Publications (8) View all
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Article: A conifer genomics resource of 200,000 spruce (Picea spp.) ESTs and 6,464 high-quality, sequence-finished full-length cDNAs for Sitka spruce (Picea sitchensis).
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ABSTRACT: Members of the pine family (Pinaceae), especially species of spruce (Picea spp.) and pine (Pinus spp.), dominate many of the world's temperate and boreal forests. These conifer forests are of critical importance for global ecosystem stability and biodiversity. They also provide the majority of the world's wood and fiber supply and serve as a renewable resource for other industrial biomaterials. In contrast to angiosperms, functional and comparative genomics research on conifers, or other gymnosperms, is limited by the lack of a relevant reference genome sequence. Sequence-finished full-length (FL)cDNAs and large collections of expressed sequence tags (ESTs) are essential for gene discovery, functional genomics, and for future efforts of conifer genome annotation. As part of a conifer genomics program to characterize defense against insects and adaptation to local environments, and to discover genes for the production of biomaterials, we developed 20 standard, normalized or full-length enriched cDNA libraries from Sitka spruce (P. sitchensis), white spruce (P. glauca), and interior spruce (P. glauca-engelmannii complex). We sequenced and analyzed 206,875 3'- or 5'-end ESTs from these libraries, and developed a resource of 6,464 high-quality sequence-finished FLcDNAs from Sitka spruce. Clustering and assembly of 147,146 3'-end ESTs resulted in 19,941 contigs and 26,804 singletons, representing 46,745 putative unique transcripts (PUTs). The 6,464 FLcDNAs were all obtained from a single Sitka spruce genotype and represent 5,718 PUTs. This paper provides detailed annotation and quality assessment of a large EST and FLcDNA resource for spruce. The 6,464 Sitka spruce FLcDNAs represent the third largest sequence-verified FLcDNA resource for any plant species, behind only rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana), and the only substantial FLcDNA resource for a gymnosperm. Our emphasis on capturing FLcDNAs and ESTs from cDNA libraries representing herbivore-, wound- or elicitor-treated induced spruce tissues, along with incorporating normalization to capture rare transcripts, resulted in a rich resource for functional genomics and proteomics studies. Sequence comparisons against five plant genomes and the non-redundant GenBank protein database revealed that a substantial number of spruce transcripts have no obvious similarity to known angiosperm gene sequences. Opportunities for future applications of the sequence and clone resources for comparative and functional genomics are discussed.BMC Genomics 11/2008; 9:484. · 4.07 Impact Factor -
Article: Analysis of 4,664 high-quality sequence-finished poplar full-length cDNA clones and their utility for the discovery of genes responding to insect feeding.
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ABSTRACT: The genus Populus includes poplars, aspens and cottonwoods, which will be collectively referred to as poplars hereafter unless otherwise specified. Poplars are the dominant tree species in many forest ecosystems in the Northern Hemisphere and are of substantial economic value in plantation forestry. Poplar has been established as a model system for genomics studies of growth, development, and adaptation of woody perennial plants including secondary xylem formation, dormancy, adaptation to local environments, and biotic interactions. As part of the poplar genome sequencing project and the development of genomic resources for poplar, we have generated a full-length (FL)-cDNA collection using the biotinylated CAP trapper method. We constructed four FLcDNA libraries using RNA from xylem, phloem and cambium, and green shoot tips and leaves from the P. trichocarpa Nisqually-1 genotype, as well as insect-attacked leaves of the P. trichocarpa x P. deltoides hybrid. Following careful selection of candidate cDNA clones, we used a combined strategy of paired end reads and primer walking to generate a set of 4,664 high-accuracy, sequence-verified FLcDNAs, which clustered into 3,990 putative unique genes. Mapping FLcDNAs to the poplar genome sequence combined with BLAST comparisons to previously predicted protein coding sequences in the poplar genome identified 39 FLcDNAs that likely localize to gaps in the current genome sequence assembly. Another 173 FLcDNAs mapped to the genome sequence but were not included among the previously predicted genes in the poplar genome. Comparative sequence analysis against Arabidopsis thaliana and other species in the non-redundant database of GenBank revealed that 11.5% of the poplar FLcDNAs display no significant sequence similarity to other plant proteins. By mapping the poplar FLcDNAs against transcriptome data previously obtained with a 15.5 K cDNA microarray, we identified 153 FLcDNA clones for genes that were differentially expressed in poplar leaves attacked by forest tent caterpillars. This study has generated a high-quality FLcDNA resource for poplar and the third largest FLcDNA collection published to date for any plant species. We successfully used the FLcDNA sequences to reassess gene prediction in the poplar genome sequence, perform comparative sequence annotation, and identify differentially expressed transcripts associated with defense against insects. The FLcDNA sequences will be essential to the ongoing curation and annotation of the poplar genome, in particular for targeting gaps in the current genome assembly and further improvement of gene predictions. The physical FLcDNA clones will serve as useful reagents for functional genomics research in areas such as analysis of gene functions in defense against insects and perennial growth. Sequences from this study have been deposited in NCBI GenBank under the accession numbers EF144175 to EF148838.BMC Genomics 02/2008; 9:57. · 4.07 Impact Factor -
Article: Genomics of hybrid poplar (Populus trichocarpa× deltoides) interacting with forest tent caterpillars (Malacosoma disstria): normalized and full‐length cDNA libraries, expressed sequence tags, and a cDNA microarray for the study of insect‐induced defences in poplar
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ABSTRACT: As part of a genomics strategy to characterize inducible defences against insect herbivory in poplar, we developed a comprehensive suite of functional genomics resources including cDNA libraries, expressed sequence tags (ESTs) and a cDNA microarray platform. These resources are designed to complement the existing poplar genome sequence and poplar (Populus spp.) ESTs by focusing on herbivore- and elicitor-treated tissues and incorporating normalization methods to capture rare transcripts. From a set of 15 standard, normalized or full-length cDNA libraries, we generated 139 007 3′- or 5′-end sequenced ESTs, representing more than one-third of the c. 385 000 publicly available Populus ESTs. Clustering and assembly of 107 519 3′-end ESTs resulted in 14 451 contigs and 20 560 singletons, altogether representing 35 011 putative unique transcripts, or potentially more than three-quarters of the predicted c. 45 000 genes in the poplar genome. Using this EST resource, we developed a cDNA microarray containing 15 496 unique genes, which was utilized to monitor gene expression in poplar leaves in response to herbivory by forest tent caterpillars (Malacosoma disstria). After 24 h of feeding, 1191 genes were classified as up-regulated, compared to only 537 down-regulated. Functional classification of this induced gene set revealed genes with roles in plant defence (e.g. endochitinases, Kunitz protease inhibitors), octadecanoid and ethylene signalling (e.g. lipoxygenase, allene oxide synthase, 1-aminocyclopropane-1-carboxylate oxidase), transport (e.g. ABC proteins, calreticulin), secondary metabolism [e.g. polyphenol oxidase, isoflavone reductase, (–)-germacrene D synthase] and transcriptional regulation [e.g. leucine-rich repeat transmembrane kinase, several transcription factor classes (zinc finger C3H type, AP2/EREBP, WRKY, bHLH)]. This study provides the first genome-scale approach to characterize insect-induced defences in a woody perennial providing a solid platform for functional investigation of plant–insect interactions in poplar.Molecular Ecology 03/2006; 15(5):1275 - 1297. · 5.52 Impact Factor -
Article: Isolation of high-quality RNA from gymnosperm and angiosperm trees.
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ABSTRACT: An improved protocol was developed for efficient and reliable extraction of high-quality total RNA and mRNA from various tissues of spruce (Picea spp.) and poplar (Populus spp.) trees, as well as other plant species. This method was specifically optimized for tissues with high content of polysaccharides, oleoresin terpenoids, and phenolic secondary metabolites, which often co-precipitate with RNA and inhibit subsequent reverse transcription. The improved protocol yielded up to 600 micrograms of total RNA per gram of tissue suitable for standard expressed sequence tags (ESTs), full-length cDNA library construction, and for microarray applications.BioTechniques 06/2004; 36(5):821-4. · 2.67 Impact Factor -
Article: (E)-beta-ocimene and myrcene synthase genes of floral scent biosynthesis in snapdragon: function and expression of three terpene synthase genes of a new terpene synthase subfamily.
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ABSTRACT: Snapdragon flowers emit two monoterpene olefins, myrcene and (E)-beta-ocimene, derived from geranyl diphosphate, in addition to a major phenylpropanoid floral scent component, methylbenzoate. Emission of these monoterpenes is regulated developmentally and follows diurnal rhythms controlled by a circadian clock. Using a functional genomics approach, we have isolated and characterized three closely related cDNAs from a snapdragon petal-specific library that encode two myrcene synthases (ama1e20 and ama0c15) and an (E)-beta-ocimene synthase (ama0a23). Although the two myrcene synthases are almost identical (98%), except for the N-terminal 13 amino acids, and are catalytically active, yielding a single monoterpene product, myrcene, only ama0c15 is expressed at a high level in flowers and contributes to floral myrcene emission. (E)-beta-Ocimene synthase is highly similar to snapdragon myrcene synthases (92% amino acid identity) and produces predominantly (E)-beta-ocimene (97% of total monoterpene olefin product) with small amounts of (Z)-beta-ocimene and myrcene. These newly isolated snapdragon monoterpene synthases, together with Arabidopsis AtTPS14 (At1g61680), define a new subfamily of the terpene synthase (TPS) family designated the Tps-g group. Members of this new Tps-g group lack the RRx(8)W motif, which is a characteristic feature of the Tps-d and Tps-b monoterpene synthases, suggesting that the reaction mechanism of Tps-g monoterpene synthase product formation does not proceed via an RR-dependent isomerization of geranyl diphosphate to 3S-linalyl diphosphate, as shown previously for limonene cyclase. Analyses of tissue-specific, developmