A new, simple, highly scalable, and efficient protocol for genomic DNA extraction from diverse plant taxa.

PREMISE: Commonly used molecular techniques such as next-generation sequencing require reliable methods to extract DNA quickly and efficiently. Secondary compounds within plant tissues make this requirement all the more challenging, often forcing researchers to test different extraction methods tailored to their particular species of interest in order to obtain large amounts of high-quality genomic DNA. The opportunities provided by high-throughput, next-generation sequencing only exacerbate these problems, especially when trying to extract DNA from multiple species at the same time. Several methods have attempted to resolve the challenges of obtaining suitable DNA from plants; however, a rapid, high-yield, high-quality, and highly scalable DNA extraction method is still needed. METHODS AND RESULTS: We present a rapid DNA extraction protocol that utilizes a buffer with relatively large amounts of cetyltrimethylammonium bromide (CTAB) and sodium chloride, combined with a silica maxi-column cleanup of the extracted DNA. The new method is easy to implement using standard equipment and inexpensive reagents. The entire procedure (from grinding to the final elution) can be completed in less than two hours for a single sample and can be easily scaled to meet desired research goals. It works on diverse green plants with highly varied secondary chemistries (e.g., ferns, gymnosperms, and phylogenetically divergent angiosperms). CONCLUSIONS: Application of the protocol to various plant species yielded DNA of high quality in less than two hours and can be adjusted to extract DNA at large (maxi-preps) or small (96-well minipreps) scales. We anticipate that our method will be of wide utility for rapidly isolating large quantities of quality genomic DNA from diverse plant species and will have broad applications in phylogenetic studies utilizing PCR and short-read DNA sequencing.

Endosperm-based incompatibilities in hybrid monkeyflowers.

Endosperm is an angiosperm innovation central to their reproduction whose development, and thus seed viability, is controlled by genomic imprinting, where expression from certain genes is parent-specific. Unsuccessful imprinting has been linked to failed inter-specific and inter-ploidy hybridization. Despite their importance in plant speciation, the underlying mechanisms behind these endosperm-based barriers remain poorly understood. Here, we describe one such barrier between diploid Mimulus guttatus and tetraploid Mimulus luteus. The two parents differ in endosperm DNA methylation, expression dynamics, and imprinted genes. Hybrid seeds suffer from underdeveloped endosperm, reducing viability, or arrested endosperm and seed abortion when M. guttatus or M. luteus is seed parent, respectively, and transgressive methylation and expression patterns emerge. The two inherited M. luteus subgenomes, genetically distinct but epigenetically similar, are expressionally dominant over the M. guttatus genome in hybrid embryos and especially their endosperm, where paternal imprints are perturbed. In aborted seeds, de novo methylation is inhibited, potentially owing to incompatible paternal instructions of imbalanced dosage from M. guttatus imprints. We suggest that diverged epigenetic/regulatory landscapes between parental genomes induce epigenetic repatterning and global shifts in expression, which, in endosperm, may uniquely facilitate incompatible interactions between divergent imprinting schemes, potentially driving rapid barriers.

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.

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 likelihood ratio test for changes in homeolog expression bias.

BACKGROUND: Gene duplications are a major source of raw material for evolution and a likely contributor to the diversity of life on earth. Duplicate genes (i.e., homeologs, in the case of a whole genome duplication) may retain their ancestral function, sub- or neofunctionalize, or be lost entirely. A primary way that duplicate genes evolve new functions is by altering their expression patterns. Comparing the expression patterns of duplicate genes gives clues as to whether any of these evolutionary processes have occurred. RESULTS: We develop a likelihood ratio test for the analysis of the expression ratios of duplicate genes across two conditions (e.g., tissues). We demonstrate an application of this test by comparing homeolog expression patterns of 1448 homeologous gene pairs using RNA-seq data generated from leaves and petals of an allotetraploid monkeyflower (Mimulus luteus). We assess the sensitivity of this test to different levels of homeolog expression bias and compare the method to several alternatives. CONCLUSIONS: The likelihood ratio test derived here is a direct, transparent, and easily implemented method for detecting changes in homeolog expression bias that outperforms alternative approaches. While our method was derived with homeolog analysis in mind, this method can be used to analyze changes in the ratio of expression levels between any two genes in any two conditions.

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.