Assessment of seed- and seedling-related traits in Santalum album (Indian sandalwood) reveals high adaptive potential.

Seed and seedling traits govern plant fitness and persistence and are influenced by the interaction between the plant and its environment. Changing climatic and edaphic conditions will drastically affect early fitnessrelated traits and can alter the demography and species distribution range. It is widely documented that trait variation among populations may increase resilience of tree communities and reduce the risk of extinction under future climates. In the present study, variation in seed and seedling traits were documented from seven populations of Santalum album representing the natural distribution range of the species in the Indian subcontinent. Significant intra-specific variation was documented in seed and seedling traits, indicating high adaptive potential of the species. Further, the measured traits were correlated with climatic variables. No significant correlation was predicted for seed-related traits, while seedling-related traits like shoot and root weight, photochemical reflectance index, relative water content, and root-shoot ratio correlated with different climatic parameters. Variance partitioning revealed predominant combined effect of environment and genotype on seed traits except seed weight, which was governed by genotypic effect. The dominance of genotypic effect was documented for all seed leachate parameters, while seedling-related traits were predominantly affected by the environment. Conservation of sandalwood genetic resources will benefit from the insights gained from the variability recorded in these fitness-related traits, which are likely to affect the adaptive potential of the species.

Wood transcriptome analysis and expression variation of lignin biosynthetic pathway transcripts in Ailanthus excelsa Roxb., a multi-purpose tropical tree species.

Safety matches are an essential consumer commodity which faces considerable shortage of raw material due to specific requirements like color, wax stability, consistent burning and splinting ability in wood. Ailanthus excelsa Roxb. is a multi-purpose tropical tree species which is predominantly used for production of match splints. It is a data deficient species with no information on molecular regulation governing the wood phenotypes. Hence, with the aim to identify the lignin biosynthetic pathway transcripts, transcriptome sequencing of pooled developing secondary wood tissues of a 22-month-old tree was conducted. The processed reads were de novo assembled and 48,493 unigenes were annotated. Quantitative real time PCR was conducted in six even-aged trees to document the natural expression profiles of nine major transcripts from the monolignol pathway. Significant differential expression of Phenylalanine ammonia-lyase (AePAL), Cinnamate 4-hydroxylase (AeC4H), Cinnamyl-alcohol dehydrogenase (AeCAD), Laccase (AeLACI) and Caffeic Acid 3-O-Methyl Transferase (AeCOMT) was documented across all genotypes studied. Transcriptome-wise microsatellites were also mined and is the first co-dominant marker resource in this species. The genomic resource generated in A. excelsa will facilitate understanding the molecular mechanisms of wood formation and accelerate trait-based breeding program through diversity assessment, population structure analysis, clonal discrimination and marker assisted selection.

Diversity of bacterial endophyte in Eucalyptus clones and their implications in water stress tolerance.

The genus Eucalyptus with over 747 species occurs in wide ecological range and is preferred for bioenergy plantations due to their short rotation, rapid growth and superior wood properties. They are planted in 22 million ha area and India is third largest planter of Eucalyptus. In the present study, the bacterial endophyte community in leaves of six Eucalyptus clones belonging to E. tereticornis and E. camaldulensis was assessed by sequencing the V3-V4 region of the bacterial 16S rRNA gene. The clones were selected based on their response to progressive water stress. A total of 4947 operational taxonomic units (OTUs) were obtained and the dominant phyla were Proteobacteria, Bacteroidetes and Firmicutes. Escherichia coli was enriched in all samples at species level. Comparison of endophyte diversity was conducted between the two species and across the water stress tolerant and susceptible clones. The alpha-diversity analysis revealed that species richness and diversity was high in E. camaldulensis and water stress susceptible clones. LefSe analysis predicted 69 and 54 significantly enriched taxonomic biomarkers between species and stress response groups respectively. A maximum of 49 taxonomic biomarkers were recorded in susceptible group and the significantly enriched species were Bacteroides thetaiotaomicron and Turicibacter sanguinis, while the tolerant group documented 5 biomarkers including oscillibacter sp. The presence of functional biomarkers was also assessed in both the groups. The findings of the present study provides an insight into the diversity of bacterial endophyte in Eucalyptus leaves and to our knowledge this is the first report on documenting the endophyte abundance in water stress responsive Eucalyptus clones.

Genetic linkage map and QTL identification for adventitious rooting traits in red gum eucalypts.

The eucalypt species, Eucalyptus tereticornis and Eucalyptus camaldulensis, show tolerance to drought and salinity conditions, respectively, and are widely cultivated in arid and semiarid regions of tropical countries. In this study, genetic linkage map was developed for interspecific cross E. tereticornis × E. camaldulensis using pseudo-testcross strategy with simple sequence repeats (SSRs), intersimple sequence repeats (ISSRs), and sequence-related amplified polymorphism (SRAP) markers. The consensus genetic map comprised totally 283 markers with 84 SSRs, 94 ISSRs, and 105 SRAP markers on 11 linkage groups spanning 1163.4 cM genetic distance. Blasting the SSR sequences against E. grandis sequences allowed an alignment of 64% and the average ratio of genetic-to-physical distance was 1.7 Mbp/cM, which strengths the evidence that high amount of synteny and colinearity exists among eucalypts genome. Blast searches also revealed that 37% of SSRs had homologies with genes, which could potentially be used in the variety of downstream applications including candidate gene polymorphism. Quantitative trait loci (QTL) analysis for adventitious rooting traits revealed six QTL for rooting percent and root length on five chromosomes with interval and composite interval mapping. All the QTL explained 12.0-14.7% of the phenotypic variance, showing the involvement of major effect QTL on adventitious rooting traits. Increasing the density of markers would facilitate the detection of more number of small-effect QTL and also underpinning the genes involved in rooting process.

