Overlap of transcriptome and proteome data among three orchid species.

<p><i>(A)</i> Venn diagram showing the species overlap in 454 reads mapped back onto the reference transcriptome. <i>(B)</i> Bar graph indicating the extent of read sharing. <i>(C)</i> Venn diagram showing the overlap of <i>Ophrys</i> proteomes (HQ data of proteins with corresponding transcripts). <i>(D)</i> Bar graph indicating the extent of proteome overlap among species. <i>(A,C)</i> E, <i>O. exaltata</i>; G, <i>O. garganica</i>; S, <i>O. sphegodes</i>.</p

Cross-validation of NGS data sets.

<p>Summary of the proportion of a given sequence data set to which reads from another NGS data set can be mapped. N_ref: number of sequences in the reference data set; N_map: number of sequences in the reference data set that is mapped by the query data set; %age (mapping): N_map expressed as a percentage. The term “3 <i>spp.</i>” refers to data from all three orchid species.</p>1<p>In this row, values in brackets are for Sanger reads.</p

Functional annotation.

<p><i>(A–C)</i> Pie charts showing the composition of 2^nd level GO terms of the <i>Ophrys</i> reference transcriptome, broken up into the three major GO categories: <i>(A)</i> biological process, <i>(B)</i> cellular components, and <i>(C)</i> molecular function. <i>(D)</i> Summary of KOG functional annotation of <i>Ophrys</i> transcripts. The KOG terms are: D: Cell cycle control, cell division, chromosome partitioning; M: Cell wall, membrane, envelope biogenesis; N: Cell motility; O: Posttranslational modification, protein turnover, chaperones; T: Signal transduction mechanisms; U: Intracellular trafficking, secretion, and vesicular transport; V: Defence mechanisms; W: Extracellular structures; Y: Nuclear structure; Z: Cytoskeleton; A: RNA processing and modification; B: Chromatin structure and dynamics; J: Translation, ribosomal structure and biogenesis; K: Transcription; L: Replication, recombination and repair; C: Energy production and conversion; E: Amino acid transport and metabolism; F: Nucleotide transport and metabolism; G: Carbohydrate transport and metabolism; H: Coenzyme transport and metabolism; I: Lipid transport and metabolism; P: Inorganic ion transport and metabolism; Q: Secondary metabolites biosynthesis, transport and catabolism; R: General function prediction only; S: Unknown function.</p

Candidate genes in biosynthetic pathways.

<p>Schematic diagrams of <i>(A)</i> hydrocarbon and <i>(B)</i> core anthocyanin biosynthesis, indicating candidate protein classes. The number of transcripts for a given candidate gene class is indicated in blue numbers, where an asterisk (*) indicates that a given candidate gene was confirmed at the protein level. Fatty acids in (<i>A</i>) are shown using C:D notation, where C is the number of carbon atoms and D is the number of double-bonds. Greyed, dotted arrows in panel <i>(B)</i> indicate metabolic reactions hypothesised to be absent. Protein abbreviations and further details on the listed candidates are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064621#pone.0064621.s008" target="_blank">Table S6</a>.</p

Summary of KEGG pathway analysis.

<p>The number of <i>Ophrys</i> transcripts in a given sub-pathway, as well as the corresponding number of distinct enzymes in the KEGG database are shown.</p

Summary of proteomics data.

<p>Data are presented for species individually and for the total of all three species combined. The protein discovery (PD) analysis was performed at a protein identification threshold of 90%, the highest quality (HQ) data set was compiled at a threshold of 99%. For spectra, numbers are given before (‘raw’) and after (‘clean’) removal of known contaminants. All other numbers were obtained from cleaned spectra only.</p>1<p>Proteins matching a sequence in the <i>Ophrys</i> reference transcriptome. In the HQ data set, 115 were 454 singleton reads, 4 Sanger singleton reads, 388 Solexa contigs, 343 pyrosequencing isotigs and 753 combined 454/Solexa contigs. Among these 1603 HQ orchid proteins, no peptide matched translations from different strands, and only 11 proteins (0.7%) had peptides matching to two reading frames of the same transcript strand (in each case due to a single frame shift).</p>2<p>Proteins not matching any <i>Ophrys</i> transcript, but with SwissProt and/or TAIR9 database hit.</p

Flow chart of transcriptome and proteome analysis.

<p>Labellum tissue from mature, unpollinated flowers was used, except where marked by an asterisk (*), indicating that additional material from sepals, petals, leaves, bracts, and flower buds was included.</p

Sequence length distributions.

<p><i>(A)</i> Distribution of 454 read lengths after filtering and adapter removal for the three <i>Ophrys</i> species. <i>(B)</i> Contig length distribution (20 bp windows) for the pooled 454 assembly of the three <i>Ophrys</i> species. <i>(C)</i> Distribution of contig/isotig lengths in the <i>Ophrys</i> reference transcriptome (20 bp windows).</p