Genome-wide profiling of histone (H3) lysine 4 (K4) tri-methylation (me3) under drought, heat, and combined stresses in switchgrass.

BACKGROUND: Switchgrass (Panicum virgatum L.) is a warm-season perennial (C4) grass identified as an important biofuel crop in the United States. It is well adapted to the marginal environment where heat and moisture stresses predominantly affect crop growth. However, the underlying molecular mechanisms associated with heat and drought stress tolerance still need to be fully understood in switchgrass. The methylation of H3K4 is often associated with transcriptional activation of genes, including stress-responsive. Therefore, this study aimed to analyze genome-wide histone H3K4-tri-methylation in switchgrass under heat, drought, and combined stress. RESULTS: In total, ~ 1.3 million H3K4me3 peaks were identified in this study using SICER. Among them, 7,342; 6,510; and 8,536 peaks responded under drought (DT), drought and heat (DTHT), and heat (HT) stresses, respectively. Most DT and DTHT peaks spanned 0 to + 2000 bases from the transcription start site [TSS]. By comparing differentially marked peaks with RNA-Seq data, we identified peaks associated with genes: 155 DT-responsive peaks with 118 DT-responsive genes, 121 DTHT-responsive peaks with 110 DTHT-responsive genes, and 175 HT-responsive peaks with 136 HT-responsive genes. We have identified various transcription factors involved in DT, DTHT, and HT stresses. Gene Ontology analysis using the AgriGO revealed that most genes belonged to biological processes. Most annotated peaks belonged to metabolite interconversion, RNA metabolism, transporter, protein modifying, defense/immunity, membrane traffic protein, transmembrane signal receptor, and transcriptional regulator protein families. Further, we identified significant peaks associated with TFs, hormones, signaling, fatty acid and carbohydrate metabolism, and secondary metabolites. qRT-PCR analysis revealed the relative expressions of six abiotic stress-responsive genes (transketolase, chromatin remodeling factor-CDH3, fatty-acid desaturase A, transmembrane protein 14C, beta-amylase 1, and integrase-type DNA binding protein genes) that were significantly (P < 0.05) marked during drought, heat, and combined stresses by comparing stress-induced against un-stressed and input controls. CONCLUSION: Our study provides a comprehensive and reproducible epigenomic analysis of drought, heat, and combined stress responses in switchgrass. Significant enrichment of H3K4me3 peaks downstream of the TSS of protein-coding genes was observed. In addition, the cost-effective experimental design, modified ChIP-Seq approach, and analyses presented here can serve as a prototype for other non-model plant species for conducting stress studies.

Dig up tall fescue plastid genomes for the identification of morphotype-specific DNA variants.

BACKGROUND: Tall fescue (Festuca arundinacea Schreb.) is an important cool-season perennial grass species. Hexaploid tall fescue has three distinct morphotypes used either as forage or turf purposes. Its chloroplast genome is conserved due to it being maternally inherited to the next generation progenies. To identify morphotype-specific DNA markers and the genetic variations, plastid genomes of all three tall fescue morphotypes, i.e., Continental cv. Texoma MaxQ II, Rhizomatous cv. Torpedo, and Mediterranean cv. Resolute, have been sequenced using Illumina MiSeq sequencing platform. RESULTS: The plastid genomes of Continental-, Rhizomatous-, and Mediterranean tall fescue were assembled into circular master molecules of 135,283 bp, 135,336 bp, and 135,324 bp, respectively. The tall fescue plastid genome of all morphotypes contained 77 protein-coding, 20 tRNAs, four rRNAs, two pseudo protein-coding, and three hypothetical protein-coding genes. We identified 630 SNPs and 124 InDels between Continental and Mediterranean, 62 SNPs and 20 InDels between Continental and Rhizomatous, and 635 SNPs and 123 InDels between Rhizomatous and Mediterranean tall fescue. Only four InDels in four genes (ccsA, rps18, accD, and ndhH-p) were identified, which discriminated Continental and Rhizomatous plastid genomes from the Mediterranean plastid genome. Here, we identified and reported eight InDel markers (NRITCHL18, NRITCHL35, NRITCHL43, NRITCHL65, NRITCHL72, NRITCHL101, NRITCHL104, and NRITCHL110) from the intergenic regions that can successfully discriminate tall fescue morphotypes. Divergence time estimation revealed that Mediterranean tall fescue evolved approximately 7.09 Mya, whereas the divergence between Continental- and Rhizomatous tall fescue occurred about 0.6 Mya. CONCLUSIONS: To our knowledge, this is the first report of the assembled plastid genomes of Rhizomatous and Mediterranean tall fescue. Our results will help to identify tall fescue morphotypes at the time of pre-breeding and will contribute to the development of lawn and forage types of commercial varieties.

