Complete mitochondrial genome of the green-lipped mussel, Perna canaliculus (Mollusca: Mytiloidea), from long nanopore sequencing reads

We describe here the first complete genome assembly of the New Zealand green-lipped mussel, Perna canaliculus, mitochondrion. The assembly was performed de novo from a mix of long nanopore sequencing reads and short sequencing reads. The genome is 16,005 bp long. Comparison to other Mytiloidea mitochondrial genomes indicates important gene rearrangements in this family

The Eucalyptus grandis NBS-LRR gene family : physical clustering and expression hotspots

Eucalyptus grandis is a commercially important hardwood species and is known to be susceptible to a number of pests and pathogens. Determining mechanisms of defense is therefore a research priority. The published genome for E. grandis has aided the identification of one important class of resistance (R) genes that incorporate nucleotide binding sites and leucine-rich repeat domains (NBS-LRR). Using an iterative search process we identified NBS-LRR gene models within the E. grandis genome. We characterized the gene models and identified their genomic arrangement. The gene expression patterns were examined in E. grandis clones, challenged with a fungal pathogen (Chrysoporthe austroafricana) and insect pest (Leptocybe invasa). One thousand two hundred and fifteen putative NBS-LRR coding sequences were located which aligned into two large classes, Toll or interleukin-1 receptor (TIR) and coiled-coil (CC) based on NB-ARC domains. NBS-LRR gene-rich regions were identified with 76% organized in clusters of three or more genes. A further 272 putative incomplete resistance genes were also identified. We determined that E. grandis has a higher ratio of TIR to CC classed genes compared to other woody plant species as well as a smaller percentage of single NBS-LRR genes. Transcriptome profiles indicated expression hotspots, within physical clusters, including expression of many incomplete genes. The clustering of putative NBS-LRR genes correlates with differential expression responses in resistant and susceptible plants indicating functional relevance for the physical arrangement of this gene family. This analysis of the repertoire and expression of E. grandis putative NBS-LRR genes provides an important resource for the identification of novel and functional R-genes; a key objective for strategies to enhance resilience.Table S1 Full list of Eucalyptus grandis putative NBS-LRR genes sorted by position on the genome. Information per gene includes the chromosomal position, class, physical cluster and phylogeny clade membership, identification method, raw expression data, log2 fold change values and ANOVA results (p-values). S_F_C, susceptible, fungal treatment, control; S_F_I, susceptible, fungal treatment, inoculated; R_F_C, resistant, fungal treatment, control; R_F_I, resistant, fungal treatment, inoculated; S_I_C, susceptible, insect treatment, control; S_I_I, susceptible, insect treatment, infested; R_I_C, resistant, insect treatment, control; R_I_I, resistant, insect treatment, infested.Table S2 Conserved amino acid sequences for NB-ARC and TIR motifs from MEME analysis with CNL-like and TNL-like gene models in Eucalyptus grandis (Eg) and Arabidopsis thaliana (At; Meyers et al., 2003). The expected amino acid tryptophan (W) is identified in the Kinase 2 subdomain for CNL sequences–underlined.Figure S1 Neighbor joining tree of 480 Eucalyptus grandis NB-ARC domains from complete NBS-LRR genes. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site. The analysis involved 495 amino acid sequences (480 E. grandis). All ambiguous positions were removed for each sequence pair.Figure S2 Neighbor joining tree of 616 Eucalyptus grandis NB-ARC domains from all non-TIR NBS-LRR-like genes. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site. The analysis involved 631 amino acid sequences (616 E. grandis). All ambiguous positions were removed for each sequence pair.Figure S3 Neighbor joining tree of 396 Eucalyptus grandis NB-ARC domains from all TIR NBS-LRR-like genes. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site. The analysis involved 411 amino acid sequences (396 E. grandis). All ambiguous positions were removed for each sequence pair.Figure S4 The definition of a (A) cluster and a (B) supercluster is illustrated using a region (starting at 13 Mb and ending at 18 Mb) on chromosome 4.Figure S5 Physical locations for all complete, partial, and incomplete NBS-LRR gene models that were expressed under challenge of Chrysoporthe austroafricana and Leptocybe invasa on Eucalyptus grandis chromosomes (Mapchart). Variation in means from treatment (ANOVA) were identified based on significance *p < 0.01, **p < 0.001, ***p < 0.0001 (*** are also underlined) and log2 gene expression ratios greater than 1 or smaller than −1 for resistant and susceptible plants. Color distinguishes between different classes (TNL = pink, CNL = green, NL = red, incomplete NL = black, BLAST homolog non-NL = black). Scale bar = Mb. Cluster and supercluster regions are indicated and E. grandis gene IDs are provided.Figure S6 NB-ARC-LRR fused domains (A) and TIR-NB-ARC-LRR fused domains (B). Conserved amino acid sequences are indicated with lines (top). The GKT (Kinase 1) conserved motif is recognized as a P-loop structure important in ATP hydrolysis while the hDD is also well conserved in NB-ARC domains (Kinase 2) as important in co-ordinating Mg2+ as a co-factor (Tameling et al., 2006). These two important sub-domains of NB-ARC are sometimes termed the Walker A and Walker B motifs (Walker et al., 1982) and are identified as A and B, respectively, within the I-Tasser protein structures (bottom) for a representative CNL (Eucgr.L01363) and TNL (Eucgr.C00020) sequence from the Eucalyptus grandis genome.Top up scholarships were generously provided for PT from the University of Sydney and Rural Industries Research and Development Corporation, Australiahttp://www.frontiersin.orgam2016Genetic

