Addressing Financial Barriers to Health Care Among People Who are Low-Income and Insured in New York City, 2014-2017.

While health care-associated financial burdens among uninsured individuals are well described, few studies have systematically characterized the array of financial and logistical complications faced by insured individuals with low household incomes. In this mixed methods paper, we conducted 6 focus groups with a total of 55 residents and analyzed programmatic administrative records to characterize the specific financial and logistic barriers faced by residents living in public housing in East and Central Harlem, New York City (NYC). Participants included individuals who enrolled in a municipal community health worker (CHW) program designed to close equity gaps in health and social outcomes. Dedicated health advocates (HAs) were explicitly paired with CHWs to provide health insurance and health care navigational assistance. We describe the needs of 150 residents with reported financial barriers to care, as well as the navigational and advocacy strategies taken by HAs to address them. Finally, we outline state-level policy recommendations to help ameliorate the problems experienced by participants. The model of paired CHW-HAs may be helpful in addressing financial barriers for insured populations with low household income and reducing health disparities in other communities.

MoChlo: A Versatile, Modular Cloning Toolbox for Chloroplast Biotechnology.

Plant synthetic biology is a rapidly evolving field with new tools constantly emerging to drive innovation. Of particular interest is the application of synthetic biology to chloroplast biotechnology to generate plants capable of producing new metabolites, vaccines, biofuels, and high-value chemicals. Progress made in the assembly of large DNA molecules, composing multiple transcriptional units, has significantly aided in the ability to rapidly construct novel vectors for genetic engineering. In particular, Golden Gate assembly has provided a facile molecular tool for standardized assembly of synthetic genetic elements into larger DNA constructs. In this work, a complete modular chloroplast cloning system, MoChlo, was developed and validated for fast and flexible chloroplast engineering in plants. A library of 128 standardized chloroplast-specific parts (47 promoters, 38 5' untranslated regions [5'UTRs], nine promoter:5'UTR fusions, 10 3'UTRs, 14 genes of interest, and 10 chloroplast-specific destination vectors) were mined from the literature and modified for use in MoChlo assembly, along with chloroplast-specific destination vectors. The strategy was validated by assembling synthetic operons of various sizes and determining the efficiency of assembly. This method was successfully used to generate chloroplast transformation vectors containing up to seven transcriptional units in a single vector (∼10.6-kb synthetic operon). To enable researchers with limited resources to engage in chloroplast biotechnology, and to accelerate progress in the field, the entire kit, as described, is available through Addgene at minimal cost. Thus, the MoChlo kit represents a valuable tool for fast and flexible design of heterologous metabolic pathways for plastid metabolic engineering.

Gene regulatory networks for lignin biosynthesis in switchgrass (Panicum virgatum).

Cell wall recalcitrance is the major challenge to improving saccharification efficiency in converting lignocellulose into biofuels. However, information regarding the transcriptional regulation of secondary cell wall biogenesis remains poor in switchgrass (Panicum virgatum), which has been selected as a biofuel crop in the United States. In this study, we present a combination of computational and experimental approaches to develop gene regulatory networks for lignin formation in switchgrass. To screen transcription factors (TFs) involved in lignin biosynthesis, we developed a modified method to perform co-expression network analysis using 14 lignin biosynthesis genes as bait (target) genes. The switchgrass lignin co-expression network was further extended by adding 14 TFs identified in this study, and seven TFs identified in previous studies, as bait genes. Six TFs (PvMYB58/63, PvMYB42/85, PvMYB4, PvWRKY12, PvSND2 and PvSWN2) were targeted to generate overexpressing and/or down-regulated transgenic switchgrass lines. The alteration of lignin content, cell wall composition and/or plant growth in the transgenic plants supported the role of the TFs in controlling secondary wall formation. RNA-seq analysis of four of the transgenic switchgrass lines revealed downstream target genes of the secondary wall-related TFs and crosstalk with other biological pathways. In vitro transactivation assays further confirmed the regulation of specific lignin pathway genes by four of the TFs. Our meta-analysis provides a hierarchical network of TFs and their potential target genes for future manipulation of secondary cell wall formation for lignin modification in switchgrass.

Autopolyploidization in switchgrass alters phenotype and flowering time via epigenetic and transcription regulation.

