De novo sequence assembly and characterisation of a partial transcriptome for an evolutionarily distinct reptile, the tuatara (Sphenodon punctatus)

BACKGROUND: The tuatara (Sphenodon punctatus) is a species of extraordinary zoological interest, being the only surviving member of an entire order of reptiles which diverged early in amniote evolution. In addition to their unique phylogenetic placement, many aspects of tuatara biology, including temperature-dependent sex determination, cold adaptation and extreme longevity have the potential to inform studies of genome evolution and development. Despite increasing interest in the tuatara genome, genomic resources for the species are still very limited. We aimed to address this by assembling a transcriptome for tuatara from an early-stage embryo, which will provide a resource for genome annotation, molecular marker development and studies of development and adaptation in tuatara. RESULTS: We obtained 30 million paired-end 50 bp reads from an Illumina Genome Analyzer and assembled them with Velvet and Oases using a range of kmers. After removing redundancy and filtering out low quality transcripts, our transcriptome dataset contained 32911 transcripts, with an N50 of 675 and a mean length of 451 bp. Almost 50% (15965) of these transcripts could be annotated by comparison with the NCBI non-redundant (NR) protein database or the chicken, green anole and zebrafish UniGene sets. A scan of candidate genes and repetitive elements revealed genes involved in immune function, sex differentiation and temperature-sensitivity, as well as over 200 microsatellite markers. CONCLUSIONS: This dataset represents a major increase in genomic resources for the tuatara, increasing the number of annotated gene sequences from just 60 to almost 16,000. This will facilitate future research in sex determination, genome evolution, local adaptation and population genetics of tuatara, as well as inform studies on amniote evolution

Within and between Whorls: Comparative Transcriptional Profiling of <i>Aquilegia</i> and <i>Arabidopsis</i>

Background: The genus Aquilegia is an emerging model system in plant evolutionary biology predominantly because of its wide variation in floral traits and associated floral ecology. The anatomy of the Aquilegia flower is also very distinct. There are two whorls of petaloid organs, the outer whorl of sepals and the second whorl of petals that form nectar spurs, as well as a recently evolved fifth whorl of staminodia inserted between stamens and carpels.Methodology/Principal Findings: We designed an oligonucleotide microarray based on EST sequences from a mixed tissue, normalized cDNA library of an A. formosa x A. pubescens F2 population representing 17,246 unigenes. We then used this array to analyze floral gene expression in late pre-anthesis stage floral organs from a natural A. formosa population. In particular, we tested for gene expression patterns specific to each floral whorl and to combinations of whorls that correspond to traditional and modified ABC model groupings. Similar analyses were performed on gene expression data of Arabidopsis thaliana whorls previously obtained using the Ath1 gene chips (data available through The Arabidopsis Information Resource).Conclusions/Significance: Our comparative gene expression analyses suggest that 1) petaloid sepals and petals of A. formosa share gene expression patterns more than either have organ-specific patterns, 2) petals of A. formosa and A. thaliana may be independently derived, 3) staminodia express B and C genes similar to stamens but the staminodium genetic program has also converged on aspects of the carpel program and 4) staminodia have unique up-regulation of regulatory genes and genes that have been implicated with defense against microbial infection and herbivory. Our study also highlights the value of comparative gene expression profiling and the Aquilegia microarray in particular for the study of floral evolution and ecology.</p

Cutoffs and k-mers: implications from a transcriptome study in allopolyploid plants

<p>Abstract</p> <p>Background</p> <p>Transcriptome analysis is increasingly being used to study the evolutionary origins and ecology of non-model plants. One issue for both transcriptome assembly and differential gene expression analyses is the common occurrence in plants of hybridisation and whole genome duplication (WGD) and hybridization resulting in allopolyploidy. The divergence of duplicated genes following WGD creates near identical homeologues that can be problematic for <it>de novo </it>assembly and also reference based assembly protocols that use short reads (35 - 100 bp).</p> <p>Results</p> <p>Here we report a successful strategy for the assembly of two transcriptomes made using 75 bp Illumina reads from <it>Pachycladon fastigiatum </it>and <it>Pachycladon cheesemanii</it>. Both are allopolyploid plant species (2n = 20) that originated in the New Zealand Alps about 0.8 million years ago. In a systematic analysis of 19 different coverage cutoffs and 20 different k-mer sizes we showed that i) none of the genes could be assembled across all of the parameter space ii) assembly of each gene required an optimal set of parameter values and iii) these parameter values could be explained in part by different gene expression levels and different degrees of similarity between genes.</p> <p>Conclusions</p> <p>To obtain optimal transcriptome assemblies for allopolyploid plants, k-mer size and k-mer coverage need to be considered simultaneously across a broad parameter space. This is important for assembling a maximum number of full length ESTs and for avoiding chimeric assemblies of homeologous and paralogous gene copies.</p

