Identification, characterization, and gene expression analysis of nucleotide binding site (NB)-type resistance gene homologues in switchgrass.
ABSTRACT: 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.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.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.
Project description:Resistance gene homologs (RGHs) were isolated from the switchgrass variety Alamo by a combination of polymerase chain reaction and expressed sequence tag (EST) database mining. Fifty-eight RGHs were isolated by polymerase chain reaction and 295 RGHs were identified in 424,545 switchgrass ESTs. Four nucleotide binding site--leucine-rich repeat RGHs were selected to investigate RGH haplotypic diversity in seven switchgrass varieties chosen for their representation of a broad range of the switchgrass germplasm. Lowland and upland ecotypes were found to be less similar, even from nearby populations, than were more distant populations with similar growth environments. Most (83.5%) of the variability in these four RGHs was found to be attributable to the within-population component. The difference in nucleotide diversity between and within populations was observed to be small, whereas this diversity is maintained to similar degrees at both population and ecotype levels. The results also revealed that the analyzed RGHs were under positive selection in the studied switchgrass accessions. Intragenic recombination was detected in switchgrass RGHs, thereby demonstrating an active genetic process that has the potential to generate new resistance genes with new specificities that might act against newly-arising pathogen races.
Project description:BACKGROUND: Cucumber is an important vegetable crop that is susceptible to many pathogens, but no disease resistance (R) genes have been cloned. The availability of whole genome sequences provides an excellent opportunity for systematic identification and characterization of the nucleotide binding and leucine-rich repeat (NB-LRR) type R gene homolog (RGH) sequences in the genome. Cucumber has a very narrow genetic base making it difficult to construct high-density genetic maps. Development of a consensus map by synthesizing information from multiple segregating populations is a method of choice to increase marker density. As such, the objectives of the present study were to identify and characterize NB-LRR type RGHs, and to develop a high-density, integrated cucumber genetic-physical map anchored with RGH loci. RESULTS: From the Gy14 draft genome, 70 NB-containing RGHs were identified and characterized. Most RGHs were in clusters with uneven distribution across seven chromosomes. In silico analysis indicated that all 70 RGHs had EST support for gene expression. Phylogenetic analysis classified 58 RGHs into two clades: CNL and TNL. Comparative analysis revealed high-degree sequence homology and synteny in chromosomal locations of these RGH members between the cucumber and melon genomes. Fifty-four molecular markers were developed to delimit 67 of the 70 RGHs, which were integrated into a genetic map through linkage analysis. A 1,681-locus cucumber consensus map including 10 gene loci and spanning 730.0 cM in seven linkage groups was developed by integrating three component maps with a bin-mapping strategy. Physically, 308 scaffolds with 193.2 Mbp total DNA sequences were anchored onto this consensus map that covered 52.6% of the 367 Mbp cucumber genome. CONCLUSIONS: Cucumber contains relatively few NB-LRR RGHs that are clustered and unevenly distributed in the genome. All RGHs seem to be transcribed and shared significant sequence homology and synteny with the melon genome suggesting conservation of these RGHs in the Cucumis lineage. The 1,681-locus consensus genetic-physical map developed and the RGHs identified and characterized herein are valuable genomics resources that may have many applications such as quantitative trait loci identification, map-based gene cloning, association mapping, marker-assisted selection, as well as assembly of a more complete cucumber genome.
Project description:BACKGROUND: Global warming predictions indicate that temperatures will increase by another 2-6°C by the end of this century. High temperature is a major abiotic stress limiting plant growth and productivity in many areas of the world. Switchgrass (Panicum virgatum L.) is a model herbaceous bioenergy crop, due to its rapid growth rate, reliable biomass yield, minimal requirements of water and nutrients, adaptability to grow on marginal lands and widespread distribution throughout North America. The effect of high temperature on switchgrass physiology, cell wall composition and biomass yields has been reported. However, there is void in the knowledge of the molecular responses to heat stress in switchgrass. RESULTS: We conducted long-term heat stress treatment (38°/30°C, day/night, for 50 days) in the switchgrass cultivar Alamo. A significant decrease in the plant height and total biomass was evident in the heat stressed plants compared to controls. Total RNA from control and heat stress samples were used for transcriptome analysis with switchgrass Affymetrix genechips. Following normalization and pre-processing, 5365 probesets were identified as differentially expressed using a 2-fold cutoff. Of these, 2233 probesets (2000 switchgrass unigenes) were up-regulated, and 3132 probesets (2809 unigenes) were down-regulated. Differential expression of 42 randomly selected genes from this list was validated using RT-PCR. Rice orthologs were retrieved for 78.7% of the heat stress responsive switchgrass probesets. Gene ontology (GOs) enrichment analysis using AgriGO program showed that genes related to ATPase regulator, chaperone binding, and protein folding was significantly up-regulated. GOs associated with protein modification, transcription, phosphorus and nitrogen metabolic processes, were significantly down-regulated by heat stress. CONCLUSIONS: Plausible connections were identified between the identified GOs, physiological responses and heat response phenotype observed in switchgrass plants. Comparative transcriptome analysis in response to heat stress among four monocots - switchgrass, rice, wheat and maize identified 16 common genes, most of which were associated with protein refolding processes. These core genes will be valuable biomarkers for identifying heat sensitive plant germplasm since they are responsive to both short duration as well as chronic heat stress treatments, and are also expressed in different plant growth stages and tissue types.