Polyploidization is a significant source of genomic and organism diversification during plant evolution, and leads to substantial alterations in plant phenotypes and natural fitness. To help understand the phenotypic and molecular impacts of autopolyploidization, we conducted epigenetic and full-transcriptomic analyses of a synthesized autopolyploid accession of switchgrass (Panicum virgatum) in order to interpret the molecular and phenotypic changes. We found that mCHH levels were decreased in both genic and transposable element (TE) regions, and that TE methylation near genes was decreased as well. Among 142 differentially expressed genes involved in cell division, cellulose biosynthesis, auxin response, growth, and reproduction processes, 75 of them were modified by 122 differentially methylated regions, 10 miRNAs, and 15 siRNAs. In addition, up-regulated PvTOE1 and suppressed PvFT probably contribute to later flowering time of the autopolyploid. The expression changes were probably associated with modification of nearby methylation sites and siRNAs. We also experimentally demonstrated that expression levels of PvFT and PvTOE1 were regulated by DNA methylation, supporting the link between alterations in methylation induced by polyploidization and the phenotypic changes that were observed. Collectively, our results show epigenetic modifications in synthetic autopolyploid switchgrass for the first time, and support the hypothesis that polyploidization-induced methylation is an important cause of phenotypic alterations and is potentially important for plant evolution and improved fitness.

Development and validation of a novel and robust cell culture system in soybean (Glycine max (L.) Merr.) for promoter screening.

KEY MESSAGE: A novel soybean cell culture was developed, establishing a reliable and rapid promoter assay to enable high-throughput automated screening in soybean protoplasts relevant to shoot tissues in whole plants. Transient reporter gene assays can be valuable to rapidly estimate expression characteristics of heterologous promoters. The challenge for maximizing the value of such screens is to combine relevant cells or tissues with methods that can be scaled for high-throughput screening, especially for crop-rather than model species. We developed a robust and novel soybean cell suspension culture derived from leaf-derived callus for protoplast production: a platform for promoter screening. The protoplasts were transfected with promoter-reporter constructs, of which were chosen and validated against known promoter expression profiles from tissue-derived protoplasts (leaves, stems, and immature cotyledons) and gene expression data from plants. The cell culture reliably produced 2.82 ± 0.94 × 108 protoplasts/g fresh culture mass with a transfection efficiency of 31.06 ± 7.69% at 48 h post-incubation. The promoter-reporter gene DNA expression levels of transfected cell culture-derived protoplasts were most similar to that of leaf- and stem-derived protoplasts (correlation coefficient of 0.99 and 0.96, respectively) harboring the same constructs. Cell culture expression was also significantly correlated to endogenous promoter-gene expression in leaf tissues as measured by qRT-PCR (correlation coefficient of 0.80). Using the manual protocols that produced these results, we performed early stage experiments to automate protoplast transformation on a robotic system. After optimizing the protocol, we achieved up to 29% transformation efficiency using our robotic system. We conclude that the soybean cell culture-to-protoplast transformation screen is amenable to automate promoter and gene screens in soybean that could be used to accelerate discoveries relevant for crop improvement. Key features of the system include low-cost, facile protoplast isolation, and transformation for soybean shoot tissue-relevant molecular screening.

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

BACKGROUND: Switchgrass (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. RESULTS: In 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. CONCLUSIONS: Our 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.

Xylem sap in cotton contains proteins that contribute to environmental stress response and cell wall development.

The xylem sap of a plant is primarily responsible for transporting molecules from the underground root system to the aboveground parts of the plant body. In order to understand the role that roots play in cotton growth and development, the components present in xylem sap must be elucidated. In this study, we used a shotgun HPLC-ESI-MS/MS proteomics approach to identify 455 peptides from the xylem sap of field-grown cotton plants at peak blooming stage. Of these peptides, 384 (84.4%) were found to be secreted proteins and 320 (70.3%) had special molecular functions. Based on Gene Ontology (GO) analysis, 348 peptides were annotated in terms of molecular function, biological process, and cellular localization, with 46.9 and 45.1% being related to catalytic activity and binding activity, respectively. Many xylem sap-containing proteins were predicted to be involved in different phases of xylem differentiation including cell wall metabolism, secondary cell wall development and patterning, and programmed cell death. The identification of starch and sucrose hydrolyzing enzymes implicated the interaction between roots and aboveground parts on the aspect of carbohydrate metabolism. Many of the proteins identified in this study are involved in defense mechanisms including pathogen-related proteins, such as peroxidases, chitinases, and germin-like proteins, proteases involved in disease resistance, and phytoalexin phenylpropanoid synthesis-related proteins. The majority of identified signaling proteins were fasciclin-like arabinogalactan proteins and kinases. The results of this study provide useful insight into the communication mechanisms between cotton roots and the rest of the cotton plant.

