A chromosome-level genome assembly reveals that a bipartite gene cluster formed via an inverted duplication controls monoterpenoid biosynthesis in Schizonepeta tenuifolia.

Biosynthetic gene clusters (BGCs) are regions of a genome where genes involved in a biosynthetic pathway are in proximity. The origin and evolution of plant BGCs as well as their role in specialized metabolism remain largely unclear. In this study, we have assembled a chromosome-scale genome of Japanese catnip (Schizonepeta tenuifolia) and discovered a BGC that contains multiple copies of genes involved in four adjacent steps in the biosynthesis of p-menthane monoterpenoids. This BGC has an unprecedented bipartite structure, with mirrored biosynthetic regions separated by 260 kilobases. This bipartite BGC includes identical copies of a gene encoding an old yellow enzyme, a type of flavin-dependent reductase. In vitro assays and virus-induced gene silencing revealed that this gene encodes the missing isopiperitenone reductase. This enzyme evolved from a completely different enzyme family to isopiperitenone reductase from closely related Mentha spp., indicating convergent evolution of this pathway step. Phylogenomic analysis revealed that this bipartite BGC has emerged uniquely in the S. tenuifolia lineage and through insertion of pathway genes into a region rich in monoterpene synthases. The cluster gained its bipartite structure via an inverted duplication. The discovered bipartite BGC for p-menthane biosynthesis in S. tenuifolia has similarities to the recently described duplicated p-menthane biosynthesis gene pairs in the Mentha longifolia genome, providing an example of the convergent evolution of gene order. This work expands our understanding of plant BGCs with respect to both form and evolution, and highlights the power of BGCs for gene discovery in plant biosynthetic pathways.

Phylogenomics of Salvia L. subgenus Calosphace (Lamiaceae).

The evolutionary relationships of Salvia have been difficult to estimate. In this study, we used the Next Generation Sequencing method Hyb-Seq to evaluate relationships among 90 Lamiaceae samples, including representatives of Mentheae, Ocimeae, Salvia subgenera Audibertia, Leonia, Salvia, and 69 species of subgenus Calosphace, representing 32 of Epling's sections. A bait set was designed in MarkerMiner using available transcriptome data to enrich 119 variable nuclear loci. Nuclear and chloroplast loci were assembled with hybphylomaker (HPM), followed by coalescent approach analyses for nuclear data (ASTRAL, BEAST) and a concatenated Maximum Likelihood analysis of chloroplast loci. The HPM assembly had an average of 1,314,368 mapped reads for the sample and 527 putative exons. Phylogenetic inferences resolved strongly supported relationships for the deep-level nodes, agreeing with previous hypotheses which assumed that subgenus Audibertia is sister to subgenus Calosphace. Within subgenus Calosphace, we recovered eight monophyletic sections sensu Epling, Cardinalis, Hastatae, Incarnatae, and Uricae in all the analyses (nDNA and cpDNA), Biflorae, Lavanduloideae, and Sigmoideae in nuclear analyses (ASTRAL, BEAST) and Curtiflorae in ASTRAL trees. Network analysis supports deep node relationships, some of the main clades, and recovers reticulation within the core Calosphace. The chloroplast phylogeny resolved deep nodes and four monophyletic Calosphace sections. Placement of S. axillaris is distinct in nuclear evidence and chloroplast, as sister to the rest of the S. subg. Calosphace in chloroplast and a clade with "Hastatae clade" sister to the rest of the subgenus in nuclear evidence. We also tested the monophyly of S. hispanica, S. polystachia, S. purpurea, and S. tiliifolia, including two samples of each, and found that S. hispanica and S. purpurea are monophyletic. Our baits can be used in future studies of Lamiaceae phylogeny to estimate relationships between genera and among species. In this study, we presented a Hyb-Seq phylogeny for complex, recently diverged Salvia, which could be implemented in other Lamiaceae.

Generation of a chromosome-scale genome assembly of the insect-repellent terpenoid-producing Lamiaceae species, Callicarpa americana.

