Unveiling stress-adapted endophytic bacteria: Characterizing plant growth-promoting traits and assessing cross-inoculation effects on Populus deltoides under abiotic stress.

In the face of the formidable environmental challenges precipitated by the ongoing climate change, Plant Growth-Promoting Bacteria (PGPB) are gaining widespread acknowledgement for their potential as biofertilizers, biocontrol agents, and microbial inoculants. However, a knowledge gap pertains to the ability of PGPB to improve stress tolerance in forestry species via cross-inoculation. To address this gap, the current investigation centres on PGPBs, namely, Acinetobacter johnsonii, Cronobacter muytjensii, and Priestia endophytica, selected from the phyllosphere of robust and healthy plants thriving in the face of stress-inducing conditions. These strains were selected based on their demonstrated adaptability to saline, arid, and nitrogen-deficient environments. The utilization of PGPB treatment resulted in an improvement of stomatal conductance (gs) and transpiration rate (E) in poplar plants exposed to both salt and drought stress. It also induced an increase in essential biochemical components such as proline (PRO), catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD). These reactions were accompanied by a decrease in leaf malonaldehyde (MDA) content and electrolyte leakage (EL). Furthermore, the PGPB treatment demonstrated a notable enhancement in nutrient absorption, particularly nitrogen and carbon, achieved through the solubilization of nutrients. The estimation of canopy temperature via thermal imaging proved to be an efficient method for distinguishing stress reactions in poplar than conventional temperature recording techniques. In summation, the utilization of PGPB especially Cronobacter muytjensii in this study, yielded profound improvements in the stress tolerance of poplar plants, manifesting in reduced membrane lipid peroxidation, enhanced photosynthesis, and bolstered antioxidant capacity within the leaves.

Exploring Steroidal Saponin Composition and Morphometric Characteristics of Rhizomes from Trillium govanianum Wall. ex D. Don: Inference for Medicinal Properties and Genetic Stock Improvement.

Trillium govanianum, a medicinal herb, exhibiting diverse morphometric traits and phytochemicals across developmental stages of plants. The changes in the chemical profile and steroidal saponin levels in the rhizome of T. govanianum across different developmental stages were previously unknown. This study categorizes rhizomes into three types based on scar presence: juvenile (5-10 scars, Type I), young (11-19 scars, Type II), and mature (21-29 scars, Type III). Rhizomes show varying sizes (length 1.2-4.7 cm, girth 0.3-1.6 cm), weight (0.18-5.0 g), and extractive yields (9.7-16.1 % w w-1), with notable differences in saponin content (5.95-21.9 mg g-1). Ultra-high performance liquid chromatography-MS/MS (UHPLC-QTOF-MS/MS)-based chemical profiling identifies 31 phytochemicals, mainly including diverse saponins. Ultra-high performance liquid chromatography coupled with evaporative light scattering detection (UHPLC-ELSD)-based quantitative analysis of seven key saponins reveals stage-specific accumulation patterns, with protodioscin (P) and dioscin (DS) predominant in mature rhizomes. Statistical analysis confirms significant variation (p=0.001) in saponin levels across developmental stages with chemical constituent protodioscin (P=4.03±0.03-15.76±0.14 mg g-1, PAve=9.79±3.03 mg g-1) and dioscin (DS=1.23±0.06-3.93±0.07 mg g-1, DSAve=2.59±0.70 mg g-1), with acceptable power (p=0.738; |δ|>0.5) statistics for effective sample size (n=27 samples used in the study) of T. govanianum. Principal Component Analysis (PCA) and Euclidean clustering further highlighted chemotype distinctions.

An improved Shorea robusta genomic DNA extraction protocol with high PCR fidelity.