Titanium dioxide nanoparticles affect the growth and microRNA expression of tobacco (Nicotiana tabacum).

Titanium dioxide (TiO(2)) is one of the most widely used pigments in the world. Due to its heavy use in industry and daily life, such as food additives, cosmetics, pharmaceuticals, and paints, many residues are released into the environment and currently TiO(2) nanoparticles are considered an emerging environmental contaminant. Although several studies have shown the effect of TiO(2) nanoparticles on a wide range of organisms including bacteria, algae, plankton, fish, mice, and rats, little research has been performed on land plants. In this study, we investigated the effect of TiO(2) nanoparticles on the growth, development, and gene expression of tobacco, an important economic and agricultural crop in the southeastern USA as well as around the world. We found that TiO(2) nanoparticles significantly inhibited the germination rates, root lengths, and biomasses of tobacco seedlings after 3 weeks of exposure to 0.1, 1, 2.5, and 5 % TiO(2) nanoparticles and that overall growth and development of the tobacco seedlings significantly decreased as TiO(2) nanoparticle concentrations increased. Overall, tobacco roots were the most sensitive to TiO(2) nanoparticle exposure. Nano-TiO(2) also significantly influenced the expression profiles of microRNAs (miRNAs), a recently discovered class of small endogenous noncoding RNAs (∼20-22 nt) that are considered important gene regulators and have been shown to play an important role in plant development as well as plant tolerance to abiotic stresses such as drought, salinity, cold, and heavy metal. Low concentrations (0.1 and 1 %) of TiO(2) nanoparticles dramatically induced miRNA expression in tobacco seedlings with miR395 and miR399 exhibiting the greatest fold changes of 285-fold and 143-fold, respectively. The results of this study show that TiO(2) nanoparticles have a negative impact on tobacco growth and development and that miRNAs may play an important role in tobacco response to heavy metals/nanoparticles by regulating gene expression.

Deep sequencing identifies novel and conserved microRNAs in peanuts (Arachis hypogaea L.).

BACKGROUND: MicroRNAs (miRNAs) are a new class of small, endogenous RNAs that play a regulatory role in the cell by negatively affecting gene expression at the post-transcriptional level. miRNAs have been shown to control numerous genes involved in various biological and metabolic processes. There have been extensive studies on discovering miRNAs and analyzing their functions in model species, such as Arabidopsis and rice. Increasing investigations have been performed on important agricultural crops including soybean, conifers, and Phaselous vulgaris but no studies have been reported on discovering peanut miRNAs using a cloning strategy. RESULTS: In this study, we employed the next generation high through-put Solexa sequencing technology to clone and identify both conserved and species-specific miRNAs in peanuts. Next generation high through-put Solexa sequencing showed that peanuts have a complex small RNA population and the length of small RNAs varied, 24-nt being the predominant length for a majority of the small RNAs. Combining the deep sequencing and bioinformatics, we discovered 14 novel miRNA families as well as 75 conserved miRNAs in peanuts. All 14 novel peanut miRNAs are considered to be species-specific because no homologs have been found in other plant species except ahy-miRn1, which has a homolog in soybean. qRT-PCR analysis demonstrated that both conserved and peanut-specific miRNAs are expressed in peanuts. CONCLUSIONS: This study led to the discovery of 14 novel and 22 conserved miRNA families from peanut. These results show that regulatory miRNAs exist in agronomically important peanuts and may play an important role in peanut growth, development, and response to environmental stress.

Identification and characterization of microRNAs and their targets in the bioenergy plant switchgrass (Panicum virgatum).

MicroRNAs (miRNAs) are a class of non-coding small endogenous RNAs with lengths of approximately 22 nucleotides (nt) that have been shown to regulate gene expression at the post-transcriptional levels by targeting mRNAs for degradation or by inhibiting protein translation. Although thousands of miRNAs have been identified in many species, miRNAs have not yet been identified in switchgrass (Panicum virgatum), one of the most important bioenergy crops in the United States and around the world. In this study, we identified 121 potential switchgrass miRNAs, belonging to 44 families, using a well-defined comparative genome-based computational approach. We also identified miRNA clusters and antisense miRNAs in switchgrass expressed sequences tags. These identified miRNAs potentially target 839 protein-coding genes, which can act as transcription factors, and take part in multiple biological and metabolic processes including sucrose and fat metabolism, signal transduction, stress response, and plant development. Gene ontology (GO) analysis, based on these targets, showed that 527 biological processes were involved. Twenty-five of these processes were demonstrated to participate in the metabolism of carbon, glucose, starch, fatty acid, and lignin and in xylem formation. According to pathway enrichment analysis based on Kyoto Encyclopedia of Genes and Genomes (KEGG), 118 metabolism networks were found. These networks are involved in sucrose metabolism, fat metabolism, carbon fixation, hormone regulation, oxidative stress response, and the processing of other secondary metabolites.

