Genome-wide analysis of cellulose synthase gene superfamily in Tectona grandis L.f.

This study aimed to explore Cellulose synthase gene superfamily of teak, and its evolutionary relationship with homologous genes of other woody species. The incidence of evolutionary events like gene duplication and gene loss, influence of the selection pressure, and consequent adaptive functional divergence of the duplicated TgCes gene were assessed alongside it's role in wood coloration. This study identified 39 full-length non-redundant proteins belonging to CesA and Csl gene families. TgCesA and TgCsl proteins with Cellulose synthase domain repeats indicated tandem gene duplication and probable genetic variability, enabling local adaptation. Further, multi-domain protein (MYB-like DNA-binding domain and CesA domain) with maximum introns was also identified indicating gene fusion and formation of complex protein with novel functions. Phylogenetic analysis grouped the genes into seven subfamilies (CesA, CslA, CslC, CslD, CslE, CslG, and CslM) with each undergoing gene duplication and loss along their evolutionary history. Post-species gene duplications and probable neofunctionalization were identified in TgCesA and TgCsl gene families. Each subfamily was found to be under strong purifying selection with a few or no sites under positive selection. Functional divergence analysis further revealed site-specific selective constraints in CesA and Csl genes of the teak Cellulose synthase gene family. Furthermore, protein-protein interaction network analysis identified co-expression of Cellulose synthase gene with flavonoid 3',5'-hydroxylase (F3'5'H, CYP75A), involved in the biosynthesis of xylem anthocyanin compounds, probably responsible for wood coloration. This study thus offers a foundation for future research in wood formation and wood property traits specific to teak and its provenances. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-03927-6.

Gender specific SNP markers in Coscinium fenestratum (Gaertn.) Colebr. for resource augmentation.

BACKGROUND: Unregulated extraction of highly traded medicinal plant species results in drastic decline of the natural resources and alters viable sex ratio of populations. Conservation and long-term survival of such species, require gender specific restoration programs to ensure reproductive success. However, it is often difficult to differentiate sex of individuals before reaching reproductive maturity. C. fenestratum is one of the medicinally important and overexploited dioecious woody liana, with a reproductive maturity of 15 years. Currently, no information is available to identify sex of C. fenestratum in seedling stage while augmenting the resources. Thus, the current study envisages to utilize transcriptomics approach for gender differentiation which is imperative for undertaking viable resource augmentation programmes. METHODS AND RESULTS: Gender specific SNPs with probable role in sexual reproduction/sex determination was located using comparative transcriptomics approach (sampling male and female individuals), alongside gene ontology and annotation. Nine sets of primers were synthesized from 7 transcripts (involved in sexual reproduction/other biological process) containing multiple SNP variants. Out of the nine primer pairs, only one SNP locus with no available information of its role in reproduction, showed consistent and accurate results (males-heterozygous and females-homozygous), in the analyzed 40 matured individuals of known sexes. Thus validated the efficiency of this SNP marker in differentiating male and female individuals. CONCLUSIONS: The study could identify SNPs linked to the loci with apparent role in gender differentiation. This SNP marker can be used for early sexing of seedlings for in-situ conservation and resource augmentation of C. fenestratum in Kerala, India.

Artificial intelligence in timber forensics employing DNA barcode database.

Extreme difficulties in species identification of illegally sourced wood with conventional tools have accelerated illicit logging activities, leading to the destruction of natural resources in India. In this regard, the study primarily focused on developing a DNA barcode database for 41 commercial timber tree species which are highly vulnerable to adulteration in south India. The developed DNA barcode database was validated using an integrated approach involving wood anatomical features of traded wood samples collected from south India. Traded wood samples were primarily identified using wood anatomical features using IAWA list of microscopic features for hardwood identification. Consortium of Barcode of Life (CBOL) recommended barcode gene regions (rbcL, matK & psbA-trnH) were employed for developing DNA barcode database. Secondly, we employed artificial intelligence (AI) analytical platform, Waikato Environment for Knowledge Analysis (WEKA) for analyzing DNA barcode sequence database which could append precision, speed, and accuracy for the entire identification process. Among the four classification algorithms implemented in the machine learning algorithm (WEKA), best performance was shown by SMO, which could clearly allocate individual samples to their respective sequence database of biological reference materials (BRM) with 100 % accuracy, indicating its efficiency in authenticating the traded timber species. Major advantage of AI is the ability to analyze huge data sets with more precision and also provides a large platform for rapid authentication of species, which subsequently reduces human labor and time. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03604-0.