Shorea robusta (Dipterocarpaceae), commonly known as Sal, is an economically and culturally important timber species, known to contain a wide spectrum of polyphenols, polysaccharides, and other secondary metabolites in the tissues, which can interfere with the extraction of high-quality genomic DNA. In order to screen simple sequence repeat (SSR) markers and carry out other DNA-based analyses for this species in our laboratory, a high-throughput DNA extraction methodology was needed. Hence, we have optimized a simple, rapid, safe, and reliable high-throughput protocol for DNA extraction suitable for both fresh and dry leaves. The standardized protocol delivered good DNA yield of ∼1500 µg from 1 g of leaf tissue, with purity indicated by a 260 nm/280 nm absorbance ratio ranging from 1.70 to 1.91, which validated the suitability of extracted DNA and revealed reduced levels of contaminants. Additionally, the protocol that we developed was found to be suitable for polymerase chain reaction (PCR) amplification using microsatellite markers. Genome-wide characterization with SSR markers has been established in S. robusta, which further validates the protocol and its usefulness in DNA-based studies across the genus and/or family.

Ecological niche modelling and population genetic analysis of Indian temperate bamboo Drepanostachyum falcatum in the western Himalayas.

The present study was conducted to understand the key ecological and biological questions of conservation importance in Drepanostachyum falcatum which aimed to map potential distribution in the western Himalayas and decipher spatial genetic structure. Eco-distribution maps were generated through ecological niche modelling using the Maximum Entropy (MaxEnt) algorithm implemented with 228 geocoordinates of species presence and 12 bioclimatic variables. Concomitantly, 26 natural populations in the western Himalayas were genetically analysed using ten genomic sequence-tagged microsatellite (STMS) markers. Model-derived distribution was adequately supported with appropriate statistical measures, such as area under the 'receiver operating characteristics (ROC)' curve (AUC; 0.917 ± 0.034)", Kappa (K; 0.418), normalized mutual information (NMI; 0.673) and true skill statistic (TSS; 0.715). Further, Jackknife test and response curves showed that the precipitation (pre- and post-monsoon) and temperature (average throughout the year and pre-monsoon) maximize the probabilistic distribution of D. falcatum. We recorded a wide and abundant (4096.86 km2) distribution of D. falcatum in the western Himalayas with maximum occurrence at 1500 to 2500 m asl. Furthermore, marker analysis exemplified high gene diversity with low genetic differentiation in D. falcatum. Relatively, the populations of Uttarakhand are more genetically diverse than Himachal Pradesh, whereas within the Uttarakhand, the Garhwal region captured a higher allelic diversity than Kumaon. Clustering and structure analysis indicated two major gene pools, where genetic admixing appeared to be controlled by long-distance gene flow, horizontal geographical distance, aspect, and precipitation. Both the species distribution map and population genetic structure derived herein may serve as valuable resources for conservation and management of Himalayan hill bamboos.

Genome survey sequencing-based SSR marker development and their validation in Dendrocalamus longispathus.

Bamboo is an important genetic resource in India, supporting rural livelihood and industries. Unfortunately, most Indian bamboo taxa are devoid of basic genomic or marker information required to comprehend the genetic processes for further conservation and management. In this study, we perform genome survey sequencing for development of de novo genomic SSRs in Dendrocalamus longispathus, a socioeconomically important bamboo species of northeast India. Using Illumina platform, 69.49 million raw reads were generated and assembled into 1,145,321 contig with GC content 43% and N50 1228 bp. In total, 46,984 microsatellite repeats were mined-out wherein di-nucleotide repeats were most abundant (54.71%) followed by mono- (31.91%) and tri-repeats (9.85%). Overall, AT-rich repeats were predominant in the genome, but GC-rich motifs were more frequent in tri-repeats. Afterwards, 21,596 SSR loci were successfully tagged with the primer pairs, and a subset of 50 were validated through polymerase chain reaction amplification. Of these, 36 SSR loci were successfully amplified, and 16 demonstrated polymorphism. Using 13 polymorphic SSRs, a moderate level of gene diversity (He = 0.480; Ar = 3.52) was recorded in the analysed populations of D. longispathus. Despite the high gene flow (Nm = 4.928) and low genetic differentiation (FST = 0.119), severe inbreeding (FIS = 0.407) was detected. Further, genetic clustering and STRUCTURE analysis revealed that the entire genetic variability is captured under two major gene pools. Conclusively, we present a comprehensive set of novel SSR markers in D. longispathus as well as other taxa of tropical woody bamboos.

Genome-wide characterization leading to simple sequence repeat (SSR) markers development in Shorea robusta.

