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.

Effect of Stock Plant Growing Medium and Density upon a Cutting Propagation System for Tea Tree, Melaleuca alternifolia.

To offer a viable alternative to seedling deployment of tea tree, clones will require the development of an efficient, robust, and vegetative propagation system for the large number of plants needed for plantations (i.e., typically 33,000 plants/ha). This study investigated the productivity of an intensive management system for tea tree stock plants and rooted cuttings grown in a subtropical environment (Lismore, NSW, Australia). Three stock plant densities (30, 100, and 200 plants/m2) were tested in coir and potting mix media (consisting of peat+perlite+vermiculite), with 11 settings of cuttings undertaken between April 2019 and March 2020. All stock plants in each media type survived 11 harvests and remained productive; however after 13 months, many plants in the coir media, appeared chlorotic and showed symptoms of iron deficiency. Rooting and cutting survival rates using the mini cutting technique were high, ranging from a maximum mean monthly setting value of 87.7% ± 4 at 84 days post-setting in potting mix, to a minimum of 80.4% ± 3.7 in coir. The most productive treatment was at high stock plant density in potting mix which had the potential to produce 13,440 plants/year/m2. Overall coir appeared less productive, but the pattern of difference among treatments was similar. For the highest system productivity, it is recommended to grow stock plants in potting mix at high densities and modulate temperatures to between 18 °C and 28 °C. Late spring and early summer were the best time for harvesting and setting tea tree mini cuttings in the subtropics.

Pests, diseases, and aridity have shaped the genome of Corymbia citriodora.

Corymbia citriodora is a member of the predominantly Southern Hemisphere Myrtaceae family, which includes the eucalypts (Eucalyptus, Corymbia and Angophora; ~800 species). Corymbia is grown for timber, pulp and paper, and essential oils in Australia, South Africa, Asia, and Brazil, maintaining a high-growth rate under marginal conditions due to drought, poor-quality soil, and biotic stresses. To dissect the genetic basis of these desirable traits, we sequenced and assembled the 408 Mb genome of Corymbia citriodora, anchored into eleven chromosomes. Comparative analysis with Eucalyptus grandis reveals high synteny, although the two diverged approximately 60 million years ago and have different genome sizes (408 vs 641 Mb), with few large intra-chromosomal rearrangements. C. citriodora shares an ancient whole-genome duplication event with E. grandis but has undergone tandem gene family expansions related to terpene biosynthesis, innate pathogen resistance, and leaf wax formation, enabling their successful adaptation to biotic/abiotic stresses and arid conditions of the Australian continent.

A high-quality draft genome for Melaleuca alternifolia (tea tree): a new platform for evolutionary genomics of myrtaceous terpene-rich species.

The economically important Melaleuca alternifolia (tea tree) is the source of a terpene-rich essential oil with therapeutic and cosmetic uses around the world. Tea tree has been cultivated and bred in Australia since the 1990s. It has been extensively studied for the genetics and biochemistry of terpene biosynthesis. Here, we report a high quality de novo genome assembly using Pacific Biosciences and Illumina sequencing. The genome was assembled into 3128 scaffolds with a total length of 362 Mb (N50  = 1.9 Mb), with significantly higher contiguity than a previous assembly (N50  = 8.7 Kb). Using a homology-based, RNA-seq evidence-based and ab initio prediction approach, 37,226 protein-coding genes were predicted. Genome assembly and annotation exhibited high completeness scores of 98.1% and 89.4%, respectively. Sequence contiguity was sufficient to reveal extensive gene order conservation and chromosomal rearrangements in alignments with Eucalyptus grandis and Corymbia citriodora genomes. This new genome advances currently available resources to investigate the genome structure and gene family evolution of M. alternifolia. It will enable further comparative genomic studies in Myrtaceae to elucidate the genetic foundations of economically valuable traits in this crop.

Annotation of the Corymbia terpene synthase gene family shows broad conservation but dynamic evolution of physical clusters relative to Eucalyptus.

Terpenes are economically and ecologically important phytochemicals. Their synthesis is controlled by the terpene synthase (TPS) gene family, which is highly diversified throughout the plant kingdom. The plant family Myrtaceae are characterised by especially high terpene concentrations, and considerable variation in terpene profiles. Many Myrtaceae are grown commercially for terpene products including the eucalypts Corymbia and Eucalyptus. Eucalyptus grandis has the largest TPS gene family of plants currently sequenced, which is largely conserved in the closely related E. globulus. However, the TPS gene family has been well studied only in these two eucalypt species. The recent assembly of two Corymbia citriodora subsp. variegata genomes presents an opportunity to examine the conservation of this important gene family across more divergent eucalypt lineages. Manual annotation of the TPS gene family in C. citriodora subsp. variegata revealed a similar overall number, and relative subfamily representation, to that previously reported in E. grandis and E. globulus. Many of the TPS genes were in physical clusters that varied considerably between Eucalyptus and Corymbia, with several instances of translocation, expansion/contraction and loss. Notably, there was greater conservation in the subfamilies involved in primary metabolism than those involved in secondary metabolism, likely reflecting different selective constraints. The variation in cluster size within subfamilies and the broad conservation between the eucalypts in the face of this variation are discussed, highlighting the potential contribution of selection, concerted evolution and stochastic processes. These findings provide the foundation to better understand terpene evolution within the ecologically and economically important Myrtaceae.

Ecotype Variation of Methyl Eugenol Content in Tea Tree (Melaleuca alternifolia and Melaleuca linariifolia).

