Identification of MYB gene family in medicinal tea tree Melaleuca alternifolia (Maiden and Betche) cheel and analysis of members regulating terpene biosynthesis.

BACKGROUND: The tea tree (Melaleuca alternifolia) is renowned for its production of tea tree oil, an essential oil primarily composed of terpenes extracted from its shoot. MYB transcription factors, which are one of the largest TF families, play a crucial role in regulating primary and secondary metabolite synthesis. However, knowledge of the MYB gene family in M. alternifolia is limited. METHODS AND RESULTS: Here, we conducted a comprehensive genome-wide analysis of MYB genes in M. alternifolia, referred to as MaMYBs, including phylogenetic relationships, structures, promoter regions, and GO annotations. Our findings classified 219 MaMYBs into four subfamilies: one 5R-MYB, four 3R-MYBs, sixty-one MYB-related, and the remaining 153 are all 2R-MYBs. Seven genes (MYB189, MYB146, MYB44, MYB29, MYB175, MYB162, and MYB160) were linked to terpenoid synthesis based on GO annotation. Phylogenetic analysis with Arabidopsis homologous MYB genes suggested that MYB193 and MYB163 may also be involved in terpenoid synthesis. Additionally, through correlation analysis of gene expression and metabolite content, we identified 42 MYB genes associated with metabolite content. CONCLUSION: The results provide valuable insights into the importance of MYB transcription factors in essential oil production in M. alternifolia. These findings lay the groundwork for a better understanding of the MYB regulatory network and the development of novel strategies to enhance essential oil synthesis in M. alternifolia.

A terpene synthase supergene locus determines chemotype in Melaleuca alternifolia (tea tree).

Leaf oil terpenes vary categorically in many plant populations, leading to discrete phenotypes of adaptive and economic significance, but for most species, a genetic explanation for the concerted fluctuation in terpene chemistry remains unresolved. To uncover the genetic architecture underlying multi-component terpene chemotypes in Melaleuca alternifolia (tea tree), a genome-wide association study was undertaken for 148 individuals representing all six recognised chemotypes. A number of single nucleotide polymorphisms in a genomic region of c. 400 kb explained large proportions of the variation in key monoterpenes of tea tree oil. The region contained a cluster of 10 monoterpene synthase genes, including four genes predicted to encode synthases for 1,8-cineole, terpinolene, and the terpinen-4-ol precursor, sabinene hydrate. Chemotype-dependent null alleles at some sites suggested structural variants within this gene cluster, providing a possible basis for linkage disequilibrium in this region. Genotyping in a separate domesticated population revealed that all alleles surrounding this gene cluster were fixed after artificial selection for a single chemotype. These observations indicate that a supergene accounts for chemotypes in M. alternifolia. A genetic model with three haplotypes, encompassing the four characterised monoterpene synthase genes, explained the six terpene chemotypes, and was consistent with available biparental cross-segregation data.

Draft genome of the medicinal tea tree Melaleuca alternifolia.

BACKGROUND: Melaleuca alternifolia is a commercially important medicinal tea tree native to Australia. Tea tree oil, the essential oil distilled from its branches and leaves, has broad-spectrum germicidal activity and is highly valued in the pharmaceutical and cosmetic industries. Thus, the study of genome, which can provide reference for the investigation of genes involved in terpinen-4-ol biosynthesis, is quite crucial for improving the productivity of Tea tree oil. METHODS AND RESULTS: In our study, the next-generation sequencing was used to investigate the whole genome of Melaleuca alternifolia. About 114 Gb high quality sequence data were obtained and assembled into 1,838,159 scafolds with an N50 length of 1021 bp. The assembled genome size is about 595 Mb, twice of that predicted by flow cytometer (300 Mb) and k-mer analysis (345 Mb). Benchmarking Universal Single-Copy Orthologs analyses indicated that only 11.3% of the conserved single-copy genes were miss. Repetitive regions cover over 40.43% of the genome. A total of 44,369 protein-coding genes were predicted and annotated against Nr, Swissprot, Refseq, COG, KOG, and KEGG database. Among these genes, 32,909 and 16,241 genes were functionally annotated in Nr and KEGG, respectively. Moreover, 29,411 and 14,435 genes were functionally annotated in COG and KOG. Additionally, 457,661 simple sequence repeats and 1109 transcription factors (TFs) form 67 TF families were identified in the assembled genome. CONCLUSION: Our findings provide a draft genome sequencing of M. alternifolia which can act as a reference for the deep sequencing strategies, and are useful for future functional and comparative genomics analyses.

