Taylor Frazier

Assessment of drought tolerance of 49 switchgrass (Panicum virgatum) genotypes using physiological and morphological parameters.

BackgroundSwitchgrass (Panicum virgatum L.) is a warm-season C4 grass that is a target lignocellulosic biofuel species. In many regions, drought stress is one of the major limiting factors for switchgrass growth. The objective of this study was to evaluate the drought tolerance of 49 switchgrass genotypes. The relative drought stress tolerance was determined based on a set of parameters including plant height, leaf length, leaf width, leaf sheath length, leaf relative water content (RWC), electrolyte leakage (EL), photosynthetic rate (Pn), stomatal conductance (gs), transpiration rate (Tr), intercellular CO2 concentration (Ci), and water use efficiency (WUE).ResultsSRAP marker analysis determined that the selected 49 switchgrass genotypes represent a diverse genetic pool of switchgrass germplasm. Principal component analysis (PCA) and drought stress indexes (DSI) of each physiological parameter showed significant differences in the drought stress tolerance among the 49 genotypes. Heatmap and PCA data revealed that physiological parameters are more sensitive than morphological parameters in distinguishing the control and drought treatments. Metabolite profiling data found that under drought stress, the five best drought-tolerant genotypes tended to have higher levels of abscisic acid (ABA), spermine, trehalose, and fructose in comparison to the five most drought-sensitive genotypes.ConclusionBased on PCA ranking value, the genotypes TEM-SEC, TEM-LoDorm, BN-13645-64, Alamo, BN-10860-61, BN-12323-69, TEM-SLC, T-2086, T-2100, T-2101, Caddo, and Blackwell-1 had relatively higher ranking values, indicating that they are more tolerant to drought. In contrast, the genotypes Grif Nebraska 28, Grenville-2, Central Iowa Germplasm, Cave-in-Rock, Dacotah, and Nebraska 28 were found to be relatively sensitive to drought stress. By analyzing physiological response parameters and different metabolic profiles, the methods utilized in this study identified drought-tolerant and drought-sensitive switchgrass genotypes. These results provide a foundation for future research directed at understanding the molecular mechanisms underlying switchgrass tolerance to drought.

Evaluation of Candidate Reference Genes for Normalization of Quantitative RT-PCR in Switchgrass Under Various Abiotic Stress Conditions

Quantitative real-time reverse transcriptase PCR (qRT-PCR) is a sensitive and powerful technique for measuring differential gene expression; however, changes in gene expression induced by abiotic stresses are complex and multifaceted. Therefore, a set of stably expressed reference genes for data normalization is required. Switchgrass (Panicum virgatum L.) is a prime candidate crop for bioenergy production. The expression stability of reference genes in switchgrass, especially under different experimental conditions, is largely unknown. In order to identify the most suitable reference genes for abiotic stress studies in switchgrass, we evaluated 14 candidate genes for their expression stability under drought, high salinity, cold, heat, and waterlogging treatments using the Delta Ct, geNorm, BestKeeper, and NormFinder approaches. Validation of reference genes indicated that the best reference genes should be selected based on the stress treatment. Actin 2 (ACT2), carotenoid-binding protein 20 (CBP20), and Tubulin (TUB) were found to have the highest expression stability to study drought stress. 18S ribosomal RNA1 (18S rRNA1), ACT2, and TUB were the most stably expressed genes under salt stress. Ubiquitin-conjugating enzyme (UBC), TUB, and cyclophilin2 (CYP2) were the most suitable reference genes across cold treatments. Likewise, 18S rRNA1, UBC, and TUB were good reference genes for studying heat stress, while ACT2, 18S rRNA1, and ubiquitin3 (UBQ3) were the top three reference genes under waterlogging treatment. Considering that reference gene expression may vary across switchgrass tissues, ACT2 and 18S ribosomal RNA2 (18S rRNA2) were shown to be the most stably expressed genes in switchgrass leaves and roots, respectively. The highly ranked reference genes that were identified in this study were shown to be capable of detecting subtle differences in the expression rates of other genes. These differences may have been missed if a less suitable reference gene was used.

De novo Transcriptome Analysis and Molecular Marker Development of Two Hemarthria Species.

Hemarthria R. Br. is an important genus of perennial forage grasses that is widely used in subtropical and tropical regions. Hemarthria grasses have made remarkable contributions to the development of animal husbandry and agro-ecosystem maintenance; however, there is currently a lack of comprehensive genomic data available for these species. In this study, we used Illumina high-throughput deep sequencing to characterize of two agriculturally important Hemarthria materials, H. compressa “Yaan” and H. altissima “1110.” Sequencing runs that used each of four normalized RNA samples from the leaves or roots of the two materials yielded more than 24 million high-quality reads. After de novo assembly, 137,142 and 77,150 unigenes were obtained for “Yaan” and “1110,” respectively. In addition, a total of 86,731 “Yaan” and 48,645 “1110” unigenes were successfully annotated. After consolidating the unigenes for both materials, 42,646 high-quality SNPs were identified in 10,880 unigenes and 10,888 SSRs were identified in 8330 unigenes. To validate the identified markers, high quality PCR primers were designed for both SNPs and SSRs. We randomly tested 16 of the SNP primers and 54 of the SSR primers and found that the majority of these primers successfully amplified the desired PCR product. In addition, high cross-species transferability (61.11–87.04%) of SSR markers was achieved for four other Poaceae species. The amount of RNA sequencing data that was generated for these two Hemarthria species greatly increases the amount of genomic information available for Hemarthria and the SSR and SNP markers identified in this study will facilitate further advancements in genetic and molecular studies of the Hemarthria genus.

Identifying differentially expressed genes under heat stress and developing molecular markers in orchardgrass (Dactylis glomerata L.) through transcriptome analysis

Orchardgrass (Dactylis glomerata L.) is a long‐lived, cool‐season forage grass that is commonly used for hay production. Despite its economic importance, orchardgrass genome remains relatively unexplored. In this study, we used Illumina RNA sequencing to identify gene‐associated molecular markers, including simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs), as well as heat stress‐induced differentially expressed genes (DEGs) in two orchardgrass genotypes, ‘Baoxing’ (heat resistant) and ‘01998’ (heat susceptible). Approximately 163 million high‐quality trimmed reads were generated from 207 million raw reads using the Illumina HiSeq 2000 platform. A total of 126 846 unigenes were obtained after de novo assembly of the trimmed reads, and 40 078 unigenes were identified as coding sequences (CDSs). Based on the assembled unigenes, 669 300 high‐quality SNPs, including 416 099 transitions and 257 736 transversions, were contained in 75 875 unigenes. In addition, a total of 8475 microsatellites were detected in 7764 unigenes. When placed under heat stress, the total number of DEGs in ‘Baoxing’ (3527) was higher than in ‘01998’ (2649), indicating that in comparison with heat‐susceptible ‘01998’, heat‐resistant ‘Baoxing’ seems to have more unigenes that respond to heat stress. The high‐throughput transcriptome sequencing of orchardgrass under heat stress provides useful information for gene identification and for the development of SNP and SSR molecular markers. The comparison of DEGs under different periods of heat stress allowed us to identify a wealth of candidate DEGs that can be further analysed in order to determine the genetic mechanisms underlying heat tolerance in orchardgrass.

Identification and validation of Asteraceae miRNAs by the expressed sequence tag analysis

MicroRNAs (miRNAs) are small non-coding RNA molecules that play a vital role in the regulation of gene expression. Despite their identification in hundreds of plant species, few miRNAs have been identified in the Asteraceae, a large family that comprises approximately one tenth of all flowering plants. In this study, we used the expressed sequence tag (EST) analysis to identify potential conserved miRNAs and their putative target genes in the Asteraceae. We applied quantitative Real-Time PCR (qRT-PCR) to confirm the expression of eight potential miRNAs in Carthamus tinctorius and Helianthus annuus. We also performed qRT-PCR analysis to investigate the differential expression pattern of five newly identified miRNAs during five different cotyledon growth stages in safflower. Using these methods, we successfully identified and characterized 151 potentially conserved miRNAs, belonging to 26 miRNA families, in 11 genus of Asteraceae. EST analysis predicted that the newly identified conserved Asteraceae miRNAs target 130 total protein-coding ESTs in sunflower and safflower, as well as 433 additional target genes in other plant species. We experimentally confirmed the existence of seven predicted miRNAs, (miR156, miR159, miR160, miR162, miR166, miR396, and miR398) in safflower and sunflower seedlings. We also observed that five out of eight miRNAs are differentially expressed during cotyledon development. Our results indicate that miRNAs may be involved in the regulation of gene expression during seed germination and the formation of the cotyledons in the Asteraceae. The findings of this study might ultimately help in the understanding of miRNA-mediated gene regulation in important crop species.

Identification, characterization, and gene expression analysis of nucleotide binding site (NB)-type resistance gene homologues in switchgrass

BackgroundSwitchgrass (Panicum virgatum L.) is a warm-season perennial grass that can be used as a second generation bioenergy crop. However, foliar fungal pathogens, like switchgrass rust, have the potential to significantly reduce switchgrass biomass yield. Despite its importance as a prominent bioenergy crop, a genome-wide comprehensive analysis of NB-LRR disease resistance genes has yet to be performed in switchgrass.ResultsIn this study, we used a homology-based computational approach to identify 1011 potential NB-LRR resistance gene homologs (RGHs) in the switchgrass genome (v 1.1). In addition, we identified 40 RGHs that potentially contain unique domains including major sperm protein domain, jacalin-like binding domain, calmodulin-like binding, and thioredoxin. RNA-sequencing analysis of leaf tissue from ‘Alamo’, a rust-resistant switchgrass cultivar, and ‘Dacotah’, a rust-susceptible switchgrass cultivar, identified 2634 high quality variants in the RGHs between the two cultivars. RNA-sequencing data from field-grown cultivar ‘Summer’ plants indicated that the expression of some of these RGHs was developmentally regulated.ConclusionsOur results provide useful insight into the molecular structure, distribution, and expression patterns of members of the NB-LRR gene family in switchgrass. These results also provide a foundation for future work aimed at elucidating the molecular mechanisms underlying disease resistance in this important bioenergy crop.

Identification and Characterization of Switchgrass Histone H3 and CENH3 Genes.

Switchgrass is one of the most promising energy crops and only recently has been employed for biofuel production. The draft genome of switchgrass was recently released; however, relatively few switchgrass genes have been functionally characterized. CENH3, the major histone protein found in centromeres, along with canonical H3 and other histones, plays an important role in maintaining genome stability and integrity. Despite their importance, the histone H3 genes of switchgrass have remained largely uninvestigated. In this study, we identified 17 putative switchgrass histone H3 genes in silico. Of these genes, 15 showed strong homology to histone H3 genes including six H3.1 genes, three H3.3 genes, four H3.3-like genes and two H3.1-like genes. The remaining two genes were found to be homologous to CENH3. RNA-seq data derived from lowland cultivar Alamo and upland cultivar Dacotah allowed us to identify SNPs in the histone H3 genes and compare their differential gene expression. Interestingly, we also found that overexpression of switchgrass histone H3 and CENH3 genes in N. benthamiana could trigger cell death of the transformed plant cells. Localization and deletion analyses of the histone H3 and CENH3 genes revealed that nuclear localization of the N-terminal tail is essential and sufficient for triggering the cell death phenotype. Our results deliver insight into the mechanisms underlying the histone-triggered cell death phenotype and provide a foundation for further studying the variations of the histone H3 and CENH3 genes in switchgrass.