Tropical rainforests in Southeast Asia are enriched by multifarious biota dominated by Dipterocarpaceae. In this family, Shorea robusta is an ecologically sensitive and economically important timber species whose genomic diversity and phylogeny remain understudied due to lack of datasets on genetic resources. Smattering availability of molecular markers impedes population genetic studies indicating a necessity to develop genomic databases and species-specific markers in S. robusta. Accordingly, the present study focused on fostering de novo low-depth genome sequencing, identification of reliable microsatellites markers, and their validation in various populations of S. robusta in Uttarakhand Himalayas. With 69.88 million raw reads assembled into 1,97,489 contigs (read mapped to 93.2%) and a genome size of 357.11 Mb (29 × coverage), Illumina paired-end sequencing technology arranged a library of sequence data of ~ 10 gigabases (Gb). From 57,702 microsatellite repeats, a total of 35,049 simple sequence repeat (SSR) primer pairs were developed. Afterward, among randomly selected 60 primer pairs, 50 showed successful amplification and 24 were found as polymorphic. Out of which, nine polymorphic loci were further used for genetic analysis in 16 genotypes each from three different geographical locations of Uttarakhand (India). Prominently, the average number of alleles per locus (Na), observed heterozygosity (Ho), expected heterozygosity (He), and the polymorphism information content (PIC) were recorded as 2.44, 0.324, 0.277 and 0.252, respectively. The accessibility of sequence information and novel SSR markers potentially enriches the current knowledge of the genomic background for S. robusta and to be utilized in various genetic studies in species under tribe Shoreae.

Engineering traits through CRISPR/cas genome editing in woody species to improve forest diversity and yield.

Dangers confronting forest ecosystems are many and the strength of these biological systems is deteriorating, thus substantially affecting tree physiology, phenology, and growth. The establishment of genetically engineered trees into degraded woodlands, which would be adaptive to changing climate, could help in subsiding ecological threats and bring new prospects. This should not be resisted due to the apprehension of transgene dispersal in forests. Consequently, it is important to have a deep insight into the genetic structure and phenotypic limits of the reproductive capability of tree stands/population(s) to endure tolerance and survival. Importantly, for a better understanding of genes and their functional mechanisms, gene editing (GeEd) technology is an excellent molecular tool to unravel adaptation progressions. Therefore, GeEd could be harnessed for resolving the allelic interactions for the creation of gene diversity, and transgene dispersal may be alleviated among the population or species in different bioclimatic zones around the globe. This review highlights the potential of the CRISPR/Cas tools in genomic, transcriptomic, and epigenomic-based assorted and programmable alterations of genes in trees that might be able to fix the trait-specific gene function. Also, we have discussed the application of diverse forms of GeEd to genetically improve several traits, such as wood density, phytochemical constituents, biotic and abiotic stress tolerance, and photosynthetic efficiency in trees. We believe that the technology encourages fundamental research in the forestry sector besides addressing key aspects, which might fasten tree breeding and germplasm improvement programs worldwide.

Global Forestry Perspective: COVID-19 Impact and Assessment.

Corona virus disease 2019 (COVID-19) caused by corona virus SARS-CoV-2 is believed to be originated in Wuhan province of China, which has spread all over the world. It has severely impacted the social, cultural, educational, research and development programmes throughout the globe. Since the inception of various lifeforms on planet earth, forests' plays an important role in the evolutionary history of organisms and fitness persuades the existence of modern-day species in different ecosystems. The visible effect of pandemic leads to the lockdown with positive implications on the earth biosphere as a whole; while on other hand, lockdown seized and reduced the urbanization and developmental projects, especially the research and development progress in the field of forestry and allied sciences. This viewpoint addresses key questions and point-out with detailed exploration of the problem - as how COVID-19 pandemic impacts forestry research all over the world? We also suggest possible solutions, and define the role and association of disciplines, such as bioinformatics, remote sensing and statistical modules along with previously explored and collected field data as a sustainable measure to resolve the problems of forestry sector of this mighty challenge.

Genome skimming-based simple sequence repeat (SSR) marker discovery and characterization in Grevillea robusta.