Methyl eugenol is a natural phenylpropanoid compound found in a wide range of plants used for food, flavouring, cosmetics, and health-care. As a suspected rodent carcinogen, methyl eugenol may also be harmful to humans when present in significant concentrations. Consequently, its level has been restricted in some foodstuffs and cosmetics for some markets. In order to assess the potential to breed uniformly low methyl eugenol cultivars for an essential oil crop, tea tree, the source of 'Oil of Melaleuca, terpinene-4-ol type', we examine levels in individual trees (n = 30) from two geographic regions and six terpene chemotypes. Overall, methyl eugenol levels were low in this species (Mean [SD] 354 [239] ppm, n = 30), much lower than levels predicted to be of toxicological concern. Within each chemotype, there was a lack of evidence for correlations between terpenoid constituents and methyl eugenol levels. Further support for the independence of methyl eugenol and terpene biosynthesis was evident from similar mean levels in selected (Mean [SD] 586 [339] ppm, n = 12) and undomesticated Melaleuca alternifolia trees (Mean [SD] 480 [299] ppm, n = 5) with terpinen-4-ol type oils. By contrast, methyl eugenol level varied by geographic origin and chemotype. Trees from the upland region, where there is a prevalence of terpinolene type trees, had lower average methyl eugenol levels than trees from the coastal region, where there is a prevalence of terpinen-4-ol and 1,8-cineole type trees.

Pectin Methylesterase genes influence solid wood properties of Eucalyptus pilularis.

This association study of Eucalyptus pilularis populations provides empirical evidence for the role of Pectin Methylesterase (PME) in influencing solid wood characteristics of Eucalyptus. PME6 was primarily associated with the shrinkage and collapse of drying timber, which are phenotypic traits consistent with the role of pectin as a hydrophilic polysaccharide. PME7 was primarily associated with cellulose and pulp yield traits and had an inverse correlation with lignin content. Selection of specific alleles in these genes may be important for improving trees as sources of high-quality wood products. A heterozygote advantage was postulated for the PME7 loci and, in combination with haplotype blocks, may explain the absence of a homozygous class at all single-nucleotide polymorphisms investigated in this gene.

Comparative mapping among subsection Australes (genus Pinus, family Pinaceae).

Comparative mapping in conifers has not yet been used to test for small-scale genomic disruptions such as inversions, duplications, and deletions occurring between closely related taxa. Using comparative mapping to probe this smaller scale of inquiry may provide clues about speciation in a phylogenetically problematic taxon, the diploxylon pine subsection Australes (genus Pinus, family Pinaceae). Genetic maps were constructed for two allopatric species of Australes, P. elliottii var. elliottii and P. caribaea var. hondurensis, using microsatellites and an F1 hybrid. A third map was generated directly from the meiotic products of an adult F1 hybrid, eliminating the need for an F2 generation. Numerous small-scale disruptions were detected in addition to synteny and collinearity, and these included (1) map shrinkage, (2) a paracentric inversion, (3) transmission ratio distortion, and (4) mild selection against a parental haplotype. Such cryptic signatures of genomic divergence between closely related interfertile species are useful in elucidating this problematic evolutionary history.

Nuclear ribosomal pseudogenes resolve a corroborated monophyly of the eucalypt genus Corymbia despite misleading hypotheses at functional ITS paralogs.

Divergent paralogs can create both obstacles and opportunities for phylogenetic reconstruction. Phylogenetic relationships among eucalypt genera have been incongruent among datasets in previous studies, where morphological characters supported monophyly of the genus Corymbia, while intergenic spacers of the nuclear ribosomes (ITS) and chloroplast loci (trnL, trnH, psbA) showed Corymbia as either equivocal or paraphyletic. Ribosomal DNA occurs in multiple copies in a genome. We cloned and sequenced the nrITS to investigate if gene duplication was the cause of incongruence among trees in the eucalypts. Three ITS riboforms, two of them widespread, were recovered within some genomes. One of the ITS riboforms recovered a robust phylogeny showing Corymbia as a monophyletic genus, corroborating the evidence from morphology, fossil data, a recent ITS/ETS dataset and microsatellites (SSRs). Compelling evidence suggested that this divergent riboform is a pseudogene, i.e., non-functional paralog: comparatively lower GC content suggesting lower structural stability, deamination-like mutations at potential methylation sites, lack of conserved helices and hairpins and conspicuously lower thermodynamic stability in secondary structures. Phylogenies from the apparently functional riboform retained Corymbia as paraphyletic. We show here that pseudogenes can recover a well-corroborated phylogeny whereas their functional paralogs show misleading hypotheses. We explain that phylogenetic signals may be obscured when functional constraints in ITS necessitate compensatory mutations in the secondary structure helices involved in RNA transcription, whereas pseudogenes mutate under neutrality.

Microsatellites retain phylogenetic signals across genera in eucalypts (Myrtaceae)

The utility of microsatellites (SSRs) in reconstructing phylogenies is largely confined to studies below the genus level, due to the potential of homoplasy resulting from allele size range constraints and poor SSR transferability among divergent taxa. The eucalypt genus Corymbia has been shown to be monophyletic using morphological characters, however, analyses of intergenic spacer sequences have resulted in contradictory hypotheses- showing the genus as either equivocal or paraphyletic. To assess SSR utility in higher order phylogeny in the family Myrtaceae, phylogenetic relationships of the bloodwood eucalypts Corymbia and related genera were investigated using eight polymorphic SSRs. Repeat size variation using the average square and Nei's distance were congruent and showed Corymbia to be a monophyletic group, supporting morphological characters and a recent combination of the internal and external transcribed spacers dataset. SSRs are selectively neutral and provide data at multiple genomic regions, thus may explain why SSRs retained informative phylogenetic signals despite deep divergences. We show that where the problems of size-range constraints, high mutation rates and size homoplasy are addressed, SSRs might resolve problematic phylogenies of taxa that have diverged for as long as three million generations or 30 million years.