Identification and characterization of microRNAs and their target genes in tobacco (Nicotiana tabacum).

microRNAs (miRNAs) are a recently discovered class of small (~21 nt) endogenous gene regulators that have been shown to play an important role in plant growth and development by aiding in organ maturation, hormone signaling, tissue differentiation, and plant tolerance to environmental stress. Since a list of miRNAs has never been generated for tobacco, we employed genome survey sequence analysis to computationally identify 259 potentially conserved tobacco miRNAs, belonging to 65 families, and validated 11 of these miRNAs using qRT-PCR. The 65 miRNA families were dramatically different in size. miRNA precursor (pre-miRNA) sequence analysis showed that tobacco pre-miRNAs greatly varied from 45 to 635 nt in length with an average of 141 ± 108 nt. We were also able to determine the presence of antisense miRNAs as well as miRNA clusters in tobacco. Using previously established protocols, a total of 1,225 potential target genes were predicted for the newly identified tobacco miRNAs. These target genes include transcription factors, DNA replication proteins, metabolic enzymes, as well as other gene targets necessary for proper plant maturation. The results of this study show that conserved miRNAs exist in tobacco and suggest that these miRNAs may play an important role in tobacco growth and development.

Generation, analysis, and transformation of macro-chloroplast Potato (Solanum tuberosum) lines for chloroplast biotechnology.

Chloroplast biotechnology is a route for novel crop metabolic engineering. The potential bio-confinement of transgenes, the high protein expression and the possibility to organize genes into operons represent considerable advantages that make chloroplasts valuable targets in agricultural biotechnology. In the last 3 decades, chloroplast genomes from a few economically important crops have been successfully transformed. The main bottlenecks that prevent efficient transformation in a greater number of crops include the dearth of proven selectable marker gene-selection combinations and tissue culture methods for efficient regeneration of transplastomic plants. The prospects of increasing organelle size are attractive from several perspectives, including an increase in the surface area of potential targets. As a proof-of-concept, we generated Solanum tuberosum (potato) macro-chloroplast lines overexpressing the tubulin-like GTPase protein gene FtsZ1 from Arabidopsis thaliana. Macro-chloroplast lines exhibited delayed growth at anthesis; however, at the time of harvest there was no significant difference in height between macro-chloroplast and wild-type lines. Macro-chloroplasts were successfully transformed by biolistic DNA-delivery and efficiently regenerated into homoplasmic transplastomic lines. We also demonstrated that macro-chloroplasts accumulate the same amount of heterologous protein than wild-type organelles, confirming efficient usage in plastid engineering. Advantages and limitations of using enlarge compartments in chloroplast biotechnology are discussed.

Rheology of transgenic switchgrass reveals practical aspects of biomass processing.

BACKGROUND: Mechanical properties of transgenic switchgrass have practical implications for biorefinery technologies. Presented are fundamentals for simple (thermo)mechanical measurements of genetically transformed switchgrass. Experimental basics are provided for the novice, where the intention is to promote collaboration between plant biologists and materials scientists. RESULTS: Stem sections were subjected to two stress modes: (1) torsional oscillation in the linear response region, and (2) unidirectional torsion to failure. Specimens were analyzed while submerged/saturated in ethylene glycol, simulating natural hydration and allowing experimental temperatures above 100 °C for an improved view of the lignin glass transition. Down-regulation of the 4-Coumarate:coenzyme A ligase gene (reduced lignin content and altered monomer composition) generally resulted in less stiff and weaker stems. These observations were associated with a reduction in the temperature and activation energy of the lignin glass transition, but surprisingly with no difference in the breadth and intensity of the tan Î´ signal. The results showed promise in further investigations of how rheological methods relate to stem lignin content, composition, and functional properties in the field and in bioprocessing. CONCLUSIONS: Measurements such as these are complicated by small specimen size; however, torsional rheometers (relatively common in polymer laboratories) are well suited for this task. As opposed to the expense and complication of relative humidity control, solvent-submersion rheological methods effectively reveal fundamental structure/property relationships in plant tissues. Demonstrated are low-strain linear methods, and also nonlinear yield and failure analysis; the latter is very uncommon for typical rheological equipment.

siRNAs regulate DNA methylation and interfere with gene and lncRNA expression in the heterozygous polyploid switchgrass.