In-silico whole-genome sequence analysis of a halotolerant filamentous mangrove cyanobacterium revealed CRISPR-Cas systems with unique properties.

Novel CRISPR systems capable of cleaving both DNA and RNA are progressively emerging as attractive tools for genome manipulation of prokaryotic and eukaryotic organisms. We report specific characteristics of CRISPR systems present in Oxynema aestuarii AP17, a halotolerant, filamentous cyanobacterium and the second known member of the Oxynema genus. In-silico analyses of its whole-genome sequence revealed the presence of multiple Type I and Type III CRISPR loci with one Type I-G system previously unreported in cyanobacteria. We further identified the leader sequences at the 5' end of multiple CRISPR loci, many of which were distinct from previously reported cyanobacterial CRISPR leaders. Phylogenetic analyses of the O. aestuarii AP17 Cas1 proteins revealed two protein sequences that were unique and distantly related to other cyanobacterial Cas1 protein sequences. Our findings are significant because novel Class 1 CRISPR systems possess multi-subunit effectors and are highly flexible for repurposing by protein domain fusions made to the effector complex. Additionally, Type III CRISPRs are particularly useful for genome editing in certain extremophiles for which mesophilic Type II CRISPRs are ineffective.

Global analysis of switchgrass (Panicum virgatum L.) transcriptomes in response to interactive effects of drought and heat stresses.

BACKGROUND: Sustainable production of high-quality feedstock has been of great interest in bioenergy research. Despite the economic importance, high temperatures and water deficit are limiting factors for the successful cultivation of switchgrass in semi-arid areas. There are limited reports on the molecular basis of combined abiotic stress tolerance in switchgrass, particularly the combination of drought and heat stress. We used transcriptomic approaches to elucidate the changes in the response of switchgrass to drought and high temperature simultaneously. RESULTS: 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. Out of a total of 32,190 genes, we identified 3912, as drought (DT) responsive genes, 2339 and 4635 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. Gene ontology annotation identified the metabolic process as the significant term enriched in DTHT genes. Other biological processes identified in DTHT responsive genes included: response to water, photosynthesis, oxidation-reduction processes, and response to stress. KEGG pathway enrichment analysis on DT and DTHT responsive genes revealed that TFs and genes controlling phenylpropanoid pathways were important for individual as well as combined stress response. For example, hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT) from the phenylpropanoid pathway was induced by single DT and combinations of DTHT stress. 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.

Comparison of prevention of parent-to-child HIV transmission programme & national biennial HIV sentinel surveillance data for tracking HIV epidemic in India.

Background & objectives: HIV sentinel surveillance (HSS) among antenatal women in India has been used to track the epidemic for many years. However, reliable tracking at the local level is not possible as ANC sentinel sites are limited in number and cover a smaller sample size at each site. Prevention of parent-to-child-transmission (PPTCT) programme data has a potential advantage due to better geographical coverage, which could provide more precise HIV case estimates; therefore, we compared HSS ANC data with PPTCT programme data for HIV tracking. Methods: Out of the 499 surveillance sites, where HSS and PPTCT programme was being conducted in 2015, 210 sites (140 urban and 70 rural) were selected using a stratified random sampling method. HSS (n=72,981) and PPTCT (n=112,832) data records were linked confidentially. The sociodemographic characteristics of HSS and PPTCT attendees were compared. HIV prevalence from HSS ANC was compared with the PPTCT programme data using Chi-square test. State- and site-level correlation of HIV prevalence was also done. Concordance between HSS and PPTCT HIV positivity was estimated using kappa statistics. Results: The age distribution of HSS and PPTCT attendees was similar (range: 23 to 27 yr); however, HSS ANC participants were better educated, whereas PPTCT recorded a higher proportion of homemakers. The correlation of HIV prevalence between HSS and PPTCT was high (r=0.9) at the State level and moderate at the site level (r=0.7). The HIV positivity agreement between HSS ANC and PPTCT was good (kappa=0.633). A similar prevalence was reported across 26 States, whereas PPTCT had a significantly lower prevalence than HSS in three States where PPTCT coverage was low. Overall HIV prevalence was 0.31 per cent in HSS and 0.22 per cent in PPTCT (P<0.001). Interpretation & conclusions: High-quality PPTCT programme data can provide reliable HIV trends in India. An operational framework for PPTCT-based surveillance should be pilot-tested in a phased manner before replacing HSS with PPTCT.

