Integrated mRNA and small RNA sequencing reveals post-transcriptional regulation of the sesquiterpene pathway in Santalum album L. (Indian sandalwood).

Key message: In sandalwood, negative pattern of regulation by miRNAs was documented in key genes from the sesquiterpene pathway, with cytochrome P450 reductase showing maximum miRNA targets, followed by sesquisabianene synthase 1. Abstract: A comprehensive knowledge of the molecular regulation of sesquiterpene biosynthetic pathway through transcriptomic studies is well established in Santalum album (Indian Sandalwood). However, the post-transcriptional regulation of the genes regulating the pathway is still elusive in this genus. In the present study, an integrated analysis of wood transcriptome and small RNA datasets was conducted to investigate the role of miRNAs in regulating the expression of transcripts involved in santalol production mediated by the sesquiterpene biosynthesis pathway. A total of 24,237 transcripts were annotated from the wood transcriptome, and 45 transcripts were mapped to the sesquiterpenoid pathway. Small RNA data analysis identified 257 conserved miRNAs belonging to 50 families and 7 novel putative miRNAs. Sa-miR156, Sa-miR396, Sa-miR166, and Sa-miR319 had the most number of members among the miRNA families. An integrated analysis predicted 69 miRNA members belonging to 12 families that targeted 12 transcripts from the sesquiterpene pathway, with a maximum of 24 miRNAs regulating cytochrome P450 reductase, followed by sesquisabianene synthase 1, which was targeted by 23 miRNAs. Validation of miRNA-mRNA interaction by qRT-PCR revealed a negative pattern of regulation in six miRNA-mRNA target pairs across wood tissues sourced from four genotypes. The present study provides the first crucial insight into the post-transcriptional regulation of the sesquiterpene pathway genes in the genus Santalum and opens up a new perspective in metabolite engineering for enhanced essential oil production in sandalwood. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03816-4.

The draft genome reveals early duplication event in Pterocarpus santalinus: an endemic timber species.

MAIN CONCLUSION: A 541 Mb draft genome of Pterocarpus santalinus is presented and evidence of whole-genome duplication in the Eocene period with expansion of drought responsive gene families is documented. Pterocarpus santalinus Linn. f., popularly known as Red Sanders, is a deciduous tree, endemic to southern parts of Eastern Ghats in India. The heartwood is highly valued in the international market due to its deep red colour, fragrant heartwood and wavy grained texture. In the present study, a high-quality draft genome of P. santalinus was assembled using short and long reads generated from Illumina and Oxford Nanopore Sequencing platforms, respectively. The haploid genome size was estimated at 541 Mb and the hybrid assembly showed 99.60% genome completeness. A total of 51,713 consensus gene set were predicted with 31,437 annotated genes. The age of the whole-genome duplication event in the species was dated at 30-39 mya with 95% confidence suggesting early genome duplication event during the Eocene period. Concurrently, phylogenomic assessment of seven Papilionoideae members including P. santalinus grouped the species based on the tribal classification and established divergence of the tribe Dalbergieae from tribe Trifolieae at ~ 54.20 mya. A significant expansion of water deprivation/drought responsive gene families documented in the study probably explains the occurrence of the species in dry rocky patches. Additionally, re-sequencing of six diverse genotypes predicted one variant every 27 bases. This report presents the first draft genome in the genus Pterocarpus and the unprecedented genomic information generated is expected to accelerate population divergence studies in the species in relation to its endemic nature, support trait-based breeding programme and aid in development of diagnostic tools for timber forensics.

Targeted re-sequencing and genome-wide association analysis for wood property traits in breeding population of Eucalyptus tereticornis × E. grandis.

Globally, Eucalyptus plantations occupy 22 million ha area and is one of the preferred hardwood species due to their short rotation, rapid growth, adaptability and wood properties. In this study, we present results of GWAS in parents and 100 hybrids of Eucalyptus tereticornis × E. grandis using 762 genes presumably involved in wood formation. Comparative analysis between parents predicted 32,202 polymorphic SNPs with high average read depth of 269-562× per individual per nucleotide. Seventeen wood related traits were phenotyped across three diverse environments and GWAS was conducted using 13,610 SNPs. A total of 45 SNP-trait associations were predicted across two locations. Seven large effect markers were identified which explained more than 80% of phenotypic variation for fibre area. This study has provided an array of candidate genes which may govern fibre morphology in this genus and has predicted potential SNPs which can guide future breeding programs in tropical Eucalyptus.

Reference-based assembly of chloroplast genome from leaf transcriptome data of Pterocarpus santalinus.