Quantitative trait loci mapping for stomatal traits in interspecific hybrids of Eucalyptus.

Eucalyptus is an important industrial species with tolerance to drought and salt stress. Genetic improvement activities including quantitative trait loci (QTL) mapping for pulping and adventitious rooting traits are in progress, but no information is available on the genomic regions on adaptive traits such as stomatal characteristics. In this study, an interspecific cross between Eucalyptus tereticornis and E. grandis was generated for the development of genetic map and QTL identification for stomatal traits. Simple sequence repeats (SSRs), inter-simple sequence repeats (ISSRs) and sequence related amplified polymorphism (SRAP)markers were used for genotyping the F1 individuals. Parent-specific geneticmaps (female, 1023.56 cM;male, 1049.64cM) and consensus map (1049.4 cM) were developed. QTL analysis was carried out to identify the chromosomal regions affecting stomatal density, area and pore length in adaxial and abaxial leaf surface. Seven QTLs were identified with phenotypic variation of 11.36 to 27.30% for stomatal density, area and pore length. Correlation of stomatal traits when combined with growth and wood properties would have greater implications for generation of stress tolerant eucalypt hybrids with higher productivity and adaptability.

Construction of co-expression network based on natural expression variation of xylogenesis-related transcripts in Eucalyptus tereticornis.

Natural genetic variation is randomly distributed and gene expression patterns vary widely in natural populations. These variations are an effect of multifactorial genetic perturbations resulting in different phenotypes. Genome-wide analysis can be used to comprehend the genetic basis governing this naturally occurring developmental variation. Secondary growth is a highly complex trait and systems genetics models are presently being applied to understand the molecular architecture of wood formation. In the present study, the natural variation in expression patterns of 18,987 transcripts expressed in the developing xylem tissues were documented across four phenotypes of Eucalyptus tereticornis with distinct holocellulose/klason lignin content. The differentially expressed genes across all the phenotypes were used to construct co-expression networks and sub-network 2 with 380 nodes and 17,711 edges was determined as the network of relevance, including 30 major cell wall biogenesis related transcripts with 2394 interactions and 10 families of transcription factors with 3360 interactions. EYE [EMBRYO YELLOW] was identified as major hub transcript with 173 degrees which interacted with known cell wall biogenesis genes. K-mean clustering was also performed for differentially expressed transcripts and two clusters discriminated the phenotypes based on their holocellulose/klason lignin content. The cluster based networks were enriched with GOs related to cell wall biogenesis and sugar metabolism. The networks developed in the present study enabled identification of critical regulators and novel transcripts whose expression variation could presumably govern the phenotypic variation in wood properties across E. tereticornis.

Development of genetic markers in Eucalyptus species by target enrichment and exome sequencing.

The advent of next-generation sequencing has facilitated large-scale discovery, validation and assessment of genetic markers for high density genotyping. The present study was undertaken to identify markers in genes supposedly related to wood property traits in three Eucalyptus species. Ninety four genes involved in xylogenesis were selected for hybridization probe based nuclear genomic DNA target enrichment and exome sequencing. Genomic DNA was isolated from the leaf tissues and used for on-array probe hybridization followed by Illumina sequencing. The raw sequence reads were trimmed and high-quality reads were mapped to the E. grandis reference sequence and the presence of single nucleotide variants (SNVs) and insertions/ deletions (InDels) were identified across the three species. The average read coverage was 216X and a total of 2294 SNVs and 479 InDels were discovered in E. camaldulensis, 2383 SNVs and 518 InDels in E. tereticornis, and 1228 SNVs and 409 InDels in E. grandis. Additionally, SNV calling and InDel detection were conducted in pair-wise comparisons of E. tereticornis vs. E. grandis, E. camaldulensis vs. E. tereticornis and E. camaldulensis vs. E. grandis. This study presents an efficient and high throughput method on development of genetic markers for family- based QTL and association analysis in Eucalyptus.

Isolation of developing secondary xylem specific cellulose synthase genes and their expression profiles during hormone signalling in Eucalyptus tereticornis.

Cellulose synthases (CesA) represent a group of ß-1, 4 glycosyl transferases involved in cellulose biosynthesis. Recent reports in higher plants have revealed that two groups of CesA gene families exist, which are associated with either primary or secondary cell wall deposition. The present study aimed at identifying developing secondary xylem specific cellulose synthase genes from Eucalyptus tereticornis, a species predominantly used in paper and pulp industries in the tropics. The differential expression analysis of the three EtCesA genes using qRT-PCR revealed 49 to 87 fold relative expression in developing secondary xylem tissues. Three full length gene sequences of EtCesA1, EtCesA2 and EtCesA3 were isolated with the size of 2940, 3114 and 3123 bp, respectively. Phytohormone regulation of all three EtCesA genes were studied by exogenous application of gibberellic acid, naphthalene acetic acid, indole acetic acid and 2, 4-epibrassinolide in internode tissues derived from three-month-old rooted cuttings. All three EtCesA transcripts were upregulated by indole acetic acid and gibberellic acid. This study demonstrates that the increased cellulose deposition in the secondary wood induced by hormones can be attributed to the upregulation of xylem specific CesAs.