Serendipita bescii promotes winter wheat growth and modulates the host root transcriptome under phosphorus and nitrogen starvation.

Serendipita vermifera ssp. bescii, hereafter referred to as S. bescii, is a root-associated fungus that promotes plant growth in both its native switchgrass host and a variety of monocots and dicots. Winter wheat (Triticum aestivum L.), a dual-purpose crop, used for both forage and grain production, significantly contributes to the agricultural economies of the Southern Great Plains, USA. In this study, we investigated the influence of S. bescii on growth and transcriptome regulation of nitrogen (N) and phosphorus (P) metabolism in winter wheat. Serendipita bescii significantly improved lateral root growth and forage biomass under a limited N or P regime. Further, S. bescii activated sets of host genes regulating N and P starvation responses. These genes include, root-specific auxin transport, strigolactone and gibberellin biosynthesis, degradation of phospholipids and biosynthesis of glycerolipid, downregulation of ammonium transport and nitrate assimilation, restriction of protein degradation by autophagy and subsequent N remobilization. All these genes are hypothesized to regulate acquisition, assimilation and remobilization of N and P. Based on transcriptional level gene regulation and physiological responses to N or P limitation, we suggest S. bescii plays a critical role in modulating stress imposed by limitation of these two critical nutrients in winter wheat.

Improved node culture methods for rapid vegetative propagation of switchgrass (Panicum virgatum L.).

BACKGROUND: Switchgrass (Panicum virgatum L.) is an important bioenergy and forage crop. The outcrossing nature of switchgrass makes it infeasible to maintain a genotype through sexual propagation. Current asexual propagation protocols in switchgrass have various limitations. An easy and highly-efficient vegetative propagation method is needed to propagate large natural collections of switchgrass genotypes for genome-wide association studies (GWAS). RESULTS: Micropropagation by node culture was found to be a rapid method for vegetative propagation of switchgrass. Bacterial and fungal contamination during node culture is a major cause for cultural failure. Adding the biocide, Plant Preservative Mixture (PPM, 0.2%), and the fungicide, Benomyl (5 mg/l), in the incubation solution after surface sterilization and in the culture medium significantly decreased bacterial and fungal contamination. In addition, "shoot trimming" before subculture had a positive effect on shoot multiplication for most genotypes tested. Using the optimized node culture procedure, we successfully propagated 330 genotypes from a switchgrass GWAS panel in three separate experiments. Large variations in shoot induction efficiency and shoot growth were observed among genotypes. Separately, we developed an in planta node culture method by stimulating the growth of aerial axillary buds into shoots directly on the parent plants, through which rooted plants can be generated within 6 weeks. By circumventing the tissue culture step and avoiding application of exterior hormones, the in planta node culture method is labor- and cost-efficient, easy to master, and has a high success rate. Plants generated by the in planta node culture method are similar to seedlings and can be used directly for various experiments. CONCLUSIONS: In this study, we optimized a switchgrass node culture protocol by minimizing bacterial and fungal contamination and increasing shoot multiplication. With this improved protocol, we successfully propagated three quarters of the genotypes in a diverse switchgrass GWAS panel. Furthermore, we established a novel and high-throughput in planta node culture method. Together, these methods provide better options for researchers to accelerate vegetative propagation of switchgrass.

Evaluation of antimicrobial activity of the extract of Streptomyces euryhalinus isolated from the Indian Sundarbans.

