Genetic Diversity analysis and population structure of tree species:

The genetic diversity estimations and population structure analysis were conducted for different tree species including teak, Casuarina, Eucalypts, Acacia, Ailanthus, Melia and Gmelina using different marker systems like RAPD, ISSR, FISSR, SSR and SNPs. The studies were conducted at different population levels including species, provenances, seed orchards and clones. Species specific ISSR and SCAR markers were also developed for Eucalypts and Casuarina.

Marker-assisted selection in Eucalyptus

 

A network program with partners from Institutes, Forest Development Corporations and Paper Industries is underway to identify DNA markers tagging industrially important traits in Eucalyptus. In this endeavor, phenotypically characterized inter-specific mapping populations were developed and multi-environmental trials were established in different agro climatic zones. Precision phenotyping of the populations for wood properties and adventitious rooting traits have been conducted. Genotyping using SNP/ SSR/InDels markers in parents and hybrids have been conducted and high density linkage maps have been constructed to identify QTLs tagging wood property and adventitious rooting traits. Genome-wide association analysis was conducted and SNPs tagging different wood property traits were identified..

Genomics Assisted Conservation of Teak and Sandal Genetic Resources

 

 

 

Tree genetic resources support the human kind for several million years and their conservation is essential to acquire the benefits continuously. The objectives of conserving forest genetic resources are to safeguard the natural genetic variations of tree species for improving the productivity and adaptation to environmental changes. Teak and Sandal genetic resources (TGRs and SGRs) are dwindling in its natural habitats and their conservation requires identification of genetic diversity hotspots and delineation of potential management units. Population genomics offer methods to quantify adaptive genetic differentiation with reference to environment and phenophase variations. Thousands of simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) were identified for genotyping of teak and sandal germplasm through whole genome sequencing. Genomic data will be utilized for teak and sandal populations available in the country to infer the spatial patterns of adaptation for conservation, management and sustainable utilization.

 

 

Genomic resources in indigenous tree species:

 

 

 

 

 

 

 

 

 

The genome sequence of indigenous species including teak, sandalwood, Korthalisia laciniosa and Calamus brandisii were generated, de novo assembled and annotated. The chloroplast genomes were re-constructed for phylogenetic positioning of the species. Additionally, transcriptome resources were generated for Casuarina equisetifolia, Ailanthus excelsa, teak and sandalwood. These genomic datasets will accelerate advanced research in population/ landscape genomics, trait based breeding, phylogenomics and facilitate understanding different developmental processes at molecular level. Reference genome assembly of teak was generated with the combination of long (Nanopore) and short read (Illumina) technology. Polymorphic microsatellites discovered from teak genome were utilized for genotyping of teak genetic resource from Southern Western Ghats region (Karnataka, Kerala and Tamil Nadu) and deciphered the population genetic structure and genecological zones for conservation purposes

 

 

 

 

 

 

 

 

Clonal propagation of industrially important species

 

 

 

The superior clones generated from the tree improvement/breeding programs of Eucalypts, Casaurinas, Melia, Acacia, Teak and Cadamba are multiplied for demonstration trials and clonal evaluation trails using minicutting techniques. Vegetative propagation facility also takes up large scale multiplication of IFGTB released clones of Casuarina junghuniana, Casuarina hybrids and Eucalyptus spp to popularize the high yielding clones developed by the institute based on the request from planting agencies, state forest departments and farmers.

 

 

 

 

 

Genetic improvement of Melia dubia

Melia dubia Cav. a fast growing short rotational indigenous tree species has captured the interest of many farmers due to its fast growth and high economic returns. To realize maximum gains by the deployment of the species, a comprehensive improvement strategy with the objective to identify natural populations and high yielding material from plantations with a long term domestication programme as the main focus is being attempted. Extensive field surveys were undertaken in the states of Kerala, Tamil Nadu and Karnataka for selecting phenotypically superior trees based on growth and stem form.  From identified candidate plus trees, plus trees were shortlisted for clonal assemblage. Establishing and managing provenance resource stands, progeny trials, seed orchards and evaluation trials to develop new high-yielding clones /seeds of Melia. Pest and disease problems of the species in nursery and natural plantations recorded and control measures being developed. Developed protocols for macro and micropropagation of Melia dubia.

 

 

 

Micropropagation of important clonal accessions

 

 

 

Teak, the premier timber species face shortage of propagules in the form of seeds and vegetative cuttings. IFGTB is producing teak from selected germplasm through tissue culture. Early growth performance of these plants in farmlands shows promisin g results. These clones can be procured from IFGTB by placing orders 90 days in advance. IFGTB also propagates germplasm of bamboos identified by RFRI, Jorhat. The species include Bambusa nutans, B. tulda and Dendrocalamus giganteus.