The Eucalyptus grandis NBS-LRR gene family : physical clustering and expression hotspots

Eucalyptus grandis is a commercially important hardwood species and is known to be susceptible to a number of pests and pathogens. Determining mechanisms of defense is therefore a research priority. The published genome for E. grandis has aided the identification of one important class of resistance (R) genes that incorporate nucleotide binding sites and leucine-rich repeat domains (NBS-LRR). Using an iterative search process we identified NBS-LRR gene models within the E. grandis genome. We characterized the gene models and identified their genomic arrangement. The gene expression patterns were examined in E. grandis clones, challenged with a fungal pathogen (Chrysoporthe austroafricana) and insect pest (Leptocybe invasa). One thousand two hundred and fifteen putative NBS-LRR coding sequences were located which aligned into two large classes, Toll or interleukin-1 receptor (TIR) and coiled-coil (CC) based on NB-ARC domains. NBS-LRR gene-rich regions were identified with 76% organized in clusters of three or more genes. A further 272 putative incomplete resistance genes were also identified. We determined that E. grandis has a higher ratio of TIR to CC classed genes compared to other woody plant species as well as a smaller percentage of single NBS-LRR genes. Transcriptome profiles indicated expression hotspots, within physical clusters, including expression of many incomplete genes. The clustering of putative NBS-LRR genes correlates with differential expression responses in resistant and susceptible plants indicating functional relevance for the physical arrangement of this gene family. This analysis of the repertoire and expression of E. grandis putative NBS-LRR genes provides an important resource for the identification of novel and functional R-genes; a key objective for strategies to enhance resilience.Table S1 Full list of Eucalyptus grandis putative NBS-LRR genes sorted by position on the genome. Information per gene includes the chromosomal position, class, physical cluster and phylogeny clade membership, identification method, raw expression data, log2 fold change values and ANOVA results (p-values). S_F_C, susceptible, fungal treatment, control; S_F_I, susceptible, fungal treatment, inoculated; R_F_C, resistant, fungal treatment, control; R_F_I, resistant, fungal treatment, inoculated; S_I_C, susceptible, insect treatment, control; S_I_I, susceptible, insect treatment, infested; R_I_C, resistant, insect treatment, control; R_I_I, resistant, insect treatment, infested.Table S2 Conserved amino acid sequences for NB-ARC and TIR motifs from MEME analysis with CNL-like and TNL-like gene models in Eucalyptus grandis (Eg) and Arabidopsis thaliana (At; Meyers et al., 2003). The expected amino acid tryptophan (W) is identified in the Kinase 2 subdomain for CNL sequences–underlined.Figure S1 Neighbor joining tree of 480 Eucalyptus grandis NB-ARC domains from complete NBS-LRR genes. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site. The analysis involved 495 amino acid sequences (480 E. grandis). All ambiguous positions were removed for each sequence pair.Figure S2 Neighbor joining tree of 616 Eucalyptus grandis NB-ARC domains from all non-TIR NBS-LRR-like genes. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site. The analysis involved 631 amino acid sequences (616 E. grandis). All ambiguous positions were removed for each sequence pair.Figure S3 Neighbor joining tree of 396 Eucalyptus grandis NB-ARC domains from all TIR NBS-LRR-like genes. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site. The analysis involved 411 amino acid sequences (396 E. grandis). All ambiguous positions were removed for each sequence pair.Figure S4 The definition of a (A) cluster and a (B) supercluster is illustrated using a region (starting at 13 Mb and ending at 18 Mb) on chromosome 4.Figure S5 Physical locations for all complete, partial, and incomplete NBS-LRR gene models that were expressed under challenge of Chrysoporthe austroafricana and Leptocybe invasa on Eucalyptus grandis chromosomes (Mapchart). Variation in means from treatment (ANOVA) were identified based on significance *p < 0.01, **p < 0.001, ***p < 0.0001 (*** are also underlined) and log2 gene expression ratios greater than 1 or smaller than −1 for resistant and susceptible plants. Color distinguishes between different classes (TNL = pink, CNL = green, NL = red, incomplete NL = black, BLAST homolog non-NL = black). Scale bar = Mb. Cluster and supercluster regions are indicated and E. grandis gene IDs are provided.Figure S6 NB-ARC-LRR fused domains (A) and TIR-NB-ARC-LRR fused domains (B). Conserved amino acid sequences are indicated with lines (top). The GKT (Kinase 1) conserved motif is recognized as a P-loop structure important in ATP hydrolysis while the hDD is also well conserved in NB-ARC domains (Kinase 2) as important in co-ordinating Mg2+ as a co-factor (Tameling et al., 2006). These two important sub-domains of NB-ARC are sometimes termed the Walker A and Walker B motifs (Walker et al., 1982) and are identified as A and B, respectively, within the I-Tasser protein structures (bottom) for a representative CNL (Eucgr.L01363) and TNL (Eucgr.C00020) sequence from the Eucalyptus grandis genome.Top up scholarships were generously provided for PT from the University of Sydney and Rural Industries Research and Development Corporation, Australiahttp://www.frontiersin.orgam2016Genetic

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

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

A phylogenomic approach reveals a low somatic mutation rate in a long-lived plant.

Somatic mutations can have important effects on the life history, ecology, and evolution of plants, but the rate at which they accumulate is poorly understood and difficult to measure directly. Here, we develop a method to measure somatic mutations in individual plants and use it to estimate the somatic mutation rate in a large, long-lived, phenotypically mosaic Eucalyptus melliodora tree. Despite being 100 times larger than Arabidopsis, this tree has a per-generation mutation rate only ten times greater, which suggests that this species may have evolved mechanisms to reduce the mutation rate per unit of growth. This adds to a growing body of evidence that illuminates the correlated evolutionary shifts in mutation rate and life history in plants

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.The research was funded by the Australian Research Council (ARC) Discovery Program (DP14101755), the Australian Government Rural Industries Research and Development Corporation (RIRDC) and the Australia Tea Tree Industry Association (ATTIA). Grants from the Go8-DAAD Research Scheme and a Humboldt Research Award from the Alexander von Humboldt Foundation to WJF underpinned this collaborative research. Each of the funding bodies granted the funds based on a research proposal. They had no influence over the experimental design, data analysis or interpretation, or writing the manuscript