Use of hydroponics-based evaluation for phenotyping tolerance/susceptibility to the aphid, Uroleucon compositae and inheritance analysis of aphid tolerance in a global germplasm collection of Carthamus tinctorius L. (Safflower).

Carthamus tinctorius L. (Safflower) is an important oilseed crop that is cultivated globally. Aphids are a serious pest of safflower and cause significant yield losses of up to 80% due to their ability to multiply rapidly by parthenogenesis. In this study, we report the identification of an aphid-tolerant accession in safflower following screening of a representative global germplasm collection of 327 accessions from 37 countries. Field-based screening methods gave inconsistent and ambiguous results for aphid tolerance between natural and controlled infestation assays and required ~ 3 months for completion. Therefore, we used a rapid, high-throughput hydroponics-based assay system that allows phenotyping of aphid tolerance/susceptibility in a large number of plants in a limited area, significantly reduces the time required to ~ 45 days and avoids inconsistencies observed in field-based studies. We identified one accession out of the 327 tested germplasm lines that demonstrated aphid tolerance in field-based natural and controlled infestation studies and also using the hydroponics approach. Inheritance analysis of the trait was conducted using the hydroponics approach on F1 and F2 progeny generated from a cross between the tolerant and susceptible lines. Aphid-tolerance was observed to be a dominant trait governed by a single locus/gene that can be mobilized after mapping into cultivated varieties of safflower. The hydroponics-based assay described in this study would be very useful for studying the molecular mechanism of aphid-tolerance in safflower and can also be used for bioassays in several other crops that are amenable to hydroponics-based growth. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01467-0.

Different GCMs yet similar outcome: predicting the habitat distribution of Shorea robusta C.F. Gaertn. in the Indian Himalayas using CMIP5 and CMIP6 climate models.

Climate change impact on the habitat distribution of umbrella species presents a critical threat to the entire regional ecosystem. This is further perilous if the species is economically important. Sal (Shorea robusta C.F. Gaertn.), a climax forest forming Central Himalayan tree species, is one of the most valuable timber species and provides several ecological services. Sal forests are under threat due to over-exploitation, habitat destruction, and climate change. Sal's poor natural regeneration and its unimodal density-diameter distribution in the region illustrate the peril to its habitat. We, modelled the current as well as future distribution of suitable sal habitats under different climate scenarios using 179 sal occurrence points and 8 bioclimatic environmental variables (non-collinear). The CMIP5-based RCP4.5 and CMIP6-based SSP245 climate models under 2041-2060 and 2061-2080 periods were used to predict the impact of climate change on sal's future potential distribution area. The niche model results predict the mean annual temperature and precipitation seasonality as the most influential sal habitat governing variables in the region. The current high suitability region for sal was 4.36% of the total geographic area, which shows a drastic decline to 1.31% and 0.07% under SSP245 for 2041-60 and 2061-80, respectively. The RCP-based models predicted more severe impact than SSP; however, both RCP and SSP models showed complete loss of high suitability regions and overall shift of species northwards in the Uttarakhand state. We could identify the current and future suitable habitats for conserving sal population through assisted regeneration and management of other regional issues.

"Is Omicron mild"? Testing this narrative with the mutational landscape of its three lineages and response to existing vaccines and therapeutic antibodies.

SARS-CoV-2 Omicron with its lineages BA.1, BA.2, and BA.3 has triggered a fresh wave of Covid-19 infections. Though, Omicron has, so far, produced mild symptoms, its genome contains 60 mutations including 37 in the spike protein and 15 in the receptor-binding domain. Thirteen sites conserved in previous SARS-CoV-2 variants carry mutations in Omicron. Many mutations have shown evolution under positive selection. Omicron's giant mutational leap has raised concerns as there are signs of higher virus infectivity rate, pathogenesis, reinfection, and immune evasion. Preliminary studies have reported waning of immunity after two-dose primary vaccine regime, need for the boosters, folds reduction in vaccine effectiveness and neutralizing antibodies even after boosting and significant neutralization resistance with the therapeutic monoclonal, polyclonal, and convalescent antibodies against Omicron. The narrative that "Omicron is mild," therefore, needs time to be tested with a deeper, scientific dwelling into the facts.