BACKGROUND: Plants exhibit wide chemical diversity due to the production of specialized metabolites that function as pollinator attractants, defensive compounds, and signaling molecules. Lamiaceae (mints) are known for their chemodiversity and have been cultivated for use as culinary herbs, as well as sources of insect repellents, health-promoting compounds, and fragrance. FINDINGS: We report the chromosome-scale genome assembly of Callicarpa americana L. (American beautyberry), a species within the early-diverging Callicarpoideae clade of Lamiaceae, known for its metallic purple fruits and use as an insect repellent due to its production of terpenoids. Using long-read sequencing and Hi-C scaffolding, we generated a 506.1-Mb assembly spanning 17 pseudomolecules with N50 contig and N50 scaffold sizes of 7.5 and 29.0 Mb, respectively. In all, 32,164 genes were annotated, including 53 candidate terpene synthases and 47 putative clusters of specialized metabolite biosynthetic pathways. Our analyses revealed 3 putative whole-genome duplication events, which, together with local tandem duplications, contributed to gene family expansion of terpene synthases. Kolavenyl diphosphate is a gateway to many of the bioactive terpenoids in C. americana; experimental validation confirmed that CamTPS2 encodes kolavenyl diphosphate synthase. Syntenic analyses with Tectona grandis L. f. (teak), a member of the Tectonoideae clade of Lamiaceae known for exceptionally strong wood resistant to insects, revealed 963 collinear blocks and 21,297 C. americana syntelogs. CONCLUSIONS: Access to the C. americana genome provides a road map for rapid discovery of genes encoding plant-derived agrichemicals and a key resource for understanding the evolution of chemical diversity in Lamiaceae.

The evolutionary origins of the cat attractant nepetalactone in catnip.

Catnip or catmint (Nepeta spp.) is a flowering plant in the mint family (Lamiaceae) famed for its ability to attract cats. This phenomenon is caused by the compound nepetalactone, a volatile iridoid that also repels insects. Iridoids are present in many Lamiaceae species but were lost in the ancestor of the Nepetoideae, the subfamily containing Nepeta. Using comparative genomics, ancestral sequence reconstructions, and phylogenetic analyses, we probed the re-emergence of iridoid biosynthesis in Nepeta. The results of these investigations revealed mechanisms for the loss and subsequent re-evolution of iridoid biosynthesis in the Nepeta lineage. We present evidence for a chronology of events that led to the formation of nepetalactone biosynthesis and its metabolic gene cluster. This study provides insights into the interplay between enzyme and genome evolution in the origins, loss, and re-emergence of plant chemical diversity.

Gene and genome duplications in the evolution of chemodiversity: perspectives from studies of Lamiaceae.

Plants are reservoirs of extreme chemical diversity, yet biosynthetic pathways remain underexplored in the majority of taxa. Access to improved, inexpensive genomic and computational technologies has recently enhanced our understanding of plant specialized metabolism at the biochemical and evolutionary levels including the elucidation of pathways leading to key metabolites. Furthermore, these approaches have provided insights into the mechanisms of chemical evolution, including neofunctionalization and subfunctionalization, structural variation, and modulation of gene expression. The broader utilization of genomic tools across the plant tree of life, and an expansion of genomic resources from multiple accessions within species or populations, will improve our overall understanding of chemodiversity. These data and knowledge will also lead to greater insight into the selective pressures contributing to and maintaining this diversity, which in turn will enable the development of more accurate predictive models of specialized metabolism in plants.

Phylotranscriptomic Analyses Reveal Asymmetrical Gene Duplication Dynamics and Signatures of Ancient Polyploidy in Mints.

Ancient duplication events and retained gene duplicates have contributed to the evolution of many novel plant traits and, consequently, to the diversity and complexity within and across plant lineages. Although mounting evidence highlights the importance of whole-genome duplication (WGD; polyploidy) and its key role as an evolutionary driver, gene duplication dynamics and mechanisms, both of which are fundamental to our understanding of evolutionary process and patterns of plant diversity, remain poorly characterized in many clades. We use newly available transcriptomic data and a robust phylogeny to investigate the prevalence, occurrence, and timing of gene duplications in Lamiaceae (mints), a species-rich and chemically diverse clade with many ecologically, economically, and culturally important species. We also infer putative WGDs-an extreme mechanism of gene duplication-using large-scale data sets from synonymous divergence (KS), phylotranscriptomic, and divergence time analyses. We find evidence for widespread but asymmetrical levels of gene duplication and ancient polyploidy in Lamiaceae that correlate with species richness, including pronounced levels of gene duplication and putative ancient WGDs (7-18 events) within the large subclade Nepetoideae and up to 10 additional WGD events in other subclades. Our results help disentangle WGD-derived gene duplicates from those produced by other mechanisms and illustrate the nonuniformity of duplication dynamics in mints, setting the stage for future investigations that explore their impacts on trait diversity and species diversification. Our results also provide a practical context for evaluating the benefits and limitations of transcriptome-based approaches to inferring WGD, and we offer recommendations for researchers interested in investigating ancient WGDs in other plant groups.