Project description:A greenhouse experiment was carried out to investigate the effects of arbuscular mycorrhizal fungi (AMF) on the growth, P and Cd concentrations and bioenergy quality-related factors of five cultivars of switchgrass, including three lowland cultivars (Alamo (Ala), Kanlow (Kan), Performer (Per)) and two highland cultivars (Blackwell (Bw), Summer (Sum)), with 0, 1 and 10 mg/kg Cd addition levels. The results showed that AMF inoculation notably increased the biomass and P concentrations of all the cultivars. The Cd concentrations in the roots were higher than those in the shoots of all cultivars irrespective of inoculation, but the AMF had different effects on Cd accumulation in highland and lowland cultivars. AMF inoculation decreased the shoot and root concentrations in Ala and Kan, increased the shoot and root concentrations of Cd in Bw and Sum, and increased shoot Cd concentrations and decreased root Cd concentrations in Per. The highest Cd concentrations were detected in the roots of Bw and in the shoots of Sum with AMF symbiosis. Bw contained the highest total extracted Cd which was primarily in the roots. Ala had the second highest extracted Cd in the shoots, reaching 32% with 1 mg/kg of added Cd, whereas Sum had the lowest extracted Cd. AMF symbiosis had varied effects on bioenergy quality-related factors: for example, AMF decreased the ash lignin content in Ala and the C/N in Sum, increased the nitrogen, gross calorie values, and maintained the hemicellulose and cellulose contents in all cultivars with all tested concentrations of Cd. A principal component analysis (PCA) showed that AMF inoculation could enhance, weaken or transform (positive-negative, PC1-PC2) the correlations of these factors with the principle components under Cd stress. Therefore, AMF symbiosis enhanced the growth of different cultivars of switchgrass, increased/decreased Cd accumulation, promoted Cd extraction, and regulated the bioenergy quality-related factors in Cd-polluted areas. Bw is a suitable cultivar for phytostabilization due to high root Cd stabilization, whereas Ala is an appropriate cultivar for phytoremediation of less polluted areas because of its high Cd extraction and excellent bioenergy quality.
Project description:<h4>Background</h4>Soil salinization is a major limiting factor for crop cultivation. Switchgrass is a perennial rhizomatous bunchgrass that is considered an ideal plant for marginal lands, including sites with saline soil. Here we investigated the physiological responses and transcriptome changes in the roots of Alamo (alkaline-tolerant genotype) and AM-314/MS-155 (alkaline-sensitive genotype) under alkaline salt stress.<h4>Results</h4>Alkaline salt stress significantly affected the membrane, osmotic adjustment and antioxidant systems in switchgrass roots, and the ASTTI values between Alamo and AM-314/MS-155 were divergent at different time points. A total of 108,319 unigenes were obtained after reassembly, including 73,636 unigenes in AM-314/MS-155 and 65,492 unigenes in Alamo. A total of 10,219 DEGs were identified, and the number of upregulated genes in Alamo was much greater than that in AM-314/MS-155 in both the early and late stages of alkaline salt stress. The DEGs in AM-314/MS-155 were mainly concentrated in the early stage, while Alamo showed greater advantages in the late stage. These DEGs were mainly enriched in plant-pathogen interactions, ubiquitin-mediated proteolysis and glycolysis/gluconeogenesis pathways. We characterized 1480 TF genes into 64 TF families, and the most abundant TF family was the C2H2 family, followed by the bZIP and bHLH families. A total of 1718 PKs were predicted, including CaMK, CDPK, MAPK and RLK. WGCNA revealed that the DEGs in the blue, brown, dark magenta and light steel blue 1 modules were associated with the physiological changes in roots of switchgrass under alkaline salt stress. The consistency between the qRT-PCR and RNA-Seq results confirmed the reliability of the RNA-seq sequencing data. A molecular regulatory network of the switchgrass response to alkaline salt stress was preliminarily constructed on the basis of transcriptional regulation and functional genes.<h4>Conclusions</h4>Alkaline salt tolerance of switchgrass may be achieved by the regulation of ion homeostasis, transport proteins, detoxification, heat shock proteins, dehydration and sugar metabolism. These findings provide a comprehensive analysis of gene expression dynamic and act network induced by alkaline salt stress in two switchgrass genotypes and contribute to the understanding of the alkaline salt tolerance mechanism of switchgrass and the improvement of switchgrass germplasm.
