Cell-type-aware regulatory landscapes governing monoterpene indole alkaloid biosynthesis in the medicinal plant Catharanthus roseus.

In plants, the biosynthetic pathways of some specialized metabolites are partitioned into specialized or rare cell types, as exemplified by the monoterpenoid indole alkaloid (MIA) pathway of Catharanthus roseus (Madagascar Periwinkle), the source of the anticancer compounds vinblastine and vincristine. In the leaf, the C. roseus MIA biosynthetic pathway is partitioned into three cell types with the final known steps of the pathway expressed in the rare cell type termed idioblast. How cell-type specificity of MIA biosynthesis is achieved is poorly understood. We generated single-cell multi-omics data from C. roseus leaves. Integrating gene expression and chromatin accessibility profiles across single cells, as well as transcription factor (TF)-binding site profiles, we constructed a cell-type-aware gene regulatory network for MIA biosynthesis. We showcased cell-type-specific TFs as well as cell-type-specific cis-regulatory elements. Using motif enrichment analysis, co-expression across cell types, and functional validation approaches, we discovered a novel idioblast-specific TF (Idioblast MYB1, CrIDM1) that activates expression of late-stage MIA biosynthetic genes in the idioblast. These analyses not only led to the discovery of the first documented cell-type-specific TF that regulates the expression of two idioblast-specific biosynthetic genes within an idioblast metabolic regulon but also provides insights into cell-type-specific metabolic regulation.

Chromosome-scale Salvia hispanica L. (Chia) genome assembly reveals rampant Salvia interspecies introgression.

Salvia hispanica L. (Chia), a member of the Lamiaceae, is an economically important crop in Mesoamerica, with health benefits associated with its seed fatty acid composition. Chia varieties are distinguished based on seed color including mixed white and black (Chia pinta) and black (Chia negra). To facilitate research on Chia and expand on comparative analyses within the Lamiaceae, we generated a chromosome-scale assembly of a Chia pinta accession and performed comparative genome analyses with a previously published Chia negra genome assembly. The Chia pinta and Chia negra genome sequences were highly similar as shown by a limited number of single nucleotide polymorphisms and extensive shared orthologous gene membership. However, there is an enrichment of terpene synthases in the Chia pinta genome relative to the Chia negra genome. We sequenced and analyzed the genomes of 20 Chia accessions with differing seed color and geographic origin revealing population structure within S. hispanica and interspecific introgressions of Salvia species. As the genus Salvia is polyphyletic, its evolutionary history remains unclear. Using large-scale synteny analysis within the Lamiaceae and orthologous group membership, we resolved the phylogeny of Salvia species. This study and its collective resources further our understanding of genomic diversity in this food crop and the extent of interspecies hybridizations in Salvia.

Targeted genotyping-by-sequencing of potato and data analysis with R/polyBreedR.

Mid-density targeted genotyping-by-sequencing (GBS) combines trait-specific markers with thousands of genomic markers at an attractive price for linkage mapping and genomic selection. A 2.5K targeted GBS assay for potato (Solanum tuberosum L.) was developed using the DArTag technology and later expanded to 4K targets. Genomic markers were selected from the potato Infinium single nucleotide polymorphism (SNP) array to maximize genome coverage and polymorphism rates. The DArTag and SNP array platforms produced equivalent dendrograms in a test set of 298 tetraploid samples, and 83% of the common markers showed good quantitative agreement, with RMSE (root mean squared error) <0.5. DArTag is suited for genomic selection candidates in the clonal evaluation trial, coupled with imputation to a higher density platform for the training population. Using the software polyBreedR, an R package for the manipulation and analysis of polyploid marker data, the RMSE for imputation by linkage analysis was 0.15 in a small half-diallel population (N = 85), which was significantly lower than the RMSE of 0.42 with the random forest method. Regarding high-value traits, the DArTag markers for resistance to potato virus Y, golden cyst nematode, and potato wart appeared to track their targets successfully, as did multi-allelic markers for maturity and tuber shape. In summary, the potato DArTag assay is a transformative and publicly available technology for potato breeding and genetics.

Single-cell multi-omics in the medicinal plant Catharanthus roseus.