Quantification of adulteration in traded ayurvedic raw drugs employing machine learning approaches with DNA barcode database.

Adulteration of expensive raw drugs with inferior taxa has become a routine practice, conceding the quality and safety of derived herbal products. In this regard, the study addresses the development of an integrated approach encompassing DNA barcode and HPTLC fingerprinting to authenticate chiefly traded ayurvedic raw drugs in south India [viz. Saraca asoca (Roxb.) Willd., Terminalia arjuna (Roxb. ex DC.) Wight and Arn., Sida alnifolia L. and Desmodium gangeticum (L.) DC.] from its adulterants. Consortium of Barcode of Life (CBOL) recommended DNA barcode gene regions viz. nuclear ribosomal-Internal Transcribed Spacer (nrDNA-ITS), maturase K (matK), ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) and psbA-trnH spacer regions along with HPTLC profiling were experimented and a reference database was created. Further, an integrated analytical approach employing genetic distance-based Maximum Likelihood phylogenetic tree and Artificial Intelligence (AI)based Machine Learning Algorithms (MLA)-Waikato Environment for Knowledge Analysis (WEKA) and Barcoding with Logic (BLOG) were employed to prove efficacy of DNA barcode tool. Even though, among the four barcodes, psbA-trnH (S. alnifolia and its adulterants, T. arjuna and its adulterants) or ITS region (S. asoca and its adulterants, D. gangeticum and its adulterants) showed highest inter specific divergences in the selected Biological Reference Materials (BRMs), rbcL or matK barcode regions alone were successful for authentication of traded samples. The automated species identification techniques, WEKA and BLOG, experimented for the first time in India for raw drug validation, could achieve rapid and precise identification. A national certification agency for raw drug authentication employing an integrated approach involving a DNA barcoding tool along with standard organoleptic and analytical methods can strengthen and ensure safety and quality of herbal medicines in India. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-03001-5.

Draft genome of Korthalsia laciniosa (Griff.) Mart., a climbing rattan elucidates its phylogenetic position.

Korthalsia laciniosa (Griff.) Mart. is a climbing rattan used as a source of durable and flexible cane. In the present study, the draft genome of K. laciniosa was sequenced, de novo assembled and annotated. Genome-wide identification of MADS-Box transcription factors revealed loss of Mß, and Mγ genes belonging to Type I subclass in the rattan lineage. Mining of the genome revealed presence of 13 families of lignin biosynthetic pathway genes and expression profiling of nine major genes documented relatively lower level of expression in cirrus when compared to leaflet and petiole. The chloroplast genome was re-constructed and analysis revealed the phylogenetic relatedness of this genus to Eugeissona, in contrast with its present taxonomic position. The genomic resource generated in the present study will accelerate population structure analysis, genetic resource conservation, phylogenomics and facilitate understanding the unique developmental processes like gender expression at molecular level.

Exploring prevalence of potential pathogens and fecal indicators in geographically distinct river systems through comparative metagenomics.