Within and between Whorls: Comparative Transcriptional Profiling of Aquilegia and Arabidopsis

Background: The genus Aquilegia is an emerging model system in plant evolutionary biology predominantly because of its wide variation in floral traits and associated floral ecology. The anatomy of the Aquilegia flower is also very distinct. There are two whorls of petaloid organs, the outer whorl of sepals and the second whorl of petals that form nectar spurs, as well as a recently evolved fifth whorl of staminodia inserted between stamens and carpels. Methodology/Principal Findings: We designed an oligonucleotide microarray based on EST sequences from a mixed tissue, normalized cDNA library of an A. formosa x A. pubescens F2 population representing 17,246 unigenes. We then used this array to analyze floral gene expression in late pre-anthesis stage floral organs from a natural A. formosa population. In particular, we tested for gene expression patterns specific to each floral whorl and to combinations of whorls that correspond to traditional and modified ABC model groupings. Similar analyses were performed on gene expression data of Arabidopsis thaliana whorls previously obtained using the Ath1 gene chips (data available through The Arabidopsis Information Resource). Conclusions/Significance: Our comparative gene expression analyses suggest that 1) petaloid sepals and petals of A. formosa share gene expression patterns more than either have organ-specific patterns, 2) petals of A. formosa and A. thaliana may be independently derived, 3) staminodia express B and C genes similar to stamens but the staminodium genetic program has also converged on aspects of the carpel program and 4) staminodia have unique up-regulation of regulatory genes and genes that have been implicated with defense against microbial infection and herbivory. Our study also highlights the value of comparative gene expression profiling and the Aquilegia microarray in particular for the study of floral evolution and ecology.Organismic and Evolutionary Biolog

Transcript and protein profiling identify candidate gene sets of potential adaptive significance in New Zealand Pachycladon

<p>Abstract</p> <p>Background</p> <p>Transcript profiling of closely related species provides a means for identifying genes potentially important in species diversification. However, the predictive value of transcript profiling for inferring downstream-physiological processes has been unclear. In the present study we use shotgun proteomics to validate inferences from microarray studies regarding physiological differences in three <it>Pachycladon </it>species. We compare transcript and protein profiling and evaluate their predictive value for inferring glucosinolate chemotypes characteristic of these species.</p> <p>Results</p> <p>Evidence from heterologous microarrays and shotgun proteomics revealed differential expression of genes involved in glucosinolate hydrolysis (myrosinase-associated proteins) and biosynthesis (methylthioalkylmalate isomerase and dehydrogenase), the interconversion of carbon dioxide and bicarbonate (carbonic anhydrases), water use efficiency (ascorbate peroxidase, 2 cys peroxiredoxin, 20 kDa chloroplastic chaperonin, mitochondrial succinyl CoA ligase) and others (glutathione-S-transferase, serine racemase, vegetative storage proteins, genes related to translation and photosynthesis). Differences in glucosinolate hydrolysis products were directly confirmed. Overall, prediction of protein abundances from transcript profiles was stronger than prediction of transcript abundance from protein profiles. Protein profiles also proved to be more accurate predictors of glucosinolate profiles than transcript profiles. The similarity of species profiles for both transcripts and proteins reflected previously inferred phylogenetic relationships while glucosinolate chemotypes did not.</p> <p>Conclusions</p> <p>We have used transcript and protein profiling to predict physiological processes that evolved differently during diversification of three <it>Pachycladon </it>species. This approach has also identified candidate genes potentially important in adaptation, which are now the focus of ongoing study. Our results indicate that protein profiling provides a valuable tool for validating transcript profiles in studies of adaptive divergence.</p