 

 

 

 

 

DNA Barcode Studies to understand species level variations between Melia accessions from South (IFGTB), North (FRI) and North East (RFRI) India.

IFGTB received accessions of Melia from FRI, Dehradun and RFRI, Jorhat. IFGTB made selections from the states of Karnataka, Andhra Pradesh, Kerala and Karnataka. Variations were observed in morphology, karyotype analysis and genetic diversity assessment using SSR markers. To understand the species level variations, Melia species were evaluated using barcode genes. Seven genotypes of Melia from IFGTB (two each from Tamil Nadu, Kerala and three from Karnataka – MD1, MD2, MD12, MD16, MD6, MD5, MD59),  two from FRI (FRI-114, FRI-234) and three from RFRI (KOH 7, DIMA 1 and DIMA 5) along with two samples of Melia azedarach (SM1 and SM2) were selected for species identification and discrimination. Four different barcode genes viz., rbcL, matK, ITS and trnH-psbA were selected for species identification and discrimination. Gene sequences and whole genome of Melia dubia and Melia azedarach available in the NCBI were used. Sequence results analyzed using rbcL, matK and ITS genes from the NCBI revealed that the accessions from FRI and RFRI are Melia azedarach, whereas the accessions of IFGTB collected from the States of Tamil Nadu, Kerala and Karnataka are Melia dubia. Though the accessions from FRI and RFRI apparently belong to the same species, Melia azedarach, there are distinct morphological variations between the accessions. The accessions of RFRI appears to be different from all others (as per the BLAST results of the rbcLII), and the possibly of it being a different ecotype, is under study.

 

 

Research in medicinal plants

• Survey and collection of Medicinal and Aromatic Plants (MAPs) from different phyto-geographical regions of the country

• Multiplication of Quality Planting Material, popularization and commercialization

• Isolation and Characterization of Chemo/bio-markers

• Quantitative determination of bioactives using HPTLC and development of chemical fingerprints

• Value addition and its impacts on quality and quantity of bioactives in myrobalans

• Genomics and transcriptomics in medicinal plants

 

Secondary metabolite production from medicinally important trees

The different activities being conducted include:

• Commercial production of teak and mass production of bamboos

• Direct regeneration systems for Aegle marmelos, Oroxylum indicum, Gmelina arborea, Premna integrifolia, Pterocarpus santalinus, Azadirachta indica

• Cell culture protocol for in vitro production of secondary metabolites from Aegle marmelos  and Azadirachta indica

• Hairy root cultures for Aegle marmelos, Oroxylum indicum and Gmelina arborea

Functional genetics and genetic engineering of trees for improved traits

The transgenic programme aims to evolve and apply tools of genetic and genome engineering (RNAi, CRISPR/Cas9) for understanding gene function and enhancing desired traits. The programme aims to evolve transgenic varieties of Eucalyptus and Casuarina for enhancing productivity in climate stressed areas like wastelands affected by salt and drought. Other traits of interest include insect tolerance and enhanced pulping efficiency. A composite transgenic strategy was developed in Eucalyptus for identifying major genes that could be used for engineering salt/drought tolerance. The composite transgenic strategy along with RNAi approach was used to identify EcHKT1:1 as a key gene regulating salt stress tolerance in Eucalyptus. To incorporate tolerance in Eucalyptus to the insect pest, Leptocybe invasa, a multigene targeting chimeric hpRNAi construct was developed based on transcriptome sequences generated from L. invasa. These RNAi constructs are now being used for evaluating their utility in generating transgenics with enhanced salt and insect tolerance. Projects on understanding salt tolerance mechanisms in mangrove species are also ongoing.

DNA Isolation kit: ArborEasy®

 

 

 

 

ArborEasy® DNA Isolation Kit provides an indigenous, non-biohazardous, low cost spin column based system for isolation of plant genomic DNA from wide range of tissue types, specifically challenging tissues from tree species. This technology was developed based on IFGTB-DBT joint patent. ArborEasy® is a registered trademark of IFGTB, Coimbatore, India. DNA isolated using this kit can be used for routine PCR analysis; high-throughput marker assays; next generation sequencing and SNaPshot analysis. The customized kit was used for Guinness World Record on “Most people conducting a DNA isolation experiment simultaneously” held at 4th India International Science Festival 2018 at Lucknow. A bipartite memorandum of agreement was signed with Kerafast Inc., USA, a Boston-based reagent company to market and sell the kit to researchers world-wide through their e-commerce portal based on a non-exclusive license.