Advances in omics technologies now permit the generation of highly contiguous genome assemblies, detection of transcripts and metabolites at the level of single cells and high-resolution determination of gene regulatory features. Here, using a complementary, multi-omics approach, we interrogated the monoterpene indole alkaloid (MIA) biosynthetic pathway in Catharanthus roseus, a source of leading anticancer drugs. We identified clusters of genes involved in MIA biosynthesis on the eight C. roseus chromosomes and extensive gene duplication of MIA pathway genes. Clustering was not limited to the linear genome, and through chromatin interaction data, MIA pathway genes were present within the same topologically associated domain, permitting the identification of a secologanin transporter. Single-cell RNA-sequencing revealed sequential cell-type-specific partitioning of the leaf MIA biosynthetic pathway that, when coupled with a single-cell metabolomics approach, permitted the identification of a reductase that yields the bis-indole alkaloid anhydrovinblastine. We also revealed cell-type-specific expression in the root MIA pathway.

Combining GWAS and TWAS to identify candidate causal genes for tocochromanol levels in maize grain.

Tocochromanols (tocopherols and tocotrienols, collectively vitamin E) are lipid-soluble antioxidants important for both plant fitness and human health. The main dietary sources of vitamin E are seed oils that often accumulate high levels of tocopherol isoforms with lower vitamin E activity. The tocochromanol biosynthetic pathway is conserved across plant species but an integrated view of the genes and mechanisms underlying natural variation of tocochromanol levels in seed of most cereal crops remains limited. To address this issue, we utilized the high mapping resolution of the maize Ames panel of ∼1,500 inbred lines scored with 12.2 million single-nucleotide polymorphisms to generate metabolomic (mature grain tocochromanols) and transcriptomic (developing grain) data sets for genetic mapping. By combining results from genome- and transcriptome-wide association studies, we identified a total of 13 candidate causal gene loci, including 5 that had not been previously associated with maize grain tocochromanols: 4 biosynthetic genes (arodeH2 paralog, dxs1, vte5, and vte7) and a plastid S-adenosyl methionine transporter (samt1). Expression quantitative trait locus (eQTL) mapping of these 13 gene loci revealed that they are predominantly regulated by cis-eQTL. Through a joint statistical analysis, we implicated cis-acting variants as responsible for colocalized eQTL and GWAS association signals. Our multiomics approach provided increased statistical power and mapping resolution to enable a detailed characterization of the genetic and regulatory architecture underlying tocochromanol accumulation in maize grain and provided insights for ongoing biofortification efforts to breed and/or engineer vitamin E and antioxidant levels in maize and other cereals.

Phased, chromosome-scale genome assemblies of tetraploid potato reveal a complex genome, transcriptome, and predicted proteome landscape underpinning genetic diversity.

Cultivated potato is a clonally propagated autotetraploid species with a highly heterogeneous genome. Phased assemblies of six cultivars including two chromosome-scale phased genome assemblies revealed extensive allelic diversity, including altered coding and transcript sequences, preferential allele expression, and structural variation that collectively result in a highly complex transcriptome and predicted proteome, which are distributed across the homologous chromosomes. Wild species contribute to the extensive allelic diversity in tetraploid cultivars, demonstrating ancestral introgressions predating modern breeding efforts. As a clonally propagated autotetraploid that undergoes limited meiosis, dysfunctional and deleterious alleles are not purged in tetraploid potato. Nearly a quarter of the loci bore mutations are predicted to have a high negative impact on protein function, complicating breeder's efforts to reduce genetic load. The StCDF1 locus controls maturity, and analysis of six tetraploid genomes revealed that 12 allelic variants of StCDF1 are correlated with maturity in a dosage-dependent manner. Knowledge of the complexity of the tetraploid potato genome with its rampant structural variation and embedded deleterious and dysfunctional alleles will be key not only to implementing precision breeding of tetraploid cultivars but also to the construction of homozygous, diploid potato germplasm containing favorable alleles to capitalize on heterosis in F1 hybrids.

Genome-wide association study suggests an independent genetic basis of zinc and cadmium concentrations in fresh sweet corn kernels.

