Brian S. Baldwin

Transcriptome-based differentiation of closely-related Miscanthus lines.

Background Distinguishing between individuals is critical to those conducting animal/plant breeding, food safety/quality research, diagnostic and clinical testing, and evolutionary biology studies. Classical genetic identification studies are based on marker polymorphisms, but polymorphism-based techniques are time and labor intensive and often cannot distinguish between closely related individuals. Illumina sequencing technologies provide the detailed sequence data required for rapid and efficient differentiation of related species, lines/cultivars, and individuals in a cost-effective manner. Here we describe the use of Illumina high-throughput exome sequencing, coupled with SNP mapping, as a rapid means of distinguishing between related cultivars of the lignocellulosic bioenergy crop giant miscanthus (Miscanthus × giganteus). We provide the first exome sequence database for Miscanthus species complete with Gene Ontology (GO) functional annotations. Results A SNP comparative analysis of rhizome-derived cDNA sequences was successfully utilized to distinguish three Miscanthus × giganteus cultivars from each other and from other Miscanthus species. Moreover, the resulting phylogenetic tree generated from SNP frequency data parallels the known breeding history of the plants examined. Some of the giant miscanthus plants exhibit considerable sequence divergence. Conclusions Here we describe an analysis of Miscanthus in which high-throughput exome sequencing was utilized to differentiate between closely related genotypes despite the current lack of a reference genome sequence. We functionally annotated the exome sequences and provide resources to support Miscanthus systems biology. In addition, we demonstrate the use of the commercial high-performance cloud computing to do computational GO annotation.

Propagation Methods for Rivercane [Arundinaria gigantea L. (Walter) Muhl.]

Abstract As awareness and interest in canebrake restoration activities in the southeastern United States continues to grow, we are faced with numerous challenges. The greatest of these challenges is obtaining enough germplasm to support restoration activities. Three propagation methods are reported in an effort to begin to elicit information regarding propagation of rivercane. Experiments to optimize micro-propagation, seed germination, and macro-propagation methods were compared. Tissue culture (rapid clonal multiplication) has great potential for production of large numbers of propagules. Production of shoots has been optimized, using 4-6 mm diameter explants on MS media, containing 0.1 µM IBA (indole-3-butyric acid ) and 0.01 µM TDZ (thidiazuron). However, the process is hampered by the current inability to generate roots. Maximizing seed germination offers the ability to exploit seed produced on existing canebrakes. Of the six temperature regimes tested on two populations, maximum germination occurred using roll towels, under a 35/25 or 30/20°C temperature regime. Removal of lemma and palea from the seed increased germination percentage for one of the populations. Macropropagation from rhizome sections derived from stock maintained in pot-in-pot container production allowed for relatively easy harvest. Rhizome sections 2-3 nodes in length soaked for 60 minutes in warm water or 1000 ppm BAP (6-benzylaminopurine) showed greatest shooting yield. Individual mother plants have the potential to yield 400 viable clones. While each method had its apparent strengths, only the macro-propagation method showed immediate promise.

Frost kill and kenaf fiber quality

Abstract Kenaf (Hibiscus cannabinus) grown in northern Mississippi and elsewhere often is injured by early frost and killed before harvest. Frost kill often is associated with fungal growth or rot, so its effect on fiber quality is a major concern. Fiber processing also affects the quality and chemical composition of fibers. Therefore, this study was aimed at determining the effects of frost kill on processing, fiber quality and chemical composition of kenaf fibers. Frost-damaged kenaf with fungal growth was decorticated by hand and divided into six sections (26.88 cm/each) from the base to tip of the stem and then retted chemically or bacterially in the laboratory. Fiber characteristics were compared between the two processes and the six locations on the plant. Ash, cellulose, hemicellulose, and lignin contents of the resultant fibers were measured. Bacterially retted (BR) fibers were stronger (11.8 g/tex) than the chemically retted fibers (CR), 7.5 g/tex, at all locations. The BR fibers from decorticated green ribbons were stronger than those from frost-killed ribbons. However, no significant differences occurred between the CR fibers from decorticated and frost-killed ribbons. Residual gum content was higher for the BR fibers (23.3%) than for the CR fibers (8.1%). The stretch properties were not affected significantly by the frost kill or fungus. The base of the stem had the weakest fibers in both processes, which may have been due to greater fungal disease. The CR process extracted more fiber than the BR process, with a consistent higher yield of clean fibers. In the BR process, the fiber extracted was higher at the tip than at the base of the stem. This may have been related to the presence of fungus, which inhibits the BR process. Analysis of chemical composition of the processed fibers indicated that CR is efficient in reducing hemicellulose and lignin contents. These results indicate that frost kill may not be the appropriate method for harvesting kenaf for quality fibers. However, fibers extracted by chemical retting were unaffected by the presence of fungus as a result of frost kill.