The discovery of new antimicrobials is the prime target in the fight against antimicrobial resistance. The continuous search for new lead compounds from bacteria of untapped and extreme ecosystems such as mangroves is currently being undertaken. This study describes the metabolite profiling of the Streptomyces euryhalinus culture extract. Previously, Streptomyces euryhalinus was isolated from the mangrove forest of Indian Sundarbans as a novel microorganism. The antimicrobial mechanism of action of Streptomyces euryhalinus culture extract against bacteria and fungi has been analyzed in this study. The gas chromatography-mass spectrometry profile of the ethyl acetate extract bacterial culture displayed the presence of several bioactive compounds with antibacterial, antifungal and antioxidant properties. The bacterial extract showed significant antimicrobial activity in terms of zone of inhibition, minimum inhibitory concentration, minimum bactericidal concentration, and minimum fungicidal concentration. Moreover, substantial capacity to alter or damage the inner membrane as well as the outer membrane of the gram-positive and gram-negative bacteria was exhibited by the bacterial extract. This membrane alteration or damaging potential of the extract is the mechanism of action. Biofilm formation inhibition property of the extract also signified its antimicrobial action, and possible use against resistant bacteria. The extract has shown notable activity against the virulence factors like prevention of hemolysis in bacteria and inhibition of secreted aspartyl proteinase in fungi. These functions of the bacterial extract have revealed the extent of its action in the prevention of infection by terminating the secretory virulence factors and by damaging the tissue.

Increased human-animal interface & emerging zoonotic diseases: An enigma requiring multi-sectoral efforts to address.

Increased human-animal interfaces impose threats on human life by creating scope for the emergence and resurgence of many infectious diseases. Over the last two decades, emergence of novel viral diseases such as SARS, influenza A/H1N1(09) pdm; MERS; Nipah virus disease; Ebola haemorrhagic fever and the current COVID-19 has resulted in massive outbreaks, epidemics and pandemics thereby causing profound losses of human life, health and economy. The current COVID-19 pandemic has affected more than 200 countries, reporting a global case load of 167,878,000 with 2 per cent mortality as on May 26, 2021. This has highlighted the importance of reducing human- animal interfaces to prevent such zoonoses. Rapid deforestation, shrinking of boundaries between human and animal, crisis for natural habitation, increasing demands for wildlife products and threat of extinction compounded by biodiversity narrowing compel to increased human-animal conflict and contact. Large quantities of animal waste generated due to animal agriculture may also allow rapid selection, amplification, dissemination of zoonotic pathogens and facilitate zoonotic pathogen adaptation and hinder host evolution for resistance. Public health system faces challenges to contain such epidemics due to inadequate understanding, poor preparedness, lack of interdisciplinary approach in surveillance and control strategy and deficient political commitments. Because the management measures are beyond the purview of health system alone, policy-level adaptation in the transdisciplinary issues are required, emphasizing the engagement of multiple stakeholders towards wildlife protection, alternative land use, community empowerment for natural resource management and regulation on business of wildlife products to ensure comprehensive one health practice.

Mother-to-child HIV transmission and its correlates in India: systematic review and meta-analysis.

BACKGROUND: In India, preventing mother-to-child transmission (PMTCT) of Human Immunodeficiency Virus (HIV) remains one of the foremost challenge in community health. Countrywide MTCT of HIV is estimated to be > 10,000 annually. Aims of present study are to find out the prevalence of HIV and correlates of HIV transmission among children given birth by HIV infected mother through systematic review along with meta-analysis. METHODS: All avaiable articles are retrieved using MEDLINE, Cochrane Library, Science Direct, EMBASE, Google Scholar and PUBMED following guidelines for Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). Joanna Briggs Institute Meta-Analysis of Statistics Assessment and Review Instrument (JBI-MAStARI) are applied to critically reviewing the selected articles. STATA 13.0 is used to preparation of forest plot for Meta-analysis. For assessment of heterogeneity and publication biases I2 statistics along with Begg and Mazumdar's test and Egger's tests are used. Odds ratio (OR) along with forest plots have been showing with 95% confidence interval (CI). RESULTS: All together 10 studies including 1537 pairs of mothers and new births are assessed in present meta-analysis. Present analysis revealed the prevalence of HIV due to MTCT in India as 8.76% (95% CI; 5.76, 12.31). Analysis of subgroups exhibit a higher pooled prevalence in eastern region of India, 10.83% (95% CI: 5.9, 17.81) and lower in in Western region in India, 6.37% (95% CI: 4.65, 8.49). Status of MTCT before and after initiation of universal ART are 10.23% (95% CI 6.61, 14.55) and 7.93% (95% CI 4.18, 12.76) respectively. Associated factors with MTCT of HIV include absence of maternal prevention of MTCT intervention, OR = 10.82 (95% CI: 5.28, 22.17), lacking in administration of infant ARV (antiretroviral), OR = 8.21 (95% CI: 4.82, 14.0) and absence of medical facility during childbirth OR = 3.73 (95% CI: 1.67, 8.33). CONCLUSIONS: In India, pooled HIV prevalence of MTCT as high as 8.78% (95% CI; 5.76, 12.31) among babies born to infected mothers warrants urgent need of focused intervention for providing ART (PMTCT intervention), ensuring proper infant ARV prophylaxis, and avoiding delivery without proper medical facility to pregnant women with HIV for reduction of occurrence in HIV transmission from mothers to children.