Chloroplast genome sequencing is an essential tool to understand genome evolution and phylogenetic relationship. The available methods for constructing chloroplast genome include chloroplast enrichment followed by long overlapping PCR or extraction and assembly of chloroplast-specific reads from whole-genome datasets. In the present study, we propose an alternate strategy of extraction and assembly of chloroplast-specific reads from leaf transcriptome data of Pterocarpus santalinus using bowtie2 aligner program. The assembled genome was compared with the published chloroplast genome of P. santalinus for genome size, number of predicted genes, microsatellite repeat motifs, and nucleotide repeats. A near-complete chloroplast genome was assembled from the transcriptome reads. The proposed method requires less computational time and know-how, limited virtual memory, and is cost-effective when compared to whole-genome sequencing. Assembly of Cp genome from transcriptome data will enhance the resolution of phylogenetic studies through comparative plastome analysis, facilitate accurate species/genotype discrimination and accelerate the development of transplastomic plants with enhanced biotic and abiotic tolerance. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02943-0.

Quantitative genetic analysis of wood property traits in biparental population of Eucalyptus camaldulensis × E. tereticornis.

Eucalyptus breeding programme mainly aims at increasing productivity associated with wood property traits which are suitable for different end uses. The principal challenge in this endeavor is to combine productivity with industrially relevant wood traits. In the present study, 23 hybrid clones derived from a biparental mapping population of Eucalyptus camaldulensis × E. tereticornis was assessed for six wood property traits across two sites in Tamil Nadu, India. The mean of most of the traits evaluated was consistently higher in Muthupettai, indicating significant site effect. Combined and location-wise analysis indicated additive genetic control of assessed traits. The stability of acoustic velocity in study sites, negligible G × E interaction and significant correlation with dynamic modulus of elasticity (DMoE) implies its use in selecting trees/logs for solid wood properties. Combined analysis of locations revealed low to moderate heritability (0.294-0.439) for all the traits with H2 being highest for cellulose per cent (0.439) followed by acoustic velocity (0.416). Genetic advance was calculated and was the highest for diameter (10.47%) followed by DMoE (9.19%). The two major chemical constituents of wood, namely total lignin and cellulose per cent showed 7.13% and 7.53% advancement in the hybrids. The out-performance of several hybrid clones when compared to the parents for different wood traits reiterates the use of Eucalyptus hybrids in plantation programmes to improve quality of raw material suitable for industrial application.

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.

Identification and expression profiling of genes governing lignin biosynthesis in Casuarina equisetifolia L.

Casuarina equisetifolia L. is an important multi-purpose, fast growing and widely planted tree species native to tropical and subtropical coastlines of Australia, Southeast Asia, Malaysia, Melanesia, Polynesia and New Caledonia. It is a nitrogen-fixing tree mainly used for charcoal making, construction poles, landscaping, timber, pulp, firewood, windbreaks, shelterbelts, soil erosion and sand dune stabilization. Casuarina wood is presently used for paper and pulp production. Raw material with reduced lignin is highly preferred to increase the pulp yield. Hence, understanding the molecular regulation of wood formation in this tree species is vital for selecting industrially suitable phenotypes for breeding programs. The lignin biosynthetic pathway has been extensively studied in tree species like Eucalypts, poplars, pines, Picea, Betula and Acacia sp. However, studies on wood formation at molecular level is presently lacking in casuarinas. Hence, in the present study, the transcriptome of the developing secondary tissues of 15 years old Casuarina equiseitfolia subsp. equisetifolia was sequenced, de novo assembled, annotated and mapped to functional pathways. Transcriptome sequencing generated a total of 26,985 transcripts mapped to 31 pathways. Mining of the annotated data identified nine genes involved in lignin biosynthesis pathway and relative expression of the transcripts in four tissues including scale-like leaves, needle-like brachlets, wood and root were documented. The expression of CeCCR1 and CeF5H were found to be significantly high in wood tissues, while maximum expression of CeHCT was documented in stem. Additionally, CeTUBA and CeH2A were identified as the most stable reference transcript for normalization of qRT-PCR data in C. equisetifolia. The present study is the first wood genomic resource in C. equisetifolia, which will be valuable for functional genomics research in this genus.

Co-expression network of transcription factors reveal ethylene-responsive element-binding factor as key regulator of wood phenotype in Eucalyptus tereticornis.

Suitability of wood biomass for pulp production is dependent on the cellular architecture and composition of secondary cell wall. Presently, systems genetics approach is being employed to understand the molecular basis of trait variation and co-expression network analysis has enabled holistic understanding of complex trait such as secondary development. Transcription factors (TFs) are reported as key regulators of meristematic growth and wood formation. The hierarchical TF network is a multi-layered system which interacts with downstream structural genes involved in biosynthesis of cellulose, hemicelluloses and lignin. Several TFs have been associated with wood formation in tree species such as Populus, Eucalyptus, Picea and Pinus. However, TF-specific co-expression networks to understand the interaction between these regulators are not reported. In the present study, co-expression network was developed for TFs expressed during wood formation in Eucalyptus tereticornis and ethylene-responsive element-binding factor, EtERF2, was identified as the major hub transcript which co-expressed with other secondary cell wall biogenesis-specific TFs such as EtSND2, EtVND1, EtVND4, EtVND6, EtMYB70, EtGRAS and EtSCL8. This study reveals a probable role of ethylene in determining natural variation in wood properties in Eucalyptus species. Understanding this transcriptional regulation underpinning the complex bio-processing trait of wood biomass will complement the Eucalyptus breeding program through selection of industrially suitable phenotypes by marker-assisted selection.