Transcriptome Profiling of Melaleuca quinquenervia Challenged by Myrtle Rust Reveals Differences in Defense Responses Among Resistant Individuals.

Plants have developed complex defense mechanisms to protect themselves against pathogens. A wide-host-range fungus, Austropuccinia psidii, which has caused severe damage to ecosystems and plantations worldwide, is a major threat to Australian ecosystems dominated by members of the family Myrtaceae. In particular, the east coast wetland foundation tree species Melaleuca quinquenervia, appears to be variably susceptible to this pathogen. Understanding the molecular basis of host resistance would enable better management of this rust disease. We identified resistant and susceptible individuals of M. quinquenervia and explored their differential gene expression in order to discover the molecular basis of resistance against A. psidii. Rust screening of germplasm showed a varying degree of response, with fully resistant to highly susceptible individuals. We used transcriptome profiling in samples collected before and at 5 days postinoculation (dpi). Differential gene expression analysis showed that numerous defense-related genes were induced in susceptible plants at 5 dpi. Mapping reads against the A. psidii genome showed that only susceptible plants contained fungal-derived transcripts. Resistant plants exhibited an overexpression of candidate A. psidii resistance-related genes such as receptor-like kinases, nucleotide-binding site leucine-rich repeat proteins, glutathione S-transferases, WRKY transcriptional regulators, and pathogenesis-related proteins. We identified large differences in the expression of defense-related genes among resistant individuals.

Four terpene synthases contribute to the generation of chemotypes in tea tree (Melaleuca alternifolia).

BACKGROUND: Terpene rich leaves are a characteristic of Myrtaceae. There is significant qualitative variation in the terpene profile of plants within a single species, which is observable as "chemotypes". Understanding the molecular basis of chemotypic variation will help explain how such variation is maintained in natural populations as well as allowing focussed breeding for those terpenes sought by industry. The leaves of the medicinal tea tree, Melaleuca alternifolia, are used to produce terpinen-4-ol rich tea tree oil, but there are six naturally occurring chemotypes; three cardinal chemotypes (dominated by terpinen-4-ol, terpinolene and 1,8-cineole, respectively) and three intermediates. It has been predicted that three distinct terpene synthases could be responsible for the maintenance of chemotypic variation in this species. RESULTS: We isolated and characterised the most abundant terpene synthases (TPSs) from the three cardinal chemotypes of M. alternifolia. Functional characterisation of these enzymes shows that they produce the dominant compounds in the foliar terpene profile of all six chemotypes. Using RNA-Seq, we investigated the expression of these and 24 additional putative terpene synthases in young leaves of all six chemotypes of M. alternifolia. CONCLUSIONS: Despite contributing to the variation patterns observed, variation in gene expression of the three TPS genes is not enough to explain all variation for the maintenance of chemotypes. Other candidate terpene synthases as well as other levels of regulation must also be involved. The results of this study provide novel insights into the complexity of terpene biosynthesis in natural populations of a non-model organism.

Transcriptome analysis of terpene chemotypes of Melaleuca alternifolia across different tissues.

Plant chemotypes or chemical polymorphisms are defined by discrete variation in secondary metabolites within a species. This variation can have consequences for ecological interactions or the human use of plants. Understanding the molecular basis of chemotypic variation can help to explain how variation of plant secondary metabolites is controlled. We explored the transcriptomes of the 3 cardinal terpene chemotypes of Melaleuca alternifolia in young leaves, mature leaves, and stem and compared transcript abundance to variation in the constitutive profile of terpenes. Leaves from chemotype 1 plants (dominated by terpinen-4-ol) show a similar pattern of gene expression when compared to chemotype 5 plants (dominated by 1,8-cineole). Only terpene synthases in young leaves were differentially expressed between these chemotypes, supporting the idea that terpenes are mainly synthetized in young tissue. Chemotype 2 plants (dominated by terpinolene) show a greater degree of differential gene expression compared to the other chemotypes, which might be related to the isolation of plant populations that exhibit this chemotype and the possibility that the terpinolene synthase gene in M. alternifolia was derived by introgression from a closely related species, Melaleuca trichostachya. By using multivariate analyses, we were able to associate terpenes with candidate terpene synthases.