Despite being one of the most consumed vegetables in the United States, the elemental profile of sweet corn (Zea mays L.) is limited in its dietary contributions. To address this through genetic improvement, a genome-wide association study was conducted for the concentrations of 15 elements in fresh kernels of a sweet corn association panel. In concordance with mapping results from mature maize kernels, we detected a probable pleiotropic association of zinc and iron concentrations with nicotianamine synthase5 (nas5), which purportedly encodes an enzyme involved in synthesis of the metal chelator nicotianamine. In addition, a pervasive association signal was identified for cadmium concentration within a recombination suppressed region on chromosome 2. The likely causal gene underlying this signal was heavy metal ATPase3 (hma3), whose counterpart in rice, OsHMA3, mediates vacuolar sequestration of cadmium and zinc in roots, whereby regulating zinc homeostasis and cadmium accumulation in grains. In our association panel, hma3 associated with cadmium but not zinc accumulation in fresh kernels. This finding implies that selection for low cadmium will not affect zinc levels in fresh kernels. Although less resolved association signals were detected for boron, nickel, and calcium, all 15 elements were shown to have moderate predictive abilities via whole-genome prediction. Collectively, these results help enhance our genomics-assisted breeding efforts centered on improving the elemental profile of fresh sweet corn kernels.

Genome assembly of Chiococca alba uncovers key enzymes involved in the biosynthesis of unusual terpenoids.

Chiococca alba (L.) Hitchc. (snowberry), a member of the Rubiaceae, has been used as a folk remedy for a range of health issues including inflammation and rheumatism and produces a wealth of specialized metabolites including terpenes, alkaloids, and flavonoids. We generated a 558 Mb draft genome assembly for snowberry which encodes 28,707 high-confidence genes. Comparative analyses with other angiosperm genomes revealed enrichment in snowberry of lineage-specific genes involved in specialized metabolism. Synteny between snowberry and Coffea canephora Pierre ex A. Froehner (coffee) was evident, including the chromosomal region encoding caffeine biosynthesis in coffee, albeit syntelogs of N-methyltransferase were absent in snowberry. A total of 27 putative terpene synthase genes were identified, including 10 that encode diterpene synthases. Functional validation of a subset of putative terpene synthases revealed that combinations of diterpene synthases yielded access to products of both general and specialized metabolism. Specifically, we identified plausible intermediates in the biosynthesis of merilactone and ribenone, structurally unique antimicrobial diterpene natural products. Access to the C. alba genome will enable additional characterization of biosynthetic pathways responsible for health-promoting compounds in this medicinal species.

Quantitative trait loci and differential gene expression analyses reveal the genetic basis for negatively associated ß-carotene and starch content in hexaploid sweetpotato [Ipomoea batatas (L.) Lam.].

KEY MESSAGE: ß-Carotene content in sweetpotato is associated with the Orange and phytoene synthase genes; due to physical linkage of phytoene synthase with sucrose synthase, ß-carotene and starch content are negatively correlated. In populations depending on sweetpotato for food security, starch is an important source of calories, while ß-carotene is an important source of provitamin A. The negative association between the two traits contributes to the low nutritional quality of sweetpotato consumed, especially in sub-Saharan Africa. Using a biparental mapping population of 315 F1 progeny generated from a cross between an orange-fleshed and a non-orange-fleshed sweetpotato variety, we identified two major quantitative trait loci (QTL) on linkage group (LG) three (LG3) and twelve (LG12) affecting starch, ß-carotene, and their correlated traits, dry matter and flesh color. Analysis of parental haplotypes indicated that these two regions acted pleiotropically to reduce starch content and increase ß-carotene in genotypes carrying the orange-fleshed parental haplotype at the LG3 locus. Phytoene synthase and sucrose synthase, the rate-limiting and linked genes located within the QTL on LG3 involved in the carotenoid and starch biosynthesis, respectively, were differentially expressed in Beauregard versus Tanzania storage roots. The Orange gene, the molecular switch for chromoplast biogenesis, located within the QTL on LG12 while not differentially expressed was expressed in developing roots of the parental genotypes. We conclude that these two QTL regions act together in a cis and trans manner to inhibit starch biosynthesis in amyloplasts and enhance chromoplast biogenesis, carotenoid biosynthesis, and accumulation in orange-fleshed sweetpotato. Understanding the genetic basis of this negative association between starch and ß-carotene will inform future sweetpotato breeding strategies targeting sweetpotato for food and nutritional security.

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.

Genome Assembly and Annotation of the Medicinal Plant Calotropis gigantea, a Producer of Anticancer and Antimalarial Cardenolides.