Motivation for adherence to antiretroviral therapy by transgender people living with hiv - A study in West Bengal, India.

The introduction of antiretroviral therapy (ART) has substantially improved the survival of persons infected with HIV. In India, the aggregated HIV prevalence among transgender (TG) community was found 7.5%. West Bengal, with over 30,000 TG population recorded HIV prevalence of 4.8%. The qualitative study was conducted in 2019 with the objective to explore the motivating factors for adherence to ART treatment among TG people living with HIV (PLHIV) in an ART center. Respondents were motivated PLHIVs on ART with suitable treatment adherence recorded. Data were inductively analyzed, using thematic analysis, to identify themes central to ART adherence. Five themes emerged with prime acknowledgment of respondents that ART medication is lifelong for their survival with receiving support. Notwithstanding the chronic nature of the disease, TG PLHIV patient stands a better chance of maintaining treatment adherence if they are involved in treatment plans with their community-based members' helping as peer navigators.

HIV Risk profile and its socio-demographic correlates among long-distance truckers in West Bengal, India: Evidence from national HIV sentinel surveillance 2017.

BACKGROUND: Long-distance truckers (LDTs) belong to a special sentinel group with potentially high risk of acquisition of HIV owing to their high mobility, sociodemographic vulnerability, and high-risk behaviors. OBJECTIVE: The objective is to estimate the prevalence of HIV and identifies its sociodemographic correlates among a representative population of LDTs in West Bengal, India. METHODS: Between May and July 2017, HIV Sentinel Surveillance (HSS) was conducted in West Bengal by the National AIDS Control Organization. A total of 749 LDTs were recruited for the study, were interviewed, and tested for HIV. Descriptive and logistic regression analysis of socio-demographics, sexual risk behavior, and HIV serostatus were performed using SAS 9.3.2. RESULTS: The prevalence of HIV among LDT was 1.2% (95% confidence interval [CI] = 0.4-2.0). Mean age was 32.8 years (standard deviation 8.5), 77.1% were currently married, 89.9% were literate, 85.7% visited HSS site for collecting condoms or seeking medical care and treatment, 53.1% were rural residents, 86.7% had sex at least once with a female partner other than wife in the past 6 months, 2.7% had sex with a male partner and 1.7% injected drugs for recreational purpose. Higher age (odds ratio [OR] = 1.1 [95% CI = 1.0-1.1]), literate (OR = 0.3 [95% CI = 0.1-0.9]), visiting HSS sites for collecting condoms or seeking medical care and treatment (adjusted OR [AOR] = 0.2 [95% CI = 0.1-0.6]), rural residence (OR = 0.2 [95% CI = 0.1-0.3]) and duration of stay in home (AOR = 1.3 [95% CI = 1.1-1.5]) were found to be significant predictors of having sex with a female partner other than wife. CONCLUSION: High HIV burden calls for urgency in the implementation of targeted intervention to minimize HIV risk among LDTs in West Bengal to fight against HIV/AIDS.

Human immunodeficiency virus prevalence and high-risk behavior of home-based and nonhome-based female sex workers in three high-prevalent North-Eastern States of India.