Predicting the potential suitable habitats of genus Nymphaea in India using MaxEnt modeling.

Modeling and mapping the distribution of suitable habitats of aquatic plants are critical for assessing the impact of factors like changing climate on species habitat range shifts, declines, and expansions. Nymphaea is an aquatic perennial herb considered valuable because of its ornamental, economic, medicinal, and ecological importance. In India, the geographical distribution of Nymphaea is diverse, and the suitable habitats of individual species are vulnerable to the changing climate and global warming effects. Despite its increased vulnerability, only a few limited conservation efforts in aquatic environments are being made to date. In several places, the distribution of Nymphaea has been impacted by both anthropogenic and climate-related disturbances. A comprehensive strategy will be needed to meet the socio-ecological challenge of Nymphaea conservation. In this study, we employed maximum entropy (MaxEnt) method to assess how climate change affects the distribution of Nymphaea suitable habitat. The occurrence records of Nymphaea were collected from primary surveys, Global Biodiversity Information Facility (GBIF), and published works. Bioclimatic variables obtained from the Coupled Model Intercomparison Project (CMIP6) were employed as predictor variables in distribution modeling. The projections were made using three SSPs (stringent mitigation scenarios) for the future period of 2050. Our results showed shifts in the suitability ranges of Nymphaea under different projection scenarios. The study provides information about the distribution of suitable habitats for Nymphaea in India, which may be helpful for ongoing efforts to conserve and manage the aquatic plants, particularly in areas that are losing suitable climate conditions.

Abiotic stress-mediated modulation of the chromatin landscape in Arabidopsis thaliana.

Limited information is available on abiotic stress-mediated alterations of chromatin conformation influencing gene expression in plants. In order to characterize the effect of abiotic stresses on changes in chromatin conformation, we employed FAIRE-seq (formaldehyde-assisted isolation of regulatory element sequencing) and DNase-seq to isolate accessible regions of chromatin from Arabidopsis thaliana seedlings exposed to either heat, cold, salt, or drought stress. Approximately 25% of regions in the Arabidopsis genome were captured as open chromatin, the majority of which included promoters and exons. A large proportion of chromatin regions apparently did not change their conformation in response to any of the four stresses. Digital footprints present within these regions had differential enrichment of motifs for binding of 43 different transcription factors. Further, in contrast to drought and salt stress, both high and low temperature treatments resulted in increased accessibility of the chromatin. Also, pseudogenes attained increased chromatin accessibility in response to cold and drought stresses. The highly accessible and inaccessible chromatin regions of seedlings exposed to drought stress correlated with the Ser/Thr protein kinases (MLK1 and MLK2)-mediated reduction and increase in H3 phosphorylation (H3T3Ph), respectively. The presented results provide a deeper understanding of abiotic stress-mediated chromatin modulation in plants.

Occurrence of subdioecy and scarcity of gender-specific markers reveal an ongoing transition to dioecy in Himalayan seabuckthorn (Hippophae rhamnoides ssp. turkestanica).

Dioecy and the dynamics of its evolution are intensely investigated aspects of plant reproduction. Seabuckthorn (Hippophae rhamnoides ssp. turkestanica) is an alpine shrub growing wild in certain parts of western Himalaya. The previous studies have reported heteromorphic sex chromosomes in the species and yet marker-based studies indicate high similarity between the male and female genomes. Lack of information on sexual system in the species has further complicated the situation. A systematic study was thus undertaken to understand the sexual system in seabuckthorn and to discern the extent of similarity/dissimilarity between the male and female genomes by generating a large number of markers using amplified fragment length polymorphism and representational difference analysis. Floral biology and regular monitoring of species revealed the presence of polygamomonoecious (PGM) plants in most populations at a low percentage (~2-4%). PGM plants showed low pollen production and overall low fertility, suggesting a monoecy-paradioecy pathway at function. The results of the marker study demonstrated that there are limited differences between male and female genomes and these differences were not uniform across the populations in the Leh-Ladakh region, especially when the geographical distance increases. Results also suggest that a dynamic partitioning of genomes is operational between the two genders of seabuckthorn and differences are not homogenized across the populations. Both reproductive biology-based and DNA marker-based studies indicate that genders have separated recently. The present study proposes seabuckthorn as a promising model system to study evolution of dioecy and sex determination.