A chromosomal-scale genome assembly of Tectona grandis reveals the importance of tandem gene duplication and enables discovery of genes in natural product biosynthetic pathways.

BACKGROUND: Teak, a member of the Lamiaceae family, produces one of the most expensive hardwoods in the world. High demand coupled with deforestation have caused a decrease in natural teak forests, and future supplies will be reliant on teak plantations. Hence, selection of teak tree varieties for clonal propagation with superior growth performance is of great importance, and access to high-quality genetic and genomic resources can accelerate the selection process by identifying genes underlying desired traits. FINDINGS: To facilitate teak research and variety improvement, we generated a highly contiguous, chromosomal-scale genome assembly using high-coverage Pacific Biosciences long reads coupled with high-throughput chromatin conformation capture. Of the 18 teak chromosomes, we generated 17 near-complete pseudomolecules with one chromosome present as two chromosome arm scaffolds. Genome annotation yielded 31,168 genes encoding 46,826 gene models, of which, 39,930 and 41,155 had Pfam domain and expression evidence, respectively. We identified 14 clusters of tandem-duplicated terpene synthases (TPSs), genes central to the biosynthesis of terpenes, which are involved in plant defense and pollinator attraction. Transcriptome analysis revealed 10 TPSs highly expressed in woody tissues, of which, 8 were in tandem, revealing the importance of resolving tandemly duplicated genes and the quality of the assembly and annotation. We also validated the enzymatic activity of four TPSs to demonstrate the function of key TPSs. CONCLUSIONS: In summary, this high-quality chromosomal-scale assembly and functional annotation of the teak genome will facilitate the discovery of candidate genes related to traits critical for sustainable production of teak and for anti-insecticidal natural products.

Genome sequences of two diploid wild relatives of cultivated sweetpotato reveal targets for genetic improvement.

Sweetpotato [Ipomoea batatas (L.) Lam.] is a globally important staple food crop, especially for sub-Saharan Africa. Agronomic improvement of sweetpotato has lagged behind other major food crops due to a lack of genomic and genetic resources and inherent challenges in breeding a heterozygous, clonally propagated polyploid. Here, we report the genome sequences of its two diploid relatives, I. trifida and I. triloba, and show that these high-quality genome assemblies are robust references for hexaploid sweetpotato. Comparative and phylogenetic analyses reveal insights into the ancient whole-genome triplication history of Ipomoea and evolutionary relationships within the Batatas complex. Using resequencing data from 16 genotypes widely used in African breeding programs, genes and alleles associated with carotenoid biosynthesis in storage roots are identified, which may enable efficient breeding of varieties with high provitamin A content. These resources will facilitate genome-enabled breeding in this important food security crop.

MarkerMiner 1.0: A new application for phylogenetic marker development using angiosperm transcriptomes.

PREMISE OF THE STUDY: Targeted sequencing using next-generation sequencing (NGS) platforms offers enormous potential for plant systematics by enabling economical acquisition of multilocus data sets that can resolve difficult phylogenetic problems. However, because discovery of single-copy nuclear (SCN) loci from NGS data requires both bioinformatics skills and access to high-performance computing resources, the application of NGS data has been limited. METHODS AND RESULTS: We developed MarkerMiner 1.0, a fully automated, open-access bioinformatic workflow and application for discovery of SCN loci in angiosperms. Our new tool identified as many as 1993 SCN loci from transcriptomic data sampled as part of four independent test cases representing marker development projects at different phylogenetic scales. CONCLUSIONS: MarkerMiner is an easy-to-use and effective tool for discovery of putative SCN loci. It can be run locally or via the Web, and its tabular and alignment outputs facilitate efficient downstream assessments of phylogenetic utility, locus selection, intron-exon boundary prediction, and primer or probe development.