Project description:Climate changes, including chronic changes in precipitation amounts, will influence plant physiology and growth. However, such precipitation effects on switchgrass, a major bioenergy crop, have not been well investigated. We conducted a two-year precipitation simulation experiment using large pots (95 L) in an environmentally controlled greenhouse in Nashville, TN. Five precipitation treatments (ambient precipitation, and -50%, -33%, +33%, and +50% of ambient) were applied in a randomized complete block design with lowland "Alamo" switchgrass plants one year after they were established from tillers. The growing season progression of leaf physiology, tiller number, height, and aboveground biomass were determined each growing season. Precipitation treatments significantly affected leaf physiology, growth, and aboveground biomass. The photosynthetic rates in the wet (+50% and +33%) treatments were significantly enhanced by 15.9% and 8.1%, respectively, than the ambient treatment. Both leaf biomass and plant height were largely increased, resulting in dramatically increases in aboveground biomass by 56.5% and 49.6% in the +50% and +33% treatments, respectively. Compared to the ambient treatment, the drought (-33% and -50%) treatments did not influence leaf physiology, but the -50% treatment significantly reduced leaf biomass by 37.8%, plant height by 16.3%, and aboveground biomass by 38.9%. This study demonstrated that while switchgrass in general is a drought tolerant grass, severe drought significantly reduces Alamo's growth and biomass, and that high precipitation stimulates its photosynthesis and growth.
Project description:Purpose: Increasing biomass yield and quality of feedstock have been a recent interest in switchgrass research. Despite the economic importance of switchgrass, increasing temperature and water deficit are limiting factors to the cultivation of bioenergy crops in the semi-arid areas. The effect of individual drought or heat stress has been studied separately in switchgrass. However, there is relatively limited or no report on the molecular basis of combined abiotic stress tolerance in switchgrass particularly the combination of drought and heat stress. We used RNA-Seq approaches to elucidate the transcriptome changes of switchgrass in response to drought and high temperatures simultaneously. Method: We conducted solely drought treatment in switchgrass plant Alamo AP13 by withholding water after 45 days of growing. For the combination of drought and heat effect, heat treatment (35 °C/25 °C day/night) was imposed after 72 h of the initiation of drought. Samples were collected at 0 h, 72 h, 96 h, 120 h, 144 h, and 168 h after treatment imposition, total RNA was extracted, and RNA-Seq conducted. Results:Out of total 32,190 genes, we identified 3,912, as DT responsive genes, 2,339 and 4,635 as , heat (HT) and drought and heat (DTHT) responsive genes, respectively. There were 209, 106, and 220 transcription factors (TFs) differentially expressed under DT, HT and DTHT respectively Conclusion: Through RNA-Seq analysis, we have identified unique and overlapping genes in response to DT and combined DTHT stress in switchgrass. The combination of DT and HT stress may affect the photosynthetic machinery and phenylpropanoid pathway of switchgrass which negatively impacts lignin synthesis and biomass production of switchgrass. The biological function of genes identified particularly in response to DTHT stress could further be confirmed by techniques such as single point mutation or RNAi. Overall design: Leaf mRNA profiling of AP13 genotype, a selection from lowland cultivar ‘Alamo’ during drought and heat stress at time points, 0h, 72h, 96h, 120h, 144h and 168h.
Project description:In this study we were interested in identifying the gene networks that are responsive to chronic heat treatment in a switchgrass cultivar that is widely grown in the south-western USA and where 38C day temperatures have been reported for more than 100 days in the recent years. Switchgrass Alamo plants were subjected to chronic heat stress for 50 days (38 C/30C; day/night) or maintained under optimal conditions (28C/20C). Leaves were collected at the end of the heat regime for transcriptome analysis.
Project description: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.
Project description:Switchgrass is an important bioenergy crop typically grown in marginal lands, where the plants must often deal with abiotic stresses such as drought and salt. Alamo is known to be more tolerant to both stress types than Dacotah, two ecotypes of switchgrass. Understanding of their stress response and adaptation programs can have important implications to engineering more stress tolerant plants. We present here a computational study by analyzing time-course transcriptomic data of the two ecotypes to elucidate and compare their regulatory systems in response to drought and salt stresses. A total of 1,693 genes (target genes or TGs) are found to be differentially expressed and possibly regulated by 143 transcription factors (TFs) in response to drought stress together in the two ecotypes. Similarly, 1,535 TGs regulated by 110 TFs are identified to be involved in response to salt stress. Two regulatory networks are constructed to predict their regulatory relationships. In addition, a time-dependent hidden Markov model is derived for each ecotype responding to each stress type, to provide a dynamic view of how each regulatory network changes its behavior over time. A few new insights about the response mechanisms are predicted from the regulatory networks and the time-dependent models. Comparative analyses between the network models of the two ecotypes reveal key commonalities and main differences between the two regulatory systems. Overall, our results provide new information about the complex regulatory mechanisms of switchgrass responding to drought and salt stresses.