Cytogenetic and molecular evidences revealing genomic changes after autopolyploidization: a case study of synthetic autotetraploid Phlox drummondii Hook.

Polyploidy is known to be common in plants; indeed most of the world's economically important crop plants are polyploids. Recent studies revealed extensive genomic changes in synthetic polyploids after genome doubling, although most of the information available is with regards to allopolyploids and little information have been generated in autopolyploids. In the present study, we used Phlox drummondii Hooker (2n = 2x = 14) as a model plant to observe genomic changes, if any, in synthetic autopolyploids. Colchitetraploids were produced and followed through different generations (C0, C1, C2 and C3). Male meiosis analysis showed differences between the frequency of both quadrivalents and bivalents from C0 to C2 generations. RAPD analysis revealed 2.8, 1.6, 2.1 and 3.2% polymorphism in C0, C1, C2 and C3 colchitetraploids respectively. The polymorphic fragments were further characterized after cloning. Dot blot assay was performed to confirm high copy/low copy nature of fragments showing variation. The analysis revealed changes in both repetitive and non-repetitive regions. Out of the six fragments only one fragment T01 was found to be of high copy, while four fragments were of the moderate copy and one fragment of the low copy nature.

Global insights into high temperature and drought stress regulated genes by RNA-Seq in economically important oilseed crop Brassica juncea.

BACKGROUND: Brassica juncea var. Varuna is an economically important oilseed crop of family Brassicaceae which is vulnerable to abiotic stresses at specific stages in its life cycle. Till date no attempts have been made to elucidate genome-wide changes in its transcriptome against high temperature or drought stress. To gain global insights into genes, transcription factors and kinases regulated by these stresses and to explore information on coding transcripts that are associated with traits of agronomic importance, we utilized a combinatorial approach of next generation sequencing and de-novo assembly to discover B. juncea transcriptome associated with high temperature and drought stresses. RESULTS: We constructed and sequenced three transcriptome libraries namely Brassica control (BC), Brassica high temperature stress (BHS) and Brassica drought stress (BDS). More than 180 million purity filtered reads were generated which were processed through quality parameters and high quality reads were assembled de-novo using SOAPdenovo assembler. A total of 77750 unique transcripts were identified out of which 69,245 (89%) were annotated with high confidence. We established a subset of 19110 transcripts, which were differentially regulated by either high temperature and/or drought stress. Furthermore, 886 and 2834 transcripts that code for transcription factors and kinases, respectively, were also identified. Many of these were responsive to high temperature, drought or both stresses. Maximum number of up-regulated transcription factors in high temperature and drought stress belonged to heat shock factors (HSFs) and dehydration responsive element-binding (DREB) families, respectively. We also identified 239 metabolic pathways, which were perturbed during high temperature and drought treatments. Analysis of gene ontologies associated with differentially regulated genes forecasted their involvement in diverse biological processes. CONCLUSIONS: Our study provides first comprehensive discovery of B. juncea transcriptome under high temperature and drought stress conditions. Transcriptome resource generated in this study will enhance our understanding on the molecular mechanisms involved in defining the response of B. juncea against two important abiotic stresses. Furthermore this information would benefit designing of efficient crop improvement strategies for tolerance against conditions of high temperature regimes and water scarcity.

Identification and characterization of miRNAome in root, stem, leaf and tuber developmental stages of potato (Solanum tuberosum L.) by high-throughput sequencing.