Microbial communities are considered as vital members to reflect the health of a riverine system. Among them, pathogenic and fecal indicators imply health risks involved with potability of river water. The present study explores the diverse microbial communities, distribution pattern of potential pathogens, and fecal indicators between the geographically distinct Himalayan and Peninsular river systems of India. It also inquires into the environmental factors associated with community variance and distribution pattern of microbial indicators. The application of high-throughput amplicon sequencing approach unveiled significant demarcation (p < 0.004, Anosim R = 0.62) of samples suggesting unique microbial diversities in these two river sediments. Random forest analysis revealed Desulfobulbulus, PSB_M_3, and Opitutus in Himalayan, while DA101, Bacillus, and Streptomyces in the Peninsular as significant contributors to develop overall dissimilarity between the river systems. Permutational multivariate analysis of variance and co-occurrence network analysis were used to study the relationships between microbial taxa and environmental factors. Amongst the various studied environmental parameters, pH, K, Ca, Mg, Ba, and Al in the Himalayan and salinity, Na, temperature, and Th in the Peninsular significantly influenced shaping of distinct microbial communities. Furthermore, the potential pathogenic genera, including Flavobacterium, Clostridium, Arcobacter, Pseudomonas, and Bacillus were highly prevalent in both the river systems. Arcobacter, Clostridium, Acinetobacter, Bacteroides, and Caloramator were the prominent fecal indicators in these river systems. Our findings provide salient information about the crucial role and interplay between various environmental factors and anthropogenic influences in framing the microbiome of the distinct river systems in India. Moreover, assessing potential pathogenic and fecal indicators suggest the public health risk associated with untreated sewage discharge into these water sources. The detection of various F/S indicators and potentially pathogenic bacteria in Himalayan and Peninsular river systems emphasize the urgent need for future monitoring and management of major riverine systems in India.

The efficacy of machine learning algorithm for raw drug authentication in Coscinium fenestratum (Gaertn.) Colebr. employing a DNA barcode database.

Medicinal plants are a valuable resource for traditional as well as modern medicine. Consequently huge demand has exerted a heavy strain on the existing natural resources. Due to over exploitation and unscientific collection most of the commercially traded ayurvedic plants are in the phase of depletion. Adulteration of expensive raw drugs with inferior taxa has become a common practice to meet the annual demand of the ayurvedic industry. Although there are several recommended methods for proper identification varying from the traditional taxonomic to organoleptic and physiochemical, it is difficult to authenticate ayurvedic raw drugs available in extremely dried, powdered or shredded forms. In this regard, the study addresses proper authentication and illicit trade in Coscinium fenestratum (Gaertn.) Colebr. using CBOL recommended standard barcode regions viz. nuclear ribosomal-Internally Transcribed Spacer (nrDNA- ITS), maturase K (matK), ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL), and psbA-trnH spacer regions. Further, an integrated analytical approach employing Maximum Likelihood phylogenetic tree and Machine Learning Approach, Waikato Environment for Knowledge Analysis was employed to prove efficacy of the method. The automated species identification technique, Artificial Intelligence uses the ability of computers to build models that can receive the input data and then conduct statistical analyses which significantly reduces the human labour. Concurrently, scientific management, restoration, cultivation and conservation measures should be given utmost priority to reduce the depletion of wild resources as well as to meet the rapidly increasing demand of the herbal industries.

Pitfalls and promises of raw drug identification techniques in the ayurvedic industry: an overview.

India, with a rich heritage of floral diversity, is well-known for its medicinal plant wealth and is the largest producer of medicinal herbs in the world. Ethnobiological Survey of Ministry of Environment and Forests (MOEF) could identify 8000 plant species utilized in various systems of medicine with approximately 25,000 effective herbal formulations. The extensive consumption to meet demand-supply ratio exerts a heavy strain on the existing resources. This subsequently led to the adulteration and substitution of medicinal plants with look-alike species. The consumer's faith on herbal medicine is in the phase of decline due to the extremities in adulteration/substitution and ensuing consequences. It is imperative to bring forth universally acceptable standard tools to authenticate raw drugs before being processed further into formulations. A vast array of techniques such as physical, chemical (analytical), biochemical, anatomical, organoleptic, and recently emerged DNA based molecular methods are widely used for plant species authentication. In recent years, DNA barcoding has made remarkable progress in the field of medicinal plants research. DNA metabarcoding is the latest development for qualitative evaluation of the herbal formulations, whereas for quantitative analysis, combination of pharmacognostic, pharmacovigilance and analytical methods are inevitable for authentication. This review addresses the overall strengths and shortcomings of the existing as well as recently emerged techniques in authenticating ayurvedic raw drugs.

The low copy nuclear region, RPB2 as a novel DNA barcode region for species identification in the rattan genus Calamus (Arecaceae).