Proteaceae, a largely southern hemisphere family consisting of 80 genera distributed in Australia and southern Africa as its centres of greatest diversity, also extends well in northern and southern America. Under this family, Grevillea robusta is a fast-growing species got popularity in farm and avenue plantations. Despite the ecological and economic importance, the species has not yet been investigated for its genetic improvement and genome-based studies. Only a few molecular markers are available for the species or its close relatives, which hinders  genomic and population genetics studies. Genetic markers have been intensively applied for the main strategies in breeding programs, especially for the economically important traits. Hence, it is of utmost priority to develop genomic database resources and species-specific markers for studying quantitative genetics in G. robusta. Given this, the present study aimed to develop de novo genome sequencing, robust microsatellites markers, sequence annotation and their validation in different stands of G. robusta in northern India. Library preparation and sequencing were carried out using Illumina paired-end sequencing technology. Approximately, ten gigabases (Gb) sequence data with 70.87 million raw reads assembled into 425,923 contigs (read mapped to 76.48%) comprising 455 Mb genome size (23 × coverage) generated through genome skimming approach. In total, 9421 simple sequence repeat (SSR) primer pairs were successfully designed from 13,335 microsatellite repeats. Afterward, a subset of 161 primer pairs was randomly selected, synthesized and validated. All the tested primers showed successful amplification but only 13 showed polymorphisms. The polymorphic SSRs were further used to estimate the measures of genetic diversity in 12 genotypes each from the states of Punjab, Haryana, Himachal Pradesh and Uttarakhand. Importantly, the average number of alleles (Na), observed heterozygosity (Ho), expected heterozygosity (He), and the polymorphism information content (PIC) were recorded as 2.69, 0.356, 0.557 and 0.388, respectively. The availability of sequence information and newly developed SSR markers could potentially be used in various genetic analyses and improvements through molecular breeding strategies for G. robusta. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01035-w.

Genome skimming-based STMS marker discovery and its validation in temperate hill bamboo Drepanostachyum falcatum.

Drepanostachyum falcatum (Nees) Keng f. is one of the most widely distributed shrubby bamboo species in the temperate region of northwest (NW) Himalayas. Along with the other three temperate bamboo species, namely Yushania anceps, Thamnocalamus spathiflorus and Himalayacalamus falconeri, commonly called as 'ringal', and utilized for making various articles of household and commercial purpose by local artisans. Despite huge ecological and socio-economic importance, they are least studied and lacks baseline genetic information. In this study, ~10 Gb genome sequence data with 70.68 million reads were generated for D. falcatum, through genome skimming approach based on high throughput next-generation sequencing technology with Illumina protocol. The high-quality reads were de novo assembled into 31,997 contigs, which comprised 1943 microsatellite repeats. The dinucleotide and trinucleotide repeats were most abundantly distributed in the genome with 52.95 and 41.17%, respectively. Depending on the sufficient flanking sequence, only 1123 repeats were successfully tagged with primer pairs and these sites were designated as sequence-tagged microsatellite (STMS) markers. Further, a subset of 106 STMS markers were validated through PCR amplification; 77 marker loci were successfully amplified, and 48 of these showed polymorphism. Same set of marker loci were also tested for their cross-amplification in other three temperate bamboo species of the NW Himalayas, which revealed good level of transferability (27-48%) but lesser polymorphism (4-12%). In addition, the genomewide in silico cross-amplification revealed poor cross-transferability in other bamboo taxa representing four different phylogenetic lineages, namely Phyllostachys edulis (10.2%), Bonia amplexicaulis (3.03%), Guadua angustifolia (1.60%), Olyra latifolia (0.89%) and Raddia guianensis (0.36%). Ten polymorphic markers were further used to estimate the measures of genetic diversity in two natural populations, which revealed high genetic diversity (polymorphic information content, PIC = 0.889; expected heterozygosity, He = 0.756) and low genetic differentiation (FST=0.061; Nm = 5.445). To the best of our knowledge, this is one of the pioneer studies carried out for the development of genomic STMS markers through genome skimming approach in Indian bamboo species. The marker information generated here is novel and of paramount importance for future genetic studies in D. falcatum as well as other temperate bamboo species through cross-transferability.

Chloroplast-based DNA barcode analysis indicates high discriminatory potential of matK locus in Himalayan temperate bamboos.