BACKGROUND: MicroRNAs (miRNAs) are ubiquitous components of endogenous plant transcriptome. miRNAs are small, single-stranded and ~21 nt long RNAs which regulate gene expression at the post-transcriptional level and are known to play essential roles in various aspects of plant development and growth. Previously, a number of miRNAs have been identified in potato through in silico analysis and deep sequencing approach. However, identification of miRNAs through deep sequencing approach was limited to a few tissue types and developmental stages. This study reports the identification and characterization of potato miRNAs in three different vegetative tissues and four stages of tuber development by high throughput sequencing. RESULTS: Small RNA libraries were constructed from leaf, stem, root and four early developmental stages of tuberization and subjected to deep sequencing, followed by bioinformatics analysis. A total of 89 conserved miRNAs (belonging to 33 families), 147 potato-specific miRNAs (with star sequence) and 112 candidate potato-specific miRNAs (without star sequence) were identified. The digital expression profiling based on TPM (Transcripts Per Million) and qRT-PCR analysis of conserved and potato-specific miRNAs revealed that some of the miRNAs showed tissue specific expression (leaf, stem and root) while a few demonstrated tuberization stage-specific expressions. Targets were predicted for identified conserved and potato-specific miRNAs, and predicted targets of four conserved miRNAs, miR160, miR164, miR172 and miR171, which are ARF16 (Auxin Response Factor 16), NAM (NO APICAL MERISTEM), RAP1 (Relative to APETALA2 1) and HAM (HAIRY MERISTEM) respectively, were experimentally validated using 5' RLM-RACE (RNA ligase mediated rapid amplification of cDNA ends). Gene ontology (GO) analysis for potato-specific miRNAs was also performed to predict their potential biological functions. CONCLUSIONS: We report a comprehensive study of potato miRNAs at genome-wide level by high-throughput sequencing and demonstrate that these miRNAs have tissue and/or developmental stage-specific expression profile. Also, predicted targets of conserved miRNAs were experimentally confirmed for the first time in potato. Our findings indicate the existence of extensive and complex small RNA population in this crop and suggest their important role in pathways involved in diverse biological processes, including tuber development.

Understanding role of roots in plant response to drought: Way forward to climate-resilient crops.

Drought stress leads to a significant amount of agricultural crop loss. Thus, with changing climatic conditions, it is important to develop resilience measures in agricultural systems against drought stress. Roots play a crucial role in regulating plant development under drought stress. In this review, we have summarized the studies on the role of roots and root-mediated plant responses. We have also discussed the importance of root system architecture (RSA) and the various structural and anatomical changes that it undergoes to increase survival and productivity under drought. Various genes, transcription factors, and quantitative trait loci involved in regulating root growth and development are also discussed. A summarization of various instruments and software that can be used for high-throughput phenotyping in the field is also provided in this review. More comprehensive studies are required to help build a detailed understanding of RSA and associated traits for breeding drought-resilient cultivars.

Variable resource allocation pattern, biased sex-ratio, and extent of sexual dimorphism in subdioecious Hippophae rhamnoides.

Evolutionary maintenance of dioecy is a complex phenomenon and varies by species and underlying pathways. Also, different sexes may exhibit variable resource allocation (RA) patterns among the vegetative and reproductive functions. Such differences are reflected in the extent of sexual dimorphism. Though rarely pursued, investigation on plant species harbouring intermediate sexual phenotypes may reveal useful information on the strategy pertaining to sex-ratios and evolutionary pathways. We studied H. rhamnoides ssp. turkestanica, a subdioecious species with polygamomonoecious (PGM) plants, in western Himalaya. The species naturally inhabits a wide range of habitats ranging from river deltas to hill slopes. These attributes of the species are conducive to test the influence of abiotic factors on sexual dimorphism, and RA strategy among different sexes. The study demonstrates sexual dimorphism in vegetative and reproductive traits. The sexual dimorphism index, aligned the traits like height, number of branches, flower production, and dry-weight of flowers with males while others including fresh-weight of leaves, number of thorns, fruit production were significantly associated with females. The difference in RA pattern is more pronounced in reproductive traits of the male and female plants, while in the PGM plants the traits overlap. In general, habitat conditions did not influence either the extent of sexual dimorphism or RA pattern. However, it seems to influence secondary sex-ratio as females show their significant association with soil moisture. Our findings on sexual dimorphism and RA pattern supports attributes of wind-pollination in the species. The observed extent of sexual dimorphism in the species reiterates limited genomic differences among the sexes and the ongoing evolution of dioecy via monoecy in the species. The dynamics of RA in the species appears to be independent of resource availability in the habitats as the species grows in a resource-limited and extreme environment.