BACKGROUND: Female sex workers (FSWs) have been identified as an important target group for human immunodeficiency virus (HIV)/sexually transmitted infections prevention. OBJECTIVES: This study aimed to describe sociodemographic and sex work characteristics and to identify the risk factors for HIV infection with special focus on the variations between home-based (HB) and non-HB (NHB) FSWs in three high-prevalent North-Eastern states of India: Manipur, Mizoram, and Nagaland. METHODS: Data from the National Integrated Bio-Behavioural Surveillance (IBBS) conducted in India during 2014-2015 were utilized in the study. IBBS is a quantitative survey conducted among identified high risk sub within India. Logistic regression analyses were performed using SAS 9.3.2 to determine the distribution and associations of sociodemographics and risk behaviors with HIV seropositivity of HB and NHB FSWs. RESULTS: HIV prevalence was found higher among NHB FSWs compared to HB FSW (7.3% vs. 4.6%). The proportions of FSW among HB (66.7%) were in sex work for longer duration are significantly higher than for NHB (60.2%) while risk of HIV infection due to injecting drug use was higher in NHB FSW (11.7% vs. 8.7%). Reference to FSW who were currently married, those who were widowed/divorced/separated had 2.73-fold risk of HIV. FSW who did not have any other income source were associated with 1.73 times more risk of HIV infection. Injecting drugs user among FSW respondents had four times higher likelihood to be HIV positive. CONCLUSION: A substantial proportion of NHB FSWs is mobile in nature. Targeted interventions are required urgently to minimize HIV risk among those FSWs especially the widowed/divorced/separated, sex work is only income source and who used injecting drugs for nonmedical purpose.

Variation in injecting drug use behavior across different North-eastern States in India.

Prevalence of adult HIV infection in India is still high in certain North-eastern (NE) states, particularly among injecting drug users (IDUs). This study aims at exploring IDU behavior profile and their variation across the different states of NE region, India. Data were drawn from a population-based, cross-sectional survey of IDU in the integrated bio-behavioral surveillance from 2014 to 2015. A total of 4272 IDUs from four states (Manipur, Meghalaya, Mizoram, and Nagaland) aged ≥15 years were interviewed. Descriptive analysis was conducted to identify the variation in demographic and IDU behavior across four states. Youth predominance in Mizoram was evident by the mean age of initiation <18 years; 74% and 65% had the first exposure of any drug and injecting drug in <20 years. In Manipur and Nagaland, 60% and 49% of IDUs, respectively, were ≥30 years of age. These specific age groups may be targeted for IDU risk mitigation addressing the state-specific determinants.

Genome-wide identification of histone methylation (H3K9me2) and acetylation (H4K12ac) marks in two ecotypes of switchgrass (Panicum virgatum L.).

BACKGROUND: Histone modifications play a significant role in the regulation of transcription and various biological processes, such as development and regeneration. Though a few genomic (including DNA methylation patterns) and transcriptomic studies are currently available in switchgrass, the genome-wide distribution of histone modifications has not yet been studied to help elucidate gene regulation and its application to switchgrass improvement. RESULTS: This study provides a comprehensive epigenomic analyses of two contrasting switchgrass ecotypes, lowland (AP13) and upland (VS16), by employing chromatin immunoprecipitation sequencing (ChIP-Seq) with two histone marks (suppressive- H3K9me2 and active- H4K12ac). In this study, most of the histone binding was in non-genic regions, and the highest enrichment was seen between 0 and 2 kb regions from the transcriptional start site (TSS). Considering the economic importance and potential of switchgrass as a bioenergy crop, we focused on genes, transcription factors (TFs), and pathways that were associated with C4-photosynthesis, biomass, biofuel production, biotic stresses, and abiotic stresses. Using quantitative real-time PCR (qPCR) the relative expression of five genes selected from the phenylpropanoid-monolignol pathway showed preferential binding of acetylation marks in AP13 rather than in VS16. CONCLUSIONS: The genome-wide histone modifications reported here can be utilized in understanding the regulation of genes important in the phenylpropanoid-monolignol biosynthesis pathway, which in turn, may help understand the recalcitrance associated with conversion of biomass to biofuel, a major roadblock in utilizing lignocellulosic feedstocks.