Biomass production of herbaceous energy crops in the United States: field trial results and yield potential maps from the multiyear regional feedstock partnership

Current knowledge of yield potential and best agronomic management practices for perennial bioenergy grasses is primarily derived from small‐scale and short‐term studies, yet these studies inform policy at the national scale. In an effort to learn more about how bioenergy grasses perform across multiple locations and years, the U.S. Department of Energy (US DOE)/Sun Grant Initiative Regional Feedstock Partnership was initiated in 2008. The objectives of the Feedstock Partnership were to (1) provide a wide range of information for feedstock selection (species choice) and management practice options for a variety of regions and (2) develop national maps of potential feedstock yield for each of the herbaceous species evaluated. The Feedstock Partnership expands our previous understanding of the bioenergy potential of switchgrass, Miscanthus, sorghum, energycane, and prairie mixtures on Conservation Reserve Program land by conducting long‐term, replicated trials of each species at diverse environments in the U.S. Trials were initiated between 2008 and 2010 and completed between 2012 and 2015 depending on species. Field‐scale plots were utilized for switchgrass and Conservation Reserve Program trials to use traditional agricultural machinery. This is important as we know that the smaller scale studies often overestimated yield potential of some of these species. Insufficient vegetative propagules of energycane and Miscanthus prohibited farm‐scale trials of these species. The Feedstock Partnership studies also confirmed that environmental differences across years and across sites had a large impact on biomass production. Nitrogen application had variable effects across feedstocks, but some nitrogen fertilizer generally had a positive effect. National yield potential maps were developed using PRISM‐ELM for each species in the Feedstock Partnership. This manuscript, with the accompanying supplemental data, will be useful in making decisions about feedstock selection as well as agronomic practices across a wide region of the country.

Nutritive Value Response of Native Warm-Season Forage Grasses to Harvest Intervals and Durations in Mixed Stands

Interest in management of native warm-season grasses for multiple uses is growing in southeastern USA. Forage quality response of early-succession mixed stands of big bluestem (BB, Andropogon gerardii), indiangrass (IG, Sorghastrum nutans), and little bluestem (SG, Schizachyrium scoparium) to harvest intervals (30-, 40-, 60-, 90 or 120-d) and durations (one or two years) were assessed in crop-field buffers. Over three years, phased harvestings were initiated in May, on sets of randomized plots, ≥90 cm apart, in five replications (blocks) to produce one-, two-, and three-year-old stands, by the third year. Whole-plot regrowths were machine-harvested after collecting species (IG and LB) sample tillers for leafiness estimates. Species-specific leaf area (SLA) and leaf-to-stem ratio (LSR) were greater for early-season harvests and shorter intervals. In a similar pattern, whole-plot crude protein concentrations were greatest for the 30-d (74 g·kg−1 DM) and the least (40 g·kg−1 DM) for the 120-d interval. Corresponding neutral detergent fiber (NDF) values were the lowest (620 g·kg−1 DM) and highest (710 g·kg−1 DM), respectively. In vitro dry matter and NDF digestibility were greater for early-season harvests at shorter intervals (63 and 720 g·kg−1 DM). With strategic harvesting, similar stands may produce quality hay for beef cattle weight gain.

EFFECT OF PLANTING DATE, NITROGEN RATE, AND HYBRID ON SUNFLOWER

Sunflower is a potential crop for the southeastern United States for production of cooking oil or biodiesel. In 2007, we evaluated the effect of planting date (PD, 20 April, 20 May, and 20 June), nitrogen (N) application rate (0, 67, 134, and 202 kg ha−1), and hybrid (‘DKF3875’, ‘DKF2990’, ‘DKF3510’, and ‘DKF3901’) on sunflower productivity and oil profile in four Mississippi locations, Newton, Starkville, and two sites in Verona. There was a trend of increased oleic acid concentration with earlier planting dates, especially in hybrids with lower oleic acid concentration. Earlier planting dates of ‘DKF3901’ and ‘DKF2990’ (the hybrid with the lowest oleic acid) actually had 200 to 300 g kg−1 higher concentration of oleic acid when grown in Mississippi vs. the original seed of the same hybrids used for planting and produced at a more northern latitude. This and a recent study in Mississippi suggest that modern hybrids could provide ample yields even when the N fertilization is relatively low.

Solid State13C NMR and Diffuse Reflectance Mid-Infrared Spectroscopic Analysis of the Effects of Retting on the Chemical Composition of KenafFibre

Bast ribbons from Tainung 1 (T 1 ) and Guatemala 45 (G 45 ) cultivars of kenaf (Hibiscus cannabinus) were subjected to a chemical retting process. The resulting changes at the base and tip regions of each cultivar were analysed using solid-state 13 C NMR and diffuse reflectance mid-infrared spectroscopies. Chemical retting was performed by boiling the substrates in NaOH (70 g litre -1 ) + NaHSO 3 (5 g litre -1 ) for 1 h, after which they were neutralised, washed and air dried. This process produced large decreases in the non-cellulosic components, though 25% of the phenolic component was left undegraded, and cellulose crystallinity was increased by ∼5% over that in unretted samples.