Contradistinctive floral attributes, pollination guilds and their consequence on the outcrossing rate in two elevational morphs of Rhododendron arboreum Sm.

Infraspecific floral trait variations may appear in response to elevational differences in alpine plant species. There is enormous information on the selection of such morphs mediated by biotic and/or abiotic variables. Whether such differences contribute to differences in reproductive strategy and mating outcomes is rarely investigated. We investigated these aspects in two distinct elevational floral morphs (Red and Pink) of Rhododendron arboreum Sm. in Western Himalaya. The red morphs occupy the lower elevations while pink morphs the higher elevations. The two morphs differ in floral traits like phenology, dimension, display, quality of floral rewards, and pollinators that happen to influence interaction with available pollinator pool at each elevation. The pink morph exhibits entomophily, while the red ones show ornithophily. Although experimental pollinations established that both the morphs are self-compatible, selfing results in significantly lower fruit-set than either cross- or open-pollinations. The outcrossing rate in the red morph, as determined by using simple sequence repeat (SSR) markers, was higher (tm=0.82) than that in the pink morph (tm=0.76), with a tendency of the latter to be shifting towards mixed-mating strategy. However, the extent of biparental inbreeding was comparable among the two morphs. It is inferred that the differences in the mating outcomes among the morphs in the tree species are linked to those emerging from floral traits and the pollination by different functional groups of floral visitors.

Identification of significant marker-trait associations for Fusarium wilt resistance in a genetically diverse core collection of safflower using AFLP and SSR markers.

Safflower (Carthamus tinctorius L.), an oilseed crop, is severely affected by Fusarium oxysporum f. sp. carthami (Foc), a fungus causing Fusarium wilt (FW) resulting in up to 80% yield loss. In the present study, we used a panel of 84 diverse accessions from the composite core collection to perform association mapping for FW-resistance. Hydroponics-based screening resulted in categorization of 84 accessions as 31 immune, 19 highly resistant, 9 moderately resistant, 4 moderately susceptible, and 21 highly susceptible. Genotyping with a combination of 155 AFLP and 144 SSR markers revealed substantial genetic differentiation and structure analysis identified three main subpopulations (K = 3) with nearly 35% of admixtures in the panel. Kinship analysis at individual and population level revealed absence of or weak relatedness between the accessions. Association mapping with General Linear Model and Mixed Linear Model identified 4 marker-trait associations (MTAs) significantly linked with the FW-resistance trait. Of these, 3 robust MTAs identified in both the models exhibited phenotypic variance ranging from 4.09 to 6.45%. Locus-128 showing a low P-value and high phenotypic variance was identified as a promising marker-trait association that will facilitate marker-assisted breeding for FW-resistance in safflower.

Contrasting Reproductive Strategies of Two Nymphaea Species Affect Existing Natural Genetic Diversity as Assessed by Microsatellite Markers: Implications for Conservation and Wetlands Restoration.

Nymphaea, commonly known as water lily, is the largest and most widely distributed genus in the order Nymphaeales. The importance of Nymphaea in wetland ecosystems and their increased vulnerability make them a great choice for conservation and management. In this work, we studied genetic diversity in a collection of 90 N. micrantha and 92 N. nouchali individuals from six different states of India, i.e., Assam, Manipur, Meghalaya, Maharashtra, Goa, and Kerala, using simple sequence repeat (SSR) markers developed by low throughput Illumina sequencing (10X coverage of genome) of N. micrantha. Nymphaea nouchali is native to India, whereas N. micrantha is suggested to be introduced to the country for its aesthetic and cultural values. The study revealed extensive polymorphism in N. nouchali, while in N. micrantha, no apparent genetic divergence was detected prompting us to investigate the reason(s) by studying the reproductive biology of the two species. The study revealed that N. micrantha predominantly reproduces asexually which has impacted the genetic diversity of the species to a great extent. This observation is of immense importance for a successful re-establishment of Nymphaea species during restoration programs of wetlands. The information generated on reproductive behaviors and their association with genotypic richness can help in strategizing genetic resource conservation, especially for species with limited distribution. The study has also generated 22,268 non-redundant microsatellite loci, out of which, 143 microsatellites were tested for polymorphism and polymorphic markers were tested for transferability in five other Nymphaea species, providing genomic resources for further studies on this important genus.