Calotropis gigantea produces specialized secondary metabolites known as cardenolides, which have anticancer and antimalarial properties. Although transcriptomic studies have been conducted in other cardenolide-producing species, no nuclear genome assembly for an Asterid cardenolide-producing species has been reported to date. A high-quality de novo assembly was generated for C. gigantea, representing 157,284,427 bp with an N50 scaffold size of 805,959 bp, for which quality assessments indicated a near complete representation of the genic space. Transcriptome data in the form of RNA-sequencing libraries from a developmental tissue series was generated to aid the annotation and construction of a gene expression atlas. Using an ab initio and evidence-driven gene annotation pipeline, 18,197 high-confidence genes were annotated. Homologous and syntenic relationships between C. gigantea and other species within the Apocynaceae family confirmed previously identified evolutionary relationships, and suggest the emergence or loss of the specialized cardenolide metabolites after the divergence of the Apocynaceae subfamilies. The C. gigantea genome assembly, annotation, and RNA-sequencing data provide a novel resource to study the cardenolide biosynthesis pathway, especially for understanding the evolutionary origin of cardenolides and the engineering of cardenolide production in heterologous organisms for existing and novel pharmaceutical applications.

Genome diversity of tuber-bearing Solanum uncovers complex evolutionary history and targets of domestication in the cultivated potato.

Cultivated potatoes (Solanum tuberosum L.), domesticated from wild Solanum species native to the Andes of southern Peru, possess a diverse gene pool representing more than 100 tuber-bearing relatives (Solanum section Petota). A diversity panel of wild species, landraces, and cultivars was sequenced to assess genetic variation within tuber-bearing Solanum and the impact of domestication on genome diversity and identify key loci selected for cultivation in North and South America. Sequence diversity of diploid and tetraploid Stuberosum exceeded any crop resequencing study to date, in part due to expanded wild introgressions following polyploidy that captured alleles outside of their geographic origin. We identified 2,622 genes as under selection, with only 14-16% shared by North American and Andean cultivars, showing that a limited gene set drove early improvement of cultivated potato, while adaptation of upland (Stuberosum group Andigena) and lowland (S. tuberosum groups Chilotanum and Tuberosum) populations targeted distinct loci. Signatures of selection were uncovered in genes controlling carbohydrate metabolism, glycoalkaloid biosynthesis, the shikimate pathway, the cell cycle, and circadian rhythm. Reduced sexual fertility that accompanied the shift to asexual reproduction in cultivars was reflected by signatures of selection in genes regulating pollen development/gametogenesis. Exploration of haplotype diversity at potato's maturity locus (StCDF1) revealed introgression of truncated alleles from wild species, particularly Smicrodontum in long-day-adapted cultivars. This study uncovers a historic role of wild Solanum species in the diversification of long-day-adapted tetraploid potatoes, showing that extant natural populations represent an essential source of untapped adaptive potential.

De novo genome assembly of Camptotheca acuminata, a natural source of the anti-cancer compound camptothecin.

Camptotheca acuminata is 1 of a limited number of species that produce camptothecin, a pentacyclic quinoline alkaloid with anti-cancer activity due to its ability to inhibit DNA topoisomerase. While transcriptome studies have been performed previously with various camptothecin-producing species, no genome sequence for a camptothecin-producing species is available to date. We generated a high-quality de novo genome assembly for C. acuminata representing 403 174 860 bp on 1394 scaffolds with an N50 scaffold size of 1752 kbp. Quality assessments of the assembly revealed robust representation of the genome sequence including genic regions. Using a novel genome annotation method, we annotated 31 825 genes encoding 40 332 gene models. Based on sequence identity and orthology with validated genes from Catharanthus roseus as well as Pfam searches, we identified candidate orthologs for genes potentially involved in camptothecin biosynthesis. Extensive gene duplication including tandem duplication was widespread in the C. acuminata genome, with 2571 genes belonging to 997 tandem duplicated gene clusters. To our knowledge, this is the first genome sequence for a camptothecin-producing species, and access to the C. acuminata genome will permit not only discovery of genes encoding the camptothecin biosynthetic pathway but also reagents that can be used for heterologous expression of camptothecin and camptothecin analogs with novel pharmaceutical applications.

Analysis of Ribosome-Associated mRNAs in Rice Reveals the Importance of Transcript Size and GC Content in Translation.