Characterization of an Insecticidal Protein from Withania somnifera Against Lepidopteran and Hemipteran Pest.

Lectins are carbohydrate-binding proteins with wide array of functions including plant defense against pathogens and insect pests. In the present study, a putative mannose-binding lectin (WsMBP1) of 1124 bp was isolated from leaves of Withania somnifera. The gene was expressed in E. coli, and the recombinant WsMBP1 with a predicted molecular weight of 31 kDa was tested for its insecticidal properties against Hyblaea puera (Lepidoptera: Hyblaeidae) and Probergrothius sanguinolens (Hemiptera: Pyrrhocoridae). Delay in growth and metamorphosis, decreased larval body mass and increased mortality was recorded in recombinant WsMBP1-fed larvae. Histological studies on the midgut of lectin-treated insects showed disrupted and diffused secretory cells surrounding the gut lumen in larvae of H. puera and P. sanguinolens, implicating its role in disruption of the digestive process and nutrient assimilation in the studied insect pests. The present study indicates that WsMBP1 can act as a potential gene resource in future transformation programs for incorporating insect pest tolerance in susceptible plant genotypes.

Identification of PEG-induced water stress responsive transcripts using co-expression network in Eucalyptus grandis.

Ecophysiological studies in Eucalyptus have shown that water is the principal factor limiting stem growth. Effect of water deficit conditions on physiological and biochemical parameters has been extensively reported in Eucalyptus. The present study was conducted to identify major polyethylene glycol induced water stress responsive transcripts in Eucalyptus grandis using gene co-expression network. A customized array representing 3359 water stress responsive genes was designed to document their expression in leaves of E. grandis cuttings subjected to -0.225MPa of PEG treatment. The differentially expressed transcripts were documented and significantly co-expressed transcripts were used for construction of network. The co-expression network was constructed with 915 nodes and 3454 edges with degree ranging from 2 to 45. Ninety four GO categories and 117 functional pathways were identified in the network. MCODE analysis generated 27 modules and module 6 with 479 nodes and 1005 edges was identified as the biologically relevant network. The major water responsive transcripts represented in the module included dehydrin, osmotin, LEA protein, expansin, arabinogalactans, heat shock proteins, major facilitator proteins, ARM repeat proteins, raffinose synthase, tonoplast intrinsic protein and transcription factors like DREB2A, ARF9, AGL24, UNE12, WLIM1 and MYB66, MYB70, MYB 55, MYB 16 and MYB 103. The coordinated analysis of gene expression patterns and coexpression networks developed in this study identified an array of transcripts that may regulate PEG induced water stress responses in E. grandis.

Characterization of Withania somnifera leaf transcriptome and expression analysis of pathogenesis-related genes during salicylic acid signaling.

Withania somnifera (L.) Dunal is a valued medicinal plant with pharmaceutical applications. The present study was undertaken to analyze the salicylic acid induced leaf transcriptome of W. somnifera. A total of 45.6 million reads were generated and the de novo assembly yielded 73,523 transcript contig with average transcript contig length of 1620 bp. A total of 71,062 transcripts were annotated and 53,424 of them were assigned GO terms. Mapping of transcript contigs to biological pathways revealed presence of 182 pathways. Seventeen genes representing 12 pathogenesis-related (PR) families were mined from the transcriptome data and their pattern of expression post 17 and 36 hours of salicylic acid treatment was documented. The analysis revealed significant up-regulation of all families of PR genes by 36 hours post treatment except WsPR10. The relative fold expression of transcripts ranged from 1 fold to 6,532 fold. The two families of peroxidases including the lignin-forming anionic peroxidase (WsL-PRX) and suberization-associated anionic peroxidase (WsS-PRX) recorded maximum expression of 377 fold and 6532 fold respectively, while the expression of WsPR10 was down-regulated by 14 fold. Additionally, the most stable reference gene for normalization of qRT-PCR data was also identified. The effect of SA on the accumulation of major secondary metabolites of W. somnifera including withanoside V, withaferin A and withanolide A was also analyzed and an increase in content of all the three metabolites were detected. This is the first report on expression patterns of PR genes during salicylic acid signaling in W. somnifera.