The study was conducted to evaluate the discriminatory potential of selected chloroplast-based DNA barcode regions for identifying and resolving phylogeny of the Indian bamboos. Among 11 chloroplast markers screened, only four, namely matK, rbcL, psbK-I and rps16-trnQ showed successful amplification in 88 genotypes of 30 Indian bamboo taxa under Bambuseae and Arundinarieae tribes. A total of 244 sequences were generated for the four chloroplast regions. Tree-based analysis demonstrated that none of the tested regions successfully discriminated the taxa under Bambuseae tribe. Importantly, our highly concerned Himalayan temperate bamboo species under Arundinarieae tribe, were successfully discriminated by matK locus with high bootstrap support (>60%). Sequence comparisons revealed that the discriminatory power demonstrated by matK region actually lies in the few unique fixed nucleotides (UFNs) despite the overall DNA polymorphism. Although, rps16-trnQ region was found to be the most polymorphic and revealed high genetic divergence among different taxonomic levels, it could not successfully discriminated the taxa with strong statistical support. In a taxonomically difficult plant group like bamboos, whose genome is relatively more complex and has a slow rate of molecular evolution, it is difficult to get a universal marker. Further, highly variable barcode regions utilized in other species may not be informative, and thus, the development of DNA barcodes for different taxonomic levels, such as lineages or tribes could be a viable approach.

First de novo genome specific development, characterization and validation of simple sequence repeat (SSR) markers in Genus Salvadora.

Salvadoraceae constitutes ecologically imperative desert families of 3 genera-Azima, Dobera and Salvadora. Under genus Salvadora of this family, S. oleoides is a keystone species of socio-economic and medicinal value. This species naturally grows in the arid zones but currently experiencing severe fragmentation due to land use change and reduced regeneration, which may have resulted in the depletion of genetic diversity. Hence, it is up-most important to develop genomic resources for studying the population genetics in S. oleoides. This study aims to develop robust microsatellites markers, which were not yet reported in genus Salvodora due to lack of genome sequence information. We developed novel microsatellites markers in S. oleoides using Illumina paired-end sequencing technology. In total, 14,552 simple sequence repeat (SSR) markers were successfully designed from 21,055 microsatellite repeats detected in the 13 Gb raw sequence data. Afterwards, a subset of 101 SSRs were randomly selected and validated, 94 primers were successfully amplified and 34 showed polymorphisms. These SSRs were used to estimate the measures of genetic diversity in three natural populations of state Rajasthan and Gujarat. Importantly, average number of alleles (Na), observed heterozygosity (Ho), expected heterozygosity (He), and polymorphism information content (PIC) were recorded as 2.4, 0.529, 0.357, and 0.326, respectively. Furthermore, 15 primers were evaluated in S. persica for cross-transferability, and all were successfully amplified but only eight showed polymorphisms. This study has been conducted first time for S. oleoides and pioneer among the native species of arid-zone in India.

First record of off-season flowering in Populus deltoides from India: paradigm of climate change indicator.

Populus deltoides is a fast-growing woody species possessing plethora of industrial applications. This species evolutionarily developed unisexual male and female catkin inflorescence on separate trees. Flowering usually occurs during early spring before the development of foliage, where buds appear near axils or at the extending shoots. In 2019, surveys were undertaken to study the flowering pattern of P. deltoides in the states of Punjab, Haryana, Uttar Pradesh and Uttarakhand in northern India. Interestingly, an anomalous flowering behaviour (appearance of off-season male catkins during autumn, i.e. October) was observed in a plantation trial at Kapurthala, Punjab. The male catkins were 2.7-3.1 ± 0.07 cm long and 0.3-0.5 ± 0.03 cm wide, which is significant for flowering and liberation of pollen grains. Preliminary results suggested that climatic factors, such as episodes of high or low temperature and the precipitation variation forcing the tree species to behave differently. Unearthing the climate-driven off-season flowering in other tree species alluded the stimulation of phytohormones, such as gibberellic and salicylic acid concentrations influencing the flowering time, therefore, needs further investigation in case of P. deltoides. Overall, this work provides early clues of changing climatic scenario altering the flowering pattern of a tropical forestry tree species.