 

 

 

 

 

 

• Obtained a Patent (a team of four researchers) on “A process for in vitro plantlet production of the bamboo, Oxytenanthera stocksii” The patent was granted on 31.08.2006 for a term of 20 years. The financial support was granted by the National Research Development Council (NRDC), New Delhi.

• The patent titled “A simple protocol for isolation of un-degraded total RNA from Eucalyptus and Casuarina and cDNA synthesis from unpurified RNA” (Patent number 272765) was granted from 13 August 2009 for a period of 20 years. The technology was subsequently upgraded and ArborEasy® DNA isolation kit was developed for commercial production and marketing.

• A Patent on “A process for in vitro plantlet production of the bamboo, Oxytenanthera stocksii” was granted on 31.08.2006 for a term of 20 years with the financial from National Research Development Council (NRDC), New Delhi.

• Filed provision patent titled “A Multilocus High Resolution Melting Analysis As Diagnostic Tool For Detection Of Viral Genes” with Application number 202041018705.

• Developed the ArborEasy® DNA isolation kit for commercial production and marketing. Trade mark for the name ‘ArborEasy’ was registered to enable branding of the DNA isolation kit.

• Executed the license agreement with Kerafast Inc., USA for worldwide marketing of ArborEasy® DNA isolation kit. The product has been catalogued (Cat No. EIF001) in the kerafast website (https://www.kerafast.com/product/3709/ arboreasy-dna-isolation-kit).

• Tissue culture protocols developed for mass multiplication of bamboos, teak, Eucalyptus and Acacias.

• DNA profiling using different dominant (RAPD/ISSR/AFLP/FISSR) and co-dominant (SSR, SCAR, SNP) for species authentication, clonal discrimination, hybrid purity, genetic fidelity, diversity and population structure assessment in species targeted for tree improvement.

• Mass clonal propagation using mini/micro cutting technology for popularization of IFGTB released clones of Casuarina and Eucalyptus.

• Developed an online database: In silico Gene bank for adaptation to abiotic stresses.

• Identified shoot to root ratio of sodium as a defining parameter for salt tolerance in Casuarina equisetifolia.

• Developed composite transgenic strategy and using RNAi, identified EcHKT1:1 as a key gene regulating salt stress tolerance in Eucalyptus.

• Based on transcriptome sequences generated from Leptocybe invasa, developed an insect-specific, multigene targeting chimeric hpRNAi for use in transgenic incorporation of tolerance to L. invasa.

• Developed an accessory for gene delivery into live plants.

• Barcodes for rbcL and ITS genes in Pterocarpus marsupium and P. santalinus were developed and deposited in BOLD (Barcode of life data system) and NCBI (National Centre for Biotechnology Information).

• Whole genome assembly of Teak was generated to support conservation and breeding activities. Repetitive elements constituted 11.18 % of the genome including several thousands of simple sequence repeats (SSRs) and 615 genes encoding for proteins responsible for the durability of timber.

• Santalum album known for its fragrant heartwood was sequenced at whole genome level, 39,800 protein coding genes identified, 574 miRNA genes and 246,522 SSRs discovered to utilize genomic tools for conservation and management efforts.

• Whole genome sequences of two threatened rattans, Korthalsia laciniosa and Calamus brandisii was assembled and annotated to accelerate conservation of the species.

• High throughput markers developed using target capture and re-sequencing strategy and genome-wide association analysis conducted to identify markers tagging different wood property traits in Eucalyptus mapping populations.
• A rapid, sensitive and cost effective methodology for high throughput validation of markers in Eucalyptus was developed using High Resolution Melting (HRM) strategy.

• A PCR based diagnostic assay was developed for detection of sandal spike disease.

• The concept of Breeding without Breeding was applied in Eucalyptus seed orchards for early prediction of orchard gains.

• Successfully developed a cell culture protocol for in vitro production of secondary metabolites under culture conditions from Aegle marmelos, the efficacy of which was tested against plant and human pathogens in comparison with extracts from the wild trees.

• Developed metabolite profiles of different age genotypes of Saraca asoca  using HPTLC and GC-MS-MS.

• Developed barcodes to differentiate Melia accessions from different locations in the country.

• IFGTB has been designated as the DUS Centre of Melia dubia.

• IFGTB has been identified as a Centre of Excellence by the Ministry of Tribal Affairs to work on Value addition of NTFPs.