Gene expression is controlled at transcriptional and post-transcriptional levels including decoding of messenger RNA (mRNA) into polypeptides via ribosome-mediated translation. Translational regulation has been intensively studied in the model dicot plant Arabidopsis thaliana, and in this study, we assessed the translational status [proportion of steady-state mRNA associated with ribosomes] of mRNAs by Translating Ribosome Affinity Purification followed by mRNA-sequencing (TRAP-seq) in rice (Oryza sativa), a model monocot plant and the most important food crop. A survey of three tissues found that most transcribed rice genes are translated whereas few transposable elements are associated with ribosomes. Genes with short and GC-rich coding regions are overrepresented in ribosome-associated mRNAs, suggesting that the GC-richness characteristic of coding sequences in grasses may be an adaptation that favors efficient translation. Transcripts with retained introns and extended 5' untranslated regions are underrepresented on ribosomes, and rice genes belonging to different evolutionary lineages exhibited differential enrichment on the ribosomes that was associated with GC content. Genes involved in photosynthesis and stress responses are preferentially associated with ribosomes, whereas genes in epigenetic regulation pathways are the least enriched on ribosomes. Such variation is more dramatic in rice than that in Arabidopsis and is correlated with the wide variation of GC content of transcripts in rice. Taken together, variation in the translation status of individual transcripts reflects important mechanisms of gene regulation, which may have a role in evolution and diversification.

Haplotype-Phased Synthetic Long Reads from Short-Read Sequencing.

Next-generation DNA sequencing has revolutionized the study of biology. However, the short read lengths of the dominant instruments complicate assembly of complex genomes and haplotype phasing of mixtures of similar sequences. Here we demonstrate a method to reconstruct the sequences of individual nucleic acid molecules up to 11.6 kilobases in length from short (150-bp) reads. We show that our method can construct 99.97%-accurate synthetic reads from bacterial, plant, and animal genomic samples, full-length mRNA sequences from human cancer cell lines, and individual HIV env gene variants from a mixture. The preparation of multiple samples can be multiplexed into a single tube, further reducing effort and cost relative to competing approaches. Our approach generates sequencing libraries in three days from less than one microgram of DNA in a single-tube format without custom equipment or specialized expertise.

Genome-guided investigation of plant natural product biosynthesis.

The medicinal plant Madagascar periwinkle, Catharanthus roseus (L.) G. Don, produces hundreds of biologically active monoterpene-derived indole alkaloid (MIA) metabolites and is the sole source of the potent, expensive anti-cancer compounds vinblastine and vincristine. Access to a genome sequence would enable insights into the biochemistry, control, and evolution of genes responsible for MIA biosynthesis. However, generation of a near-complete, scaffolded genome is prohibitive to small research communities due to the expense, time, and expertise required. In this study, we generated a genome assembly for C. roseus that provides a near-comprehensive representation of the genic space that revealed the genomic context of key points within the MIA biosynthetic pathway including physically clustered genes, tandem gene duplication, expression sub-functionalization, and putative neo-functionalization. The genome sequence also facilitated high resolution co-expression analyses that revealed three distinct clusters of co-expression within the components of the MIA pathway. Coordinated biosynthesis of precursors and intermediates throughout the pathway appear to be a feature of vinblastine/vincristine biosynthesis. The C. roseus genome also revealed localization of enzyme-rich genic regions and transporters near known biosynthetic enzymes, highlighting how even a draft genome sequence can empower the study of high-value specialized metabolites.

Carbohydrate-active enzymes in pythium and their role in plant cell wall and storage polysaccharide degradation.

Carbohydrate-active enzymes (CAZymes) are involved in the metabolism of glycoconjugates, oligosaccharides, and polysaccharides and, in the case of plant pathogens, in the degradation of the host cell wall and storage compounds. We performed an in silico analysis of CAZymes predicted from the genomes of seven Pythium species (Py. aphanidermatum, Py. arrhenomanes, Py. irregulare, Py. iwayamai, Py. ultimum var. ultimum, Py. ultimum var. sporangiiferum and Py. vexans) using the "CAZymes Analysis Toolkit" and "Database for Automated Carbohydrate-active Enzyme Annotation" and compared them to previously published oomycete genomes. Growth of Pythium spp. was assessed in a minimal medium containing selected carbon sources that are usually present in plants. The in silico analyses, coupled with our in vitro growth assays, suggest that most of the predicted CAZymes are involved in the metabolism of the oomycete cell wall with starch and sucrose serving as the main carbohydrate sources for growth of these plant pathogens. The genomes of Pythium spp. also encode pectinases and cellulases that facilitate degradation of the plant cell wall and are important in hyphal penetration; however, the species examined in this study lack the requisite genes for the complete saccharification of these carbohydrates for use as a carbon source. Genes encoding for xylan, xyloglucan, (galacto)(gluco)mannan and cutin degradation were absent or infrequent in Pythium spp.. Comparative analyses of predicted CAZymes in oomycetes indicated distinct evolutionary histories. Furthermore, CAZyme gene families among Pythium spp. were not uniformly distributed in the genomes, suggesting independent gene loss events, reflective of the polyphyletic relationships among some of the species.