MbEOMT1 regulates methyleugenol biosynthesis in Melaleuca bracteata F. Muell.

Methyleugenol, a bioactive compound in the phenylpropene family, undergoes its final and crucial biosynthetic transformation when eugenol O-methyltransferase (EOMT) converts eugenol into methyleugenol. While Melaleuca bracteata F. Muell essential oil is particularly rich in methyleugenol, it contains only trace amounts of its precursor, eugenol. This suggests that the EOMT enzyme in M. bracteata is highly efficient, although it has not yet been characterized. In this study, we isolated and identified an EOMT gene from M. bracteata, termed MbEOMT1, which is primarily expressed in the flowers and leaves and is inducible by methyl jasmonate (MeJA). Subcellular localization of MbEOMT1 in the cytoplasm was detected. Through transient overexpression experiments, we found that MbEOMT1 significantly elevates the concentration of methyleugenol in M. bracteata leaves. Conversely, silencing of MbEOMT1 via virus-induced gene silencing led to a marked reduction in methyleugenol levels. Our in vitro enzymatic assays further confirmed that MbEOMT1 specifically catalyzes the methylation of eugenol. Collectively, these findings establish that the MbEOMT1 gene is critical for methyleugenol biosynthesis in M. bracteata. This study enriches the understanding of phenylpropene biosynthesis and suggests that MbEOMT1 could serve as a valuable catalyst for generating bioactive compounds in the future.

Sprouting and genetic structure vary with flood disturbance in the tropical riverine paperbark tree, Melaleuca leucadendra (Myrtaceae).

PREMISE OF THE STUDY: Sprouting in woody plants promotes persistence in the face of disturbance, ultimately influencing population structure. Different disturbance regimes drive variable population responses, but there have been few direct tests of the relative differences in population structure to specific drivers. We measured population structure as genotypic diversity (clonality) as a function of hydrological regime for a riverine tree, Melaleuca leucadendra, a major structural component in flood landscapes in the Australian dry tropics. METHODS: We estimated clonality, genotypic richness, and population allelic diversity. The relationship among disturbance, genetic estimates of clonality, and population distinctiveness was compared with flood regime, characterized by return frequencies and hydrological stress at individual river reaches. KEY RESULTS: Two contrasting patterns of genotypic structure were detected and corresponded to order-of-magnitude differences in flood regime between sites. At mainstem locations characterized by greatest flood intensity, sprouting generated clonal structure to 17 m (30% ramets clonal). By contrast, clonality was atypical at lower-disturbance tributaries (0% clonal). Population allelic distributions showed extensive genetic exchange among mainstem locations, but strong genetic differentiation between mainstem and tributaries. CONCLUSIONS: Population structure and distinctiveness in riverine Melaleuca are determined by differences in sprouting and recruitment responses that depend on localized hydrological regime. Sprouting contributes to population persistence via localized clonal growth. Resprouting following disturbance in M. leucadendra may help explain its numerical dominance in tropical river systems. This study, although preliminary, suggests that flood ecosystems may represent excellent experimental systems to develop a better understanding of whole-organism responses to environmental drivers.

Genome assembly of wild tea tree DASZ reveals pedigree and selection history of tea varieties.

Wild teas are valuable genetic resources for studying domestication and breeding. Here we report the assembly of a high-quality chromosome-scale reference genome for an ancient tea tree. The further RNA sequencing of 217 diverse tea accessions clarifies the pedigree of tea cultivars and reveals key contributors in the breeding of Chinese tea. Candidate genes associated with flavonoid biosynthesis are identified by genome-wide association study. Specifically, diverse allelic function of CsANR, CsF3'5'H and CsMYB5 is verified by transient overexpression and enzymatic assays, providing comprehensive insights into the biosynthesis of catechins, the most important bioactive compounds in tea plants. The inconspicuous differentiation between ancient trees and cultivars at both genetic and metabolic levels implies that tea may not have undergone long-term artificial directional selection in terms of flavor-related metabolites. These genomic resources provide evolutionary insight into tea plants and lay the foundation for better understanding the biosynthesis of beneficial natural compounds.