BACKGROUND: Understanding the DNA methylome and its relationship with non-coding RNAs, including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), is essential for elucidating the molecular mechanisms underlying key biological processes in plants. Few studies have examined the functional roles of the DNA methylome in grass species with highly heterozygous polyploid genomes. RESULTS: We performed genome-wide DNA methylation profiling in the tetraploid switchgrass (Panicum virgatum L.) cultivar 'Alamo' using bisulfite sequencing. Single-base-resolution methylation patterns were observed in switchgrass leaf and root tissues, which allowed for characterization of the relationship between DNA methylation and mRNA, miRNA, and lncRNA populations. The results of this study revealed that siRNAs positively regulate DNA methylation of the mCHH sites surrounding genes, and that DNA methylation interferes with gene and lncRNA expression in switchgrass. Ninety-six genes covered by differentially methylated regions (DMRs) were annotated by GO analysis as being involved in stimulus-related processes. Functionally, 82% (79/96) of these genes were found to be hypomethylated in switchgrass root tissue. Sequencing analysis of lncRNAs identified two lncRNAs that are potential precursors of miRNAs, which are predicted to target genes that function in cellulose biosynthesis, stress regulation, and stem and root development. CONCLUSIONS: This study characterized the DNA methylome in switchgrass and elucidated its relevance to gene and non-coding RNAs. These results provide valuable genomic resources and references that will aid further epigenetic research in this important biofuel crop.

Genomic differentiation among natural populations of orang-utan (Pongo pygmaeus).

BACKGROUND: Orang-utans exist today in small isolated populations on the islands of Borneo (subspecies Pongo pygmaeus pygmaeus) and Sumatra (subspecies P. p. abelii). Although, on the basis of their morphological, behavioral and cytogenetical characteristics, the Bornean and Sumatran orang-utan populations are generally considered as two separate subspecies, there is no universal agreement as to whether their genetic differentiation is sufficient to consider and manage them as species, subspecies or population level taxonomic units. A more precise phylogenetic description would affect many conservation management decisions about captive and free-ranging orang-utans. RESULTS: We analyzed the amount and patterns of molecular genetic variation in orang-utan populations using cellular DNA from orang-utans from two locations in Sumatra and nine locations-representing four isolated populations-in Borneo. Genetic and phylogenetic analyses of mitochondrial DNA restriction fragment length polymorphisms, nuclear minisatellite (or variable number tandem repeat) loci and mitochondrial 16S ribosomal RNA sequences led to three major findings. First, the genetic distance and phylogenetic differentiation between Sumatran and Bornean orang-utans is large, greater than that between the common chimpanzee, Pan troglodytes, and the pygmy chimpanzee or bonobo, Pan paniscus. The genetic distance suggests that the two island subspecies diverged approximately 1.5-1.7 million years ago, well before the two islands separated and long enough for species-level differentiation. Second, there is considerable endemic genetic diversity within the Bornean and Sumatran orang-utan populations, suggesting that they have not experienced recent bottlenecks or founder effects. And third, there is little genetic differentiation among four geographically isolated populations of Bornean orang-utans, consistent with gene flow having occurred between them until recently. CONCLUSIONS: Our results are consistent with the view that the genetic differentiation between Sumatran and Bornean orang-utans has reached the level of distinct species. Furthermore, our findings indicate that there is not a genetic imperative for the separate management of geographically isolated Bornean populations.

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.

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.

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

BACKGROUND: Switchgrass (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 (g s), transpiration rate (Tr), intercellular CO2 concentration (Ci), and water use efficiency (WUE). RESULTS: SRAP 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. CONCLUSION: Based 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.

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 of plant microRNAs using expressed sequence tag analysis.

microRNAs (miRNAs) are a new class of small endogenous noncoding regulatory RNAs, which play an important function in plant growth, development, phase change, and response to environmental stress. Identifying miRNAs is the first step for investigating miRNA-mediated gene regulation and miRNA function. In this chapter, we describe a comprehensive comparative genomics-based expressed sequence tag (EST) analysis for identifying miRNAs from a wide range of plant species. EST analysis is based on the conservation of miRNA sequences and the stem-loop hairpin secondary structures of miRNAs. In this method, potential miRNAs will first be identified by EST analysis followed by confirmation using TaqMan(®) MicroRNA qRT-PCR. This method is simple and reliable with high efficiency. This method has also been widely adopted by many scientists around the world and several hundreds of miRNAs have been identified in many plant species using this method.