignin composition and tricin content using 2-5 mg of dry samples. The modified method eliminates the solvent extraction and drastically improves the throughput; 80 samples can be processed in one day per person. Our results indicate that there is no significant difference in the determination of lignin S/G ratio and tricin content between the original and modified methods. CONCLUSIONS: A modified thioacidolysis protocol was established. The results demonstrate that the modified method can be used for rapid, high-throughput, and reliable lignin composition and tricin content analyses for screening transgenic plants for cell wall modifications or in large-scale genome-wide association studies (GWAS).

Soil microbial communities alter leaf chemistry and influence allelopathic potential among coexisting plant species.

While both plant-soil feedbacks and allelochemical interactions are key drivers of plant community dynamics, the potential for these two drivers to interact with each other remains largely unexplored. If soil microbes influence allelochemical production, this would represent a novel dimension of heterogeneity in plant-soil feedbacks. To explore the linkage between soil microbial communities and plant chemistry, we experimentally generated soil microbial communities and evaluated their impact on leaf chemical composition and allelopathic potential. Four native perennial old-field species (two each of Aster and Solidago) were grown in pairwise combination with each species' soil microbial community as well as a sterilized inoculum. We demonstrated unequivocally that variation in soil microbial communities altered leaf chemical fingerprints for all focal plant species and also changed their allelopathic potential. Soil microbes reduced allelopathic potential in bioassays by increasing germination 25-54% relative to sterile control soils in all four species. Plants grown with their own microbial communities had the lowest allelopathic potential, suggesting that allelochemical production may be lessened when growing with microbes from conspecifics. The allelopathic potential of plants grown in congener and confamilial soils was indistinguishable from each other, indicating an equivalent response to all non-conspecific microbial communities within these closely related genera. Our results clearly demonstrated that soil microbial communities cause changes in leaf tissue chemistry that altered their allelopathic properties. These findings represent a new mechanism of plant-soil feedbacks that may structure perennial plant communities over very small spatial scales that must be explored in much more detail.

MycoDB, a global database of plant response to mycorrhizal fungi.

Plants form belowground associations with mycorrhizal fungi in one of the most common symbioses on Earth. However, few large-scale generalizations exist for the structure and function of mycorrhizal symbioses, as the nature of this relationship varies from mutualistic to parasitic and is largely context-dependent. We announce the public release of MycoDB, a database of 4,010 studies (from 438 unique publications) to aid in multi-factor meta-analyses elucidating the ecological and evolutionary context in which mycorrhizal fungi alter plant productivity. Over 10 years with nearly 80 collaborators, we compiled data on the response of plant biomass to mycorrhizal fungal inoculation, including meta-analysis metrics and 24 additional explanatory variables that describe the biotic and abiotic context of each study. We also include phylogenetic trees for all plants and fungi in the database. To our knowledge, MycoDB is the largest ecological meta-analysis database. We aim to share these data to highlight significant gaps in mycorrhizal research and encourage synthesis to explore the ecological and evolutionary generalities that govern mycorrhizal functioning in ecosystems.

A mutualistic endophyte alters the niche dimensions of its host plant.

Mutualisms can play important roles in influencing species coexistence and determining community composition. However, few studies have tested whether such interactions can affect species distributions by altering the niches of partner species. In subalpine meadows of the Rocky Mountains, USA, we explored whether the presence of a fungal endophyte (genus Epichloë) may shift the niche of its partner plant, marsh bluegrass (Poa leptocoma) relative to a closely related but endophyte-free grass species, nodding bluegrass (Poa reflexa). Using observations and a 3-year field experiment, we tested two questions: (i) Do P. leptocoma and P. reflexa occupy different ecological niches? and (ii) Does endophyte presence affect the relative fitness of P. leptocoma versus P. reflexa in the putative niches of these grass species? The two species were less likely to co-occur than expected by chance. Specifically, P. leptocoma grew closer to water sources and in wetter soils than P. reflexa, and also had higher root colonization by mycorrhizal fungi. Endophyte-symbiotic P. leptocoma seeds germinated with greater frequency in P. leptocoma niches relative to P. reflexa niches, whereas neither endophyte-free (experimentally removed) P. leptocoma seeds nor P. reflexa seeds showed differential germination between the two niche types. Thus, endophyte presence constrained the germination and early survival of host plants to microsites occupied by P. leptocoma. However, endophyte-symbiotic P. leptocoma ultimately showed greater growth than endophyte-free plants across all microsites, indicating a net benefit of the symbiosis at this life history stage. Differential effects of endophyte symbiosis on different host life history stages may thus contribute to niche partitioning between the two congeneric plant species. Our study therefore identifies a symbiotic relationship as a potential mechanism facilitating the coexistence of two species, suggesting that symbiont effects on host niche may have community-level consequences.