Identifying genetic markers for a range of phylogenetic utility-From species to family level.

Resolving the phylogenetic relationships of closely related species using a small set of loci is challenging as sufficient information may not be captured from a limited sample of the genome. Relying on few loci can also be problematic when conflict between gene-trees arises from incomplete lineage sorting and/or ongoing hybridization, problems especially likely in recently diverged lineages. Here, we developed a method using limited genomic resources that allows identification of many low copy candidate loci from across the nuclear and chloroplast genomes, design probes for target capture and sequence the captured loci. To validate our method we present data from Eucalyptus and Melaleuca, two large and phylogenetically problematic genera within the Myrtaceae family. With one annotated genome, one transcriptome and two whole-genome shotgun sequences of one Eucalyptus and four Melaleuca species, respectively, we identified 212 loci representing 263 kbp for targeted sequence capture and sequencing. Of these, 209 were successfully tested from 47 samples across five related genera of Myrtaceae. The average percentage of reads mapped back to the reference was 57.6% with coverage of more than 20 reads per position across 83.5% of the data. The methods developed here should be applicable across a large range of taxa across all kingdoms. The core methods are very flexible, providing a platform for various genomic resource availabilities and are useful from shallow to deep phylogenies.

Selection on herbivory resistance and growth rate in an invasive plant.

The evolution of increased competitive ability (EICA) hypothesis proposes that invasive species evolve decreased defense and increased competitive ability following natural enemy release. Previous tests of EICA examined the result of evolution by comparing individuals from home and introduced ranges, but no previous study of this hypothesis has examined the process of evolution by analyzing patterns of selection. On the basis of EICA, there should be selection for competitive ability without herbivores and selection for defense with herbivores. Selection on competitive ability should be stronger for genotypes accustomed to herbivores (home range genotypes), and selection on defense should be stronger for genotypes unaccustomed to herbivores (introduced range genotypes). Using a field experiment, we tested these hypotheses for the invasive plant Melaleuca quinquenervia. There was a negative genetic correlation between resistance and growth, indicating a trade-off. However, selection for stem elongation (an indicator of competitive ability) was always positive, and selection on resistance was always negative and did not depend on genotype source or the presence of herbivores. The patterns of selection found in this study contrast with predictions from EICA and accurately predict the lack of evolutionary change in growth and resistance following the introduction of this species from Australia to Florida.

Isolation and partial characterisation of a putative monoterpene synthase from Melaleuca alternifolia.

Melaleuca alternifolia (Cheel) is an Australia native tree harvested for its monoterpene-rich, essential oil. Monoterpene synthases (E.C. 4.2.3.20) were partially purified from the flush growth of the commercially important, high terpinen-4-ol chemotype of M. alternifolia. The purified fractions produced an acyclic monoterpene, linalool that is not present in the essential oil. To further characterise the monoterpene synthase, a cDNA library was constructed and 500 expressed sequence tags (ESTs) were sequenced to isolate putative terpene synthases. A single clone with similarity to the TspB gene sub-family of angiosperm monoterpene and isoprene synthases was isolated but was truncated at the 5' end. This single clone was used to design a probe for a cDNA library and was applied to isolate a full-length clone. This gene encoded a polypeptide 583 amino acids in length (67 kDa) including a putative transit peptide. Heterologous expression of the gene in Escherichia coli and subsequent assay of the recombinant enzyme did not result in the production of terpinen-4-ol, the major constituent of tea tree oil, or of its precursor sabinene hydrate. Significant quantities of linalool were observed in these assays, and in the assays of monoterpene synthase activity of a native enzyme in vitro, but the racemic nature of the linalool means that it may have a non-enzymatic origin.

The yield of essential oils in Melaleuca alternifolia (Myrtaceae) is regulated through transcript abundance of genes in the MEP pathway.