Herbivore-induced changes in the transcriptome of Nicotiana attenuata

Pflanzen begegnen Schwankungen ihrer Umwelt durch ein hohes Maß an phenotypischer Plastizität. Gegenstand dieser Arbeit waren Veränderungen im Transkriptom einer wilden Tabakart, ausgelöst durch Befall verschiedener natürlicher Herbivoren, in Abhängigkeit von deren Fraßverhalten, Abstammung und Wirtspektrum. Mittels unvoreingenommener Verfahren (Differential Display, Subtraktive Hybridisierung) wurden herbivor-induzierte Gene kloniert, deren Expressionsmuster anschließend mittels Microarray Technologie und multivariater Statistik (PCA) analysiert wurden. Ein wiederkehrendes Muster beinhaltet einen Anstieg in der Expression von Sekundärstoffwechsel- und Signalgenen und einen Abfall in der Expression von Primärstoffwechsel-, Photosynthese - und Pathogenabwehrgenen. Die Expressionmuster ändern sich in Abhängigkeit von der Spezialisierung und der Nahrungsgilde des Herbivoren, der Kinetik der Induktion und dem gleichzeitigen oder aufeinander folgenden Befall verschiedener Herbivoren. Außerdem wurden Gene identifiziert, deren Regulationsverhalten auf eine ökologische Relevanz schließen läßt und welche in funktionellen Studien mittels transgener Verfahren weiter charakterisiert werden

Herbivore-induced changes in the transcriptome of Nicotiana attenuata

Pflanzen begegnen Schwankungen ihrer Umwelt durch ein hohes Maß an phenotypischer Plastizität. Gegenstand dieser Arbeit waren Veränderungen im Transkriptom einer wilden Tabakart, ausgelöst durch Befall verschiedener natürlicher Herbivoren, in Abhängigkeit von deren Fraßverhalten, Abstammung und Wirtspektrum. Mittels unvoreingenommener Verfahren (Differential Display, Subtraktive Hybridisierung) wurden herbivor-induzierte Gene kloniert, deren Expressionsmuster anschließend mittels Microarray Technologie und multivariater Statistik (PCA) analysiert wurden. Ein wiederkehrendes Muster beinhaltet einen Anstieg in der Expression von Sekundärstoffwechsel- und Signalgenen und einen Abfall in der Expression von Primärstoffwechsel-, Photosynthese - und Pathogenabwehrgenen. Die Expressionmuster ändern sich in Abhängigkeit von der Spezialisierung und der Nahrungsgilde des Herbivoren, der Kinetik der Induktion und dem gleichzeitigen oder aufeinander folgenden Befall verschiedener Herbivoren. Außerdem wurden Gene identifiziert, deren Regulationsverhalten auf eine ökologische Relevanz schließen läßt und welche in funktionellen Studien mittels transgener Verfahren weiter charakterisiert werden

Herbivore-induced plant vaccination. Part II. Array-studies reveal the transience of herbivore-specific transcriptional imprints and a distinct imprint from stress combinations

Microarray technology has given plant biologists the ability to simultaneously monitor changes in the expression of hundreds of genes, and yet, to date, this technology has not been applied to ecological phenomena. In native tobacco (Nicotiana attenuata), prior attack of sap-feeding mirids (Tupiocoris notatus) results in vaccination of the plant against subsequent attacks by chewing hornworms (Manduca sexta). This vaccination is mediated by a combination of direct and indirect defenses and tolerance responses, which act in concert with the attack preferences of a generalist predator. Here, we use microarrays enriched in herbivore-elicited genes with a principal components analysis (PCA) to characterize transcriptional 'imprints' of single, sequential, or simultaneous attacks by these two main herbivores of N. attenuata. The PCA identified distinctly different imprints left by individual attack from the two species after 24 h, but not after 5 days. Moreover, imprints of sequential or simultaneous attacks differed significantly from those of single attack, suggesting the existence of a distinct gene expression program responsive to the combination of biological stressors. A dissection of the transcriptional imprints revealed responses in direct and indirect defense genes that were well correlated with observed increases in defense metabolites. Attack from both herbivores elicits a switch from growth- to defense-related transcriptional processes, and herbivore-specific changes occur largely in primary metabolism and signaling cascades. PCA of these polygenic transcriptional imprints characterizes the ephemeral changes in the transcriptome that occur during the maturation of ecologically relevant phenotypic responses. [References: 47