Taxonomic complexities, like environmental plasticity and homoplasy, make precise identification challenging in Calamus, the genus of spiny climbing palms of the subfamily Calamoideae (Arecaceae). In the present study, the species discriminatory power of twelve potential DNA barcode regions (rbcL, matK, psbA-trnH, rpoC, rpoB, psbK-psbI, atpF-atpH, psbZ-trnfM, ITS1, ITS2, PRK, and RPB2) were evaluated in 21 species of Calamus from the Western Ghats region of India, using distance, tree, and similarity based statistical methods. Except for the low copy nuclear region, RPB2, none of the tested plastid loci or nuclear loci ITS, either singly or in combinations, could discriminate all the species of Calamus due to low substitution rate of plastid regions and multiple copies of ITS respectively. The RPB2 locus showed highest species resolution with 96% accuracy in similarity based analysis, indicating its potential and efficiency as a barcode locus for the genus. The putative "Calamus gamblei complex" based on overlapping morphology was successfully resolved as six distinct, though closely related, species. The analysis also indicates that C. delessertianus is a morphological variant of C. dransfieldii. In spite of being a low copy nuclear gene region, RPB2 provided an efficient barcode to delineate Calamus species and has the potential to further extend its use as a prospective barcode to other Palm genera.

DNA barcoding as a valuable molecular tool for the certification of planting materials in bamboo.

DNA barcodes developed for selected commercially important bamboo species can be utilized for the certification of planting stock in bamboo nurseries in absence of discriminatory features at the juvenile stage. Planting materials such as micropropagated plantlets, rhizome transplants and culm cuttings, generated at nursery level are directly procured for establishment of commercial plantations without any further verification. Very often misidentification and mixing up occur at nursery level and the error is not discovered until several years have passed. The present study evaluated the potentiality of seven Consortium for Barcode of Life (CBOL) recommended standard DNA barcode regions in commercially important bamboo species of India. Among the analyzed barcode regions, multiple sequence alignment (MSA) of psbA-trnH barcode region showed species-specific nucleotide differences in the studied bamboo taxa. The major nucleotide changes observed were transitions/transversions as well as insertions/deletions of nucleotides. Even though species-specific mononucleotide differences could be identified for most of the studied bamboo taxa, a small amount of sequence similarities were found in some of the Dendrocalamus and Bambusa species, which were grouped together in tree-based analysis. In subtribe Melocanninae, Ochlandra travancorica, Melocanna baccifera and M. clarkei showed unique species-specific psbA-trnH barcodes. Similarly, in the genus Oxytenanthera, unique species-specific psbA-trnH barcodes were obtained for O. monadelpha and O. parvifolia. Thus psbA-trnH barcode region generated distinct species-specific barcodes for commercial bamboo species in genera Bambusa, Dendrocalamus, Melocanna, Oxytenanthera as well as Ochlandra. Any national certification agency set up for the purpose can utilize psbA-trnH DNA barcode region to tag species identity and to establish the authenticity of multiplied planting materials in bamboos.

Narrow gene pool can threaten the survival of Calamus nagbettai R. R. Fernald & Dey: a highly, endemic dioecious rattan species in the Western Ghats of India.

Rattans, the spiny climbing palms of Arecaceae (Palmae) family exhibit high endemism to the biodiversity hot spots in India. Of the five rattan genera, Calamus is the only genus found in peninsular India with 15 of 21 species, endemic to the Western Ghats. The extensive utilization of rattans owing to their strength, durability and huge demand has resulted in depletion of their natural resources. Of the 15 endemic species, C. nagbettai is the most affected species on account of endemism, low population size and restricted distribution with fragmented populations. The present study revealed high amount of genetic diversity in the surviving scattered populations of the species using microsatellite markers. High gene flow (Nm = 1.498) observed across the populations resulted in low genetic differentiation (14%). A clear genetic admixture could be seen in Kerala as well as one of the Karnataka's populations while the remaining two populations were genetically distinct. UPGMA, PCoA and STRUCTURE analyses showed significantly different genetic composition in Kerala population compared to other populations. Kerala and Karnataka populations of C. nagbettai were also unique in their genetic structure and allelic composition. Therefore, effective management and conservation strategies have to be implemented to preserve the rarealleles with adaptive potential to protect this economically valuable Calamus species from endangerment. Over exploitation, low seed set and poor regeneration, as well as habitat fragmentation can further threaten the survival of this endemic, narrowly distributed dioecious rattan species in the Western Ghats region.