Alternative splicing of a multi-drug transporter from Pseudoperonospora cubensis generates an RXLR effector protein that elicits a rapid cell death.

Pseudoperonospora cubensis, an obligate oomycete pathogen, is the causal agent of cucurbit downy mildew, a foliar disease of global economic importance. Similar to other oomycete plant pathogens, Ps. cubensis has a suite of RXLR and RXLR-like effector proteins, which likely function as virulence or avirulence determinants during the course of host infection. Using in silico analyses, we identified 271 candidate effector proteins within the Ps. cubensis genome with variable RXLR motifs. In extending this analysis, we present the functional characterization of one Ps. cubensis effector protein, RXLR protein 1 (PscRXLR1), and its closest Phytophthora infestans ortholog, PITG_17484, a member of the Drug/Metabolite Transporter (DMT) superfamily. To assess if such effector-non-effector pairs are common among oomycete plant pathogens, we examined the relationship(s) among putative ortholog pairs in Ps. cubensis and P. infestans. Of 271 predicted Ps. cubensis effector proteins, only 109 (41%) had a putative ortholog in P. infestans and evolutionary rate analysis of these orthologs shows that they are evolving significantly faster than most other genes. We found that PscRXLR1 was up-regulated during the early stages of infection of plants, and, moreover, that heterologous expression of PscRXLR1 in Nicotiana benthamiana elicits a rapid necrosis. More interestingly, we also demonstrate that PscRXLR1 arises as a product of alternative splicing, making this the first example of an alternative splicing event in plant pathogenic oomycetes transforming a non-effector gene to a functional effector protein. Taken together, these data suggest a role for PscRXLR1 in pathogenicity, and, in total, our data provide a basis for comparative analysis of candidate effector proteins and their non-effector orthologs as a means of understanding function and evolutionary history of pathogen effectors.

Development of transcriptomic resources for interrogating the biosynthesis of monoterpene indole alkaloids in medicinal plant species.

The natural diversity of plant metabolism has long been a source for human medicines. One group of plant-derived compounds, the monoterpene indole alkaloids (MIAs), includes well-documented therapeutic agents used in the treatment of cancer (vinblastine, vincristine, camptothecin), hypertension (reserpine, ajmalicine), malaria (quinine), and as analgesics (7-hydroxymitragynine). Our understanding of the biochemical pathways that synthesize these commercially relevant compounds is incomplete due in part to a lack of molecular, genetic, and genomic resources for the identification of the genes involved in these specialized metabolic pathways. To address these limitations, we generated large-scale transcriptome sequence and expression profiles for three species of Asterids that produce medicinally important MIAs: Camptotheca acuminata, Catharanthus roseus, and Rauvolfia serpentina. Using next generation sequencing technology, we sampled the transcriptomes of these species across a diverse set of developmental tissues, and in the case of C. roseus, in cultured cells and roots following elicitor treatment. Through an iterative assembly process, we generated robust transcriptome assemblies for all three species with a substantial number of the assembled transcripts being full or near-full length. The majority of transcripts had a related sequence in either UniRef100, the Arabidopsis thaliana predicted proteome, or the Pfam protein domain database; however, we also identified transcripts that lacked similarity with entries in either database and thereby lack a known function. Representation of known genes within the MIA biosynthetic pathway was robust. As a diverse set of tissues and treatments were surveyed, expression abundances of transcripts in the three species could be estimated to reveal transcripts associated with development and response to elicitor treatment. Together, these transcriptomes and expression abundance matrices provide a rich resource for understanding plant specialized metabolism, and promotes realization of innovative production systems for plant-derived pharmaceuticals.