Nanoparticle-induced enzyme pretreatment method for increased glucose production from lignocellulosic biomass under cold conditions.

BACKGROUND: Appropriate pretreatment strategies that fractionate sugarcane bagasse (SB) are essential for the successful use of this feedstock in ethanol production. In this paper, we investigate a purely enzymatic process to achieve increased production of reducing sugars (RS) from SB in the presence of MgO nanoparticles (MgN) subjected to a three-step sequential enzyme treatment. RESULT: Pretreatment of SB with protease activated by magnesium oxide nanoparticles (MgN-pro) at 95 °C showed an increase in amino acid production by 6.18-fold compared to the untreated enzyme set at the same temperature. High-performance liquid chromatography (HPLC) studies showed an 18-fold removal of lignin from the samples subjected to protease (+ MgN) treatment compared to untreated samples. When the MgN-pro pretreated samples were subjected to pretreatment with xylanase activated by magnesium oxide nanoparticles (MgN-xyl), more than 30-fold increased RS was produced at 8 °C compared to cellulase (cel) pretreated samples. Xylanase pretreated SB samples produced 1.82- and 1.91-fold increased reducing sugar and glucose respectively at 8 °C in the presence of MgN compared to untreated samples at the same temperature. CONCLUSION: The results presented here show the efficiency of the proposed method for improving the enzymatic digestibility of SB and explain the pretreatment action mechanism. These findings have potential implications in bio-ethanol, bio-fuel, and agro industries. © 2018 Society of Chemical Industry.

Analyses of methylomes of upland and lowland switchgrass (Panicum virgatum) ecotypes using MeDIP-seq and BS-seq.

BACKGROUND: Switchgrass is a crop with many desirable traits for bioenergy production. Plant genomes have high DNA methylation levels throughout genes and transposable elements and DNA methylation is known to play a role in silencing transposable elements. Here we analyzed methylomes in two switchgrass genotypes AP13 and VS16. AP13 is derived from a lowland ecotype and VS16, typically considered drought-tolerant, is derived from an upland ecotype, both genotypes are tetraploid (2n = 4× = 36). RESULTS: Methylated DNA immunoprecipitation-sequencing (MeDIP-seq) and bisulfite-sequencing (BS-seq) were used to profile DNA methylation in genomic features of AP13 and VS16. The methylation patterns in genes and transposable elements were similar to other plants, however, overall CHH methylation levels were comparatively low. Differentially methylated regions (DMRs) were assessed and a total of 1777 CG-DMRs, 573 CHG-DMRs, and 3 CHH-DMRs were detected between the two genotypes. TEs and their flanking regions were higher than that of genic regions. Different types of TEs had different methylation patterns, but the two LTRs (Copia and Gypsy) were similarly methylated, while LINEs and DNA transposons typically had different methylation patterns. MeDIP-seq data was compared to BS-seq data and most of the peaks generated by MeDIP-seq were confirmed to be highly methylated by BS-seq. CONCLUSIONS: DNA methylation in switchgrass genotypes obtained from the two ecotypes were found similar. Collinear gene pairs in two subgenomes (A and B) were not significantly differentially methylated. Both BS-seq and MeDIP-seq methodologies were found effective. Methylation levels were highest at CG and least in CHH. Increased DNA methylation was seen in TEs compared to genic regions. Exploitation of TE methylations can be a viable option in future crop improvement.

Comparative transcriptome profiling of upland (VS16) and lowland (AP13) ecotypes of switchgrass.