Draft genome of a high value tropical timber tree, Teak (Tectona grandis L. f): insights into SSR diversity, phylogeny and conservation.

Teak (Tectona grandis L. f.) is one of the precious bench mark tropical hardwood having qualities of durability, strength and visual pleasantries. Natural teak populations harbour a variety of characteristics that determine their economic, ecological and environmental importance. Sequencing of whole nuclear genome of teak provides a platform for functional analyses and development of genomic tools in applied tree improvement. A draft genome of 317 Mb was assembled at 151× coverage and annotated 36, 172 protein-coding genes. Approximately about 11.18% of the genome was repetitive. Microsatellites or simple sequence repeats (SSRs) are undoubtedly the most informative markers in genotyping, genetics and applied breeding applications. We generated 182,712 SSRs at the whole genome level, of which, 170,574 perfect SSRs were found; 16,252 perfect SSRs showed in silico polymorphisms across six genotypes suggesting their promising use in genetic conservation and tree improvement programmes. Genomic SSR markers developed in this study have high potential in advancing conservation and management of teak genetic resources. Phylogenetic studies confirmed the taxonomic position of the genus Tectona within the family Lamiaceae. Interestingly, estimation of divergence time inferred that the Miocene origin of the Tectona genus to be around 21.4508 million years ago.

Identification and Characterization of Lipopeptides from Bacillus subtilis B1 Against Sapstain Fungus of Rubberwood Through MALDI-TOF-MS and RT-PCR.

Bacillus subtilis is a potent biocontrol agent producing a wide array of antifungal lipopeptides for the inhibition of fungal growth. B. subtilis B1 isolated from market-available compost provided an efficient control of rubberwood sapstain fungus, Lasiodiplodia theobromae. The current study is aimed to identify and characterize the lipopeptides responsible for the biocontrol of rubberwood sapstain fungus by Bacillus subtilis B1. The bacterial whole-cell surface extract from the dual culture of B. subtilis B1 and sapstain fungus (L. theobromae) was analysed using MALDI-TOF-MS. The protonated as well as sodium, potassium adducts of homologues of iturin C, surfactin, bacillomycin D and fengycin A and B were identified and expression of the lipopeptide biosynthetic genes could be confirmed through RT-PCR. This is the first report of mycobacillin and trimethylsilyl derivative of bacilysin during antagonism through MALDI-TOF-MS. MALDI-TOF-MS with RT-PCR offered easy platforms to characterize the antifungal lipopeptides. The identification of antifungal lipopeptides can lead to the formulation of prospective biocontrol by-products which have wide-scale utility.

Screening of bacterial biocontrols against sapstain fungus (Lasiodiplodia theobromae Pat.) of rubberwood (Hevea brasiliensis Muell.Arg.).

Diverse bacterial biocontrol agents from various sources of aerobic composts against the sapstain fungus Lasiodiplodia theobromae in rubberwood (Hevea brasiliensis) were isolated, screened and identified by various morphological, biochemical and molecular techniques. The inhibitory effect of seventeen bacterial isolates was examined and seven exhibited inhibition towards the sapstain fungus. Among the seven antagonists, six were conclusively identified as Bacillus subtilis and one as Paenibacillus polymyxa using 16S rRNA-encoding gene sequencing. This is the first report on the occurrence of P. polymyxa, a potent biofertilizer and antagonist in vermicompost. HiCrome Bacillus agar was identified as an effective medium for differentiation of B. subtilis from other Bacillus species. The present work demonstrates the efficacy of the antagonistic property of B. subtilis strains against rubberwood sapstain fungus. Culture-based antagonistic inhibition displayed by B. subtilis can be extended to cater to the biocontrol requirements of wood-based industries against the stain fungus. The study showed the utility of an integrated approach, employing morphological, biochemical and molecular tools for conclusive identification of several bacterial isolates present in aerobic composts from diverse sources.