Medicinal tea tree (Melaleuca alternifolia) leaves contain large amounts of an essential oil, dominated by monoterpenes. Several enzymes of the chloroplastic methylerythritol phosphate (MEP) pathway are hypothesised to act as bottlenecks to the production of monoterpenes. We investigated, whether transcript abundance of genes encoding for enzymes of the MEP pathway were correlated with foliar terpenes in M. alternifolia using a population of 48 individuals that ranged in their oil concentration from 39 -122 mg x g DM(-1). Our study shows that most genes in the MEP pathway are co-regulated and that the expression of multiple genes within the MEP pathway is correlated with oil yield. Using multiple regression analysis, variation in expression of MEP pathway genes explained 87% of variation in foliar monoterpene concentrations. The data also suggest that sesquiterpenes in M. alternifolia are synthesised, at least in part, from isopentenyl pyrophosphate originating from the plastid via the MEP pathway.

Genetic variation and evolution of secondary compounds in native and introduced populations of the invasive plant Melaleuca quinquenervia.

We examined multivariate evolution of 20 leaf terpenoids in the invasive plant Melaleuca quinquenervia in a common garden experiment. Although most compounds, including 1,8-Cineole and Viridiflorol, were reduced in home compared with invaded range genotypes, consistent with an evolutionary decrease in defense, one compound (E-Nerolidol) was greater in invaded than home range genotypes. Nerolidol was negatively genetically correlated with Cineole and Viridiflorol, and the increase in this compound in the new range may have been driven by this negative correlation. There was positive selection on all three focal compounds, and a loss of genetic variation in introduced range genotypes. Selection skewers analysis predicted an increase in Cineole and Viridiflorol and a decrease or no change in Nerolidol, in direct contrast to the observed changes in the new range. This discrepancy could be due to differences in patterns of selection, genetic correlations, or the herbivore communities in the home versus introduced ranges. Although evolutionary changes in most compounds were consistent with the evolution of increased competitive ability hypothesis, changes in other compounds as well as selection patterns were not, indicating that it is important to understand selection and the nature of genetic correlations to predict evolutionary change in invasive species.

Reticulate evolution in the natural range of the invasive wetland tree species Melaleuca quinquenervia.

The Melaleuca leucadendra complex (broad-leaf paperbarks; Myrtaceae) is a dominant component of the tropical and sub-tropical biota of Australia, particularly in wetlands of high conservation significance. In Florida and other parts of the Americas, however, one member of the group (Melaleuca quinquenervia) is a serious ecological and economic weed. Understanding the relationships and evolution of the group is integral to both conservation and biocontrol efforts. Although the complex is currently considered to include up to 14 species, there has been some concern over taxonomic boundaries within the complex because most species are circumscribed only by combinations of characters, each of which also occurs in other species. Here, DNA sequence data derived from the chloroplast and two nuclear regions are used to explore the relationships of M. quinquenervia. We find little evidence for clear species boundaries within the M. leucadendra complex in general, with regional sharing of chloroplast haplotypes across morphologically defined taxa, indicating asymmetrical introgression or retention of ancestral haplotypes (lineage sorting). Phylogenies were further confounded by the recovery of multiple copies of both nuclear regions sequenced (ITS and rpb2) from many individuals. There was no clear evidence of polyploidy or pseudogenes, but multiple duplications of rpb2 could not be ruled out. Parsimony networks of the nuclear ITS region show some clustering of haplotypes by morphospecies but there is also evidence of both hybridisation and recombination. Signals of introgression were also evident in rpb2, supporting an hypothesis of recent or ongoing gene flow between M. quinquenervia and other members of the M. leucadendra complex. Both relaxed and fixed molecular-clock dating estimate the introgression to have occurred sometime within the past seven million years (95% CI: 0.7-18). The New Caledonian population of M. quinquenervia appears to have been established by dispersal from Australia during this period. M. quinquenervia is found to have alleles closely related to multiple different morphotaxa within the M. leucadendra complex, suggesting considerable past introgression into this taxon from some other members of the M. leucadendra complex, and this has implications for biocontrol efforts. The M. leucadendra complex appears to reflect early to intermediate stages of speciation, possibly driven by different ecologies.