Soil and Variety Effects on Energy Use and Carbon Emissions Associated with Switchgrass-Based Ethanol Production in Mississippi

High biomass production potential, wide adaptability, low input requirement, and low environmental risk make switchgrass an economically and ecologically viable energy crop. The inherent variability in switchgrass productivity due to variations in soil and variety could affect the sustainability and eco-friendliness of switchgrass-based ethanol production. This study examined the soil and variety effects on these variables. Three locations in Mississippi were selected based on latitude and potential acreage. Using ALMANAC, switchgrass biomass yields were simulated for several scenarios of soils and varieties. The simulated yields were fed to IBSAL to compute energy use and CO2 emissions in various operations in the biomass supply chain. From the energy and emissions values, the sustainability and eco-friendliness of ethanol production were determined using net energy value (NEV) and carbon credit balance (CCB) as indicators, respectively. Soil and variety effects on NEV and CCB were analyzed using the Kruskal-Wallis test. Results showed significant differences in NEV and CCB across soils and varieties. Both NEV and CCB increased in the direction of heavier to lighter soils and on the order of north-upland, south-upland, north-lowland, and south-lowland varieties. Only north-upland and south-lowland varieties were significantly different because they were different in both cytotype and ecotype. Gaps between lowland and upland varieties were smaller in a dry year than in a wet year. The NEV and CCB increased in the direction of dry to wet year. From south to north, they decreased for lowland cytotypes but increased for upland cytotypes. Thus, the differences among varieties decreased northwards.

Transcriptome profiles of sunflower reveal the potential role of microsatellites in gene expression divergence

The mechanisms by which natural populations generate adaptive genetic variation are not well understood. Some studies propose that microsatellites can function as drivers of adaptive variation. Here, we tested a potentially adaptive role for transcribed microsatellites with natural populations of the common sunflower (Helianthus annuus L.) by assessing the enrichment of microsatellites in genes that show expression divergence across latitudes. Seeds collected from six populations at two distinct latitudes in Kansas and Oklahoma were planted and grown in a common garden. Morphological measurements from the common garden demonstrated that phenotypic variation among populations is largely explained by underlying genetic variation. An RNA-Seq experiment was conducted with 96 of the individuals grown in the common garden and differentially expressed (DE) transcripts between the two latitudes were identified. A total number of 825 DE transcripts were identified. DE transcripts and nondifferentially expressed (NDE) transcripts were then scanned for microsatellites. The abundance of different motif lengths and types in both groups were estimated. Our results indicate that DE transcripts are significantly enriched with mononucleotide repeats and significantly depauperate in trinucleotide repeats. Further, the standardized mononucleotide repeat motif A and dinucleotide repeat motif AG were significantly enriched within DE transcripts while motif types, C, AT, ACC and AAC in DE transcripts, are significantly differentiated in microsatellite tract length between the two latitudes. The tract length differentiation at specific microsatellite motif types across latitudes and their enrichment within DE transcripts indicate a potential functional role for transcribed microsatellites in gene expression divergence in sunflower.

Evaluating Physiological and Growth Responses of Arundinaria Species to Inundation

Abstract Dense stands of Arundinaria species, or canebrakes, once were a dominant landscape feature along floodplains of the southeastern United States. However, human activities have reduced canebrakes to fragmented remnants representing <2% of their extent prior to European settlement. Canebrake restoration thus is a top priority for preserving and improving wetland biodiversity in the United States. Successful restoration requires an understanding of factors influencing establishment of the two most common United States Arundinaria species; therefore, this greenhouse study examined effects of inundation on A. gigantea and A. tecta. Both Arundinaria species were subjected to 0, 2, 4, or 6 weeks of inundation under long-day, warm temperature growing season conditions. Plant growth, mean net photosynthesis (Pn), and stomatal conductance (Gs) were measured on a weekly basis, and at the conclusion of the experiment, above- and belowground biomass were measured. We found significant correlations of Pn and Gs with duration of flooding and duration of post-inundation recovery, as well as a significant interaction among time, species, and flood duration, with both species responding similarly to flooded conditions. Once flooding was arrested, both Pn and Gs rates were higher in A. tecta than A. gigantea; the same was true for growth rates across the duration of the study. In conclusion, A. tecta appeared to be more flood tolerant than A. gigantea, reflecting habitats in which these species are known to occur. Future canebrake restoration projects may benefit from this information on differential flooding tolerance in selecting restoration sites or restoration species, based on hydrologic conditions.