KEY MESSAGE: Transcriptomes of two switchgrass genotypes representing the upland and lowland ecotypes will be key tools in switchgrass genome annotation and biotic and abiotic stress functional genomics. Switchgrass (Panicum virgatum L.) is an important bioenergy feedstock for cellulosic ethanol production. We report genome-wide transcriptome profiling of two contrasting tetraploid switchgrass genotypes, VS16 and AP13, representing the upland and lowland ecotypes, respectively. A total of 268 million Illumina short reads (50 nt) were generated, of which, 133 million were obtained in AP13 and the rest 135 million in VS16. More than 90% of these reads were mapped to the switchgrass reference genome (V1.1). We identified 6619 and 5369 differentially expressed genes in VS16 and AP13, respectively. Gene ontology and KEGG pathway analysis identified key genes that regulate important pathways including C4 photosynthesis, photorespiration and phenylpropanoid metabolism. A series of genes (33) involved in photosynthetic pathway were up-regulated in AP13 but only two genes showed higher expression in VS16. We identified three dicarboxylate transporter homologs that were highly expressed in AP13. Additionally, genes that mediate drought, heat, and salinity tolerance were also identified. Vesicular transport proteins, syntaxin and signal recognition particles were seen to be up-regulated in VS16. Analyses of selected genes involved in biosynthesis of secondary metabolites, plant-pathogen interaction, membrane transporters, heat, drought and salinity stress responses confirmed significant variation in the relative expression reflected in RNA-Seq data between VS16 and AP13 genotypes. The phenylpropanoid pathway genes identified here are potential targets for biofuel conversion.

Transcriptome analysis in switchgrass discloses ecotype difference in photosynthetic efficiency.

BACKGROUND: Switchgrass, a warm-season perennial grass studied as a potential dedicated biofuel feedstock, is classified into two main taxa - lowland and upland ecotypes - that differ in morphology and habitat of adaptation. But there is limited information on their inherent molecular variations. RESULTS: Transcriptome analysis by RNA-sequencing (RNA-Seq) was conducted for lowland and upland ecotypes to document their gene expression variations. Mapping of transcriptome to the reference genome (Panicum virgatum v1.1) revealed that the lowland and upland ecotypes differ substantially in sets of genes transcribed as well as levels of expression. Differential gene expression analysis exhibited that transcripts related to photosynthesis efficiency and development and photosystem reaction center subunits were upregulated in lowlands compared to upland genotype. On the other hand, catalase isozymes, helix-loop-helix, late embryogenesis abundant group I, photosulfokinases, and S-adenosyl methionine synthase gene transcripts were upregulated in the upland compared to the lowlands. At ≥100x coverage and ≥5% minor allele frequency, a total of 25,894 and 16,979 single nucleotide polymorphism (SNP) markers were discovered for VS16 (upland ecotype) and K5 (lowland ecotype) against the reference genome. The allele combination of the SNPs revealed that the transition mutations are more prevalent than the transversion mutations. CONCLUSIONS: The gene ontology (GO) analysis of the transcriptome indicated lowland ecotype had significantly higher representation for cellular components associated with photosynthesis machinery controlling carbon fixation. In addition, using the transcriptome data, SNP markers were detected, which were distributed throughout the genome. The differentially expressed genes and SNP markers detected in this study would be useful resources for traits mapping and gene transfer across ecotypes in switchgrass breeding for increased biomass yield for biofuel conversion.

Development of an integrated transcript sequence database and a gene expression atlas for gene discovery and analysis in switchgrass (Panicum virgatum L.).

Switchgrass (Panicum virgatum L.) is a perennial C4 grass with the potential to become a major bioenergy crop. To help realize this potential, a set of RNA-based resources were developed. Expressed sequence tags (ESTs) were generated from two tetraploid switchgrass genotypes, Alamo AP13 and Summer VS16. Over 11.5 million high-quality ESTs were generated with 454 sequencing technology, and an additional 169 079 Sanger sequences were obtained from the 5' and 3' ends of 93 312 clones from normalized, full-length-enriched cDNA libraries. AP13 and VS16 ESTs were assembled into 77 854 and 30 524 unique transcripts (unitranscripts), respectively, using the Newbler and pave programs. Published Sanger-ESTs (544 225) from Alamo, Kanlow, and 15 other cultivars were integrated with the AP13 and VS16 assemblies to create a universal switchgrass gene index (PviUT1.2) with 128 058 unitranscripts, which were annotated for function. An Affymetrix cDNA microarray chip (Pvi_cDNAa520831) containing 122 973 probe sets was designed from PviUT1.2 sequences, and used to develop a Gene Expression Atlas for switchgrass (PviGEA). The PviGEA contains quantitative transcript data for all major organ systems of switchgrass throughout development. We developed a web server that enables flexible, multifaceted analyses of PviGEA transcript data. The PviGEA was used to identify representatives of all known genes in the phenylpropanoid-monolignol biosynthesis pathway.