Belowground biotic complexity drives aboveground dynamics: a test of the soil community feedback model.

Feedbacks between soil communities and plants may determine abundance and diversity in plant communities by influencing fitness and competitive outcomes. We tested the core hypotheses of soil community feedback theory: plant species culture distinct soil communities that alter plant performance and the outcome of interspecific competition. We applied this framework to inform the repeated dominance of Solidago canadensis in old-field communities. In glasshouse experiments, we examined the effects of soil communities on four plant species' performance in monoculture and outcomes of interspecific competition. We used terminal restriction fragment length polymorphism (TRFLP) analysis to infer differences in the soil communities associated with these plant species. Soil community origin had strong effects on plant performance, changed the intensity of interspecific competition and even reversed whether plant species were limited by conspecifics or heterospecifics. These plant-soil feedbacks are strong enough to upend winners and losers in classic competition models. Plant species cultured significantly different mycorrhizal fungal and bacterial soil communities, indicating that these feedbacks are likely microbiotic in nature. In old-fields and other plant communities, these soil feedbacks appear common, fundamentally alter the intensity and nature of plant competition and potentially maintain diversity while facilitating the dominance of So. canadensis.

Extremely long-distance seed dispersal by an overfished Amazonian frugivore.

Throughout Amazonia, overfishing has decimated populations of fruit-eating fishes, especially the large-bodied characid, Colossoma macropomum. During lengthy annual floods, frugivorous fishes enter vast Amazonian floodplains, consume massive quantities of fallen fruits and egest viable seeds. Many tree and liana species are clearly specialized for icthyochory, and seed dispersal by fish may be crucial for the maintenance of Amazonian wetland forests. Unlike frugivorous mammals and birds, little is known about seed dispersal effectiveness of fishes. Extensive mobility of frugivorous fish could result in extremely effective, multi-directional, long-distance seed dispersal. Over three annual flood seasons, we tracked fine-scale movement patterns and habitat use of wild Colossoma, and seed retention in the digestive tracts of captive individuals. Our mechanistic model predicts that Colossoma disperses seeds extremely long distances to favourable habitats. Modelled mean dispersal distances of 337-552 m and maximum of 5495 m are among the longest ever reported. At least 5 per cent of seeds are predicted to disperse 1700-2110 m, farther than dispersal by almost all other frugivores reported in the literature. Additionally, seed dispersal distances increased with fish size, but overfishing has biased Colossoma populations to smaller individuals. Thus, overexploitation probably disrupts an ancient coevolutionary relationship between Colossoma and Amazonian plants.

Fructose variation in the dengue vector, Aedes aegypti, during high and low transmission seasons in the Mae Sot region of Thailand.

To understand the potential for facultative sugar feeding by Aedes aegypti, an important vector of dengue viruses, we evaluated seasonal and villagewide variation in sugar feeding during high- and low-dengue transmission seasons (rainy and dry seasons, respectively) in the Mae Sot region of Thailand. These seasons in Thailand are represented by different periods of flowering plant phenology. Although overall sugar feeding among female and male mosquitoes was low (ranging from 0.60 to 7.53 microg fructose per mosquito), sugar feeding among females was significantly greater during the dry, low-dengue transmission season. This variation could reflect specific preferences for flowering plants that were abundant in and around village homes during the dry season, such as Bougainvillea, Hibiscus, and Euphorbia.