Mapping of SARS-CoV-2 spike protein evolution during first and second waves of COVID-19 infections in India.

Aim: The aim of this study was to investigate the SARS-CoV-2 spike protein evolution during the first and second wave of COVID-19 infections in India. Materials & Methods: Detailed mutation analysis was done in 763 samples taken from GISAID for the ten most affected Indian states between March 2020 to August 2021. Results: The study revealed 242 mutations corresponding to 207 sites. Fifty one novel mutations emerged during the assessment period, including many with higher transmissibility and immune evasion functions. Highest number of mutations per spike protein also rose from 5 (first wave) to 13 (second wave). Conclusion: The study identified mutation-rich and no mutation regions in the spike protein. The conserved spike regions can be useful for designing future diagnostics, vaccines and therapeutics.

A comprehensive account of SARS-CoV-2 genome structure, incurred mutations, lineages and COVID-19 vaccination program.

This review collates information on the onset of COVID-19, SARS-CoV-2 genome architecture, emergence of novel viral lineages that drove multiple waves of infection around the world and standard and fast track development of vaccines. With the passage of time, the continuously evolving SARS-CoV-2 has acquired an expanded mutational landscape. The functional characterization of spike protein mutations, the primary target of diagnostics, therapeutics and vaccines has revealed increased transmission, pathogenesis and immune escape potential in the variant lineages of the virus. The incurred mutations have also resulted in substantial viral neutralization escape to vaccines, monoclonal, polyclonal and convalescent antibodies presently in use. The present situation suggests the need for development of precise next-generation vaccines and therapeutics by targeting the more conservative genomic viral regions for providing adequate protection.

Random distribution of nucleotide polymorphism throughout the genome of tomato-infecting begomovirus species occurring in India: implication in PCR based diagnosis.

Multiple begomovirus species are known to cause leaf curl disease in tomato in India. In order to develop specific and generic PCR based diagnostics for the tomato-infecting begomoviruses, in this study, we attempted to design primers initially based on the multiple alignment of the complete genome sequence of DNA-A component. However, the specific nucleotide stretches adequate for preparing specific primers could not be obtained. Alternatively, the online Primer-BLAST tool that offers designing of target-specific PCR primers was attempted to prepare specific primers targeting three clones (DNA-A) of tomato-infecting begomovirus species (Tomato leaf curl New Delhi virus, Tomato leaf curl Palampur virus and Tomato leaf curl Joydebpur virus) selected based on their sequence identity and phylogenetic relatedness. The primers derived from Primer-BLAST tool showed high level of cross-reaction among these begomovirus species and therefore were not able to differentiate these target begomovirus species. In order to understand the reason of cross-reactivity further sequence analysis revealed the high occurrence of single nucleotide variations (SNVs) compared to the multi-nucleotide stretches. There was no SNV hot-spot in the genome, rather the SNVs were randomly distributed throughout the genome of these begomovirus species. This pattern of nucleotide diversities among these tomato-infecting begomoviruses seriously implicated on developing specific PCR diagnostics. On the contrary, sequence analysis showed high sequence conservancy, which enabled to develop a generic PCR diagnostic for these begomoviruses. Our study, thus showed that the genome sequence diversity pattern among the tomato-infecting begomoviruses in India poses challenges in developing PCR based specific diagnostics. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-022-00785-9.

Epigenetic variation: A major player in facilitating plant fitness under changing environmental conditions.

Recent research in plant epigenetics has increased our understanding of how epigenetic variability can contribute to adaptive phenotypic plasticity in natural populations. Studies show that environmental changes induce epigenetic switches either independently or in complementation with the genetic variation. Although most of the induced epigenetic variability gets reset between generations and is short-lived, some variation becomes transgenerational and results in heritable phenotypic traits. The short-term epigenetic responses provide the first tier of transient plasticity required for local adaptations while transgenerational epigenetic changes contribute to stress memory and help the plants respond better to recurring or long-term stresses. These transgenerational epigenetic variations translate into an additional tier of diversity which results in stable epialleles. In recent years, studies have been conducted on epigenetic variation in natural populations related to various biological processes, ecological factors, communities, and habitats. With the advent of advanced NGS-based technologies, epigenetic studies targeting plants in diverse environments have increased manifold to enhance our understanding of epigenetic responses to environmental stimuli in facilitating plant fitness. Taking all points together in a frame, the present review is a compilation of present-day knowledge and understanding of the role of epigenetics and its fitness benefits in diverse ecological systems in natural populations.

Aberrant promoter hypermethylation regulates thrombomodulin in high altitude induced deep vein thrombosis.

BACKGROUND: DNA methylation regulates gene expression by inhibiting transcription factor binding to promoter and regulatory regions. Acute hypoxia during altitude exposure is associated with decreased natural anticoagulants and morbid thrombotic events. Thrombomodulin (TM) is a high affinity thrombin binding receptor protein, vital for vascular homeostasis. The purpose of this study is to determine gene expression regulation via methylation of TM gene in high altitude hypoxia induced deep vein thrombosis (DVT) patients. MATERIALS AND RESULTS: Percent 5-methyl cytosine analysis showed increased methylation in high altitude DVT patients (HAP) as compared to high altitude control (HAC) and seal level control (Control) subjects, while TM protein and mRNA levels were decreased in high altitude DVT patients as compared to other two groups. Bisulfite sequencing analysis indicated increased methylation in TM promoter in high altitude DVT patients compared to high altitude controls. Flow cytometry analysis showed decreased TM expression in hypoxia induced primary human umbilical vein endothelial cells (HUVECs). Treatment with specific DNA methyltransferase (DNMT) inhibitor-decitabine during hypoxia, restored TM expression. in vitro global methylation assay showed increased methylation in hypoxia group. Specific concentration of decitabine in hypoxia decreased global methylation showing a direct correlation between DNMTs and methylation. Selective dose of decitabine restored TM levels in HUVECs. DNMT1 and DNMT3B proteins showed to mediate the overall expression of TM. CONCLUSION: TM emerged as a potential candidate for methylation in high altitude DVT patients, regulated by hypoxia-induced epigenetic mechanism. Hypoxia culminates in methylation of DNA sequences in the promoter region of TM gene and increased the expression of DNMT1 and DNMT3B per se in primary HUVECs. Critical DNA methylation events were found to be compromised in high altitude DVT patients.

Long-read-based draft genome sequence of Indian black gram IPU-94-1 'Uttara': Insights into disease resistance and seed storage protein genes.

Black gram [Vigna mungo (L.) Hepper var. mungo] is a warm-season legume highly prized for its protein content along with significant folate and iron proportions. To expedite the genetic enhancement of black gram, a high-quality draft genome from the center of origin of the crop is indispensable. Here, we established a draft genome sequence of an Indian black gram cultivar, 'Uttara' (IPU 94-1), known for its high resistance to mungbean yellow mosaic virus. Pacific Biosciences of California, Inc. (PacBio) single-molecule real-time (SMRT) and Illumina sequencing assembled a draft reference-guided assembly with a cumulative size of ∼454.4 Mb, of which, 444.4 Mb was anchored on 11 pseudomolecules corresponding to 11 chromosomes. Uttara assembly denotes features of a high-quality draft genome illustrated through high N50 value (42.88 Mb), gene completeness (benchmarking universal single-copy ortholog [BUSCO] score 94.17%) and low levels of ambiguous nucleotides (N) percent (0.0005%). Gene discovery using transcript evidence predicted 28,881 protein-coding genes, from which, ∼95% were functionally annotated. A global survey of genes associated with disease resistance revealed 119 nucleotide binding site-leucine rich repeat (NBS-LRR) proteins, while 23 genes encoding seed storage proteins (SSPs) were discovered in black gram. A large set of microsatellite loci were discovered for marker development in the crop. Our draft genome of an Indian black gram provides the foundational genomic resources for the improvement of important agronomic traits and ultimately will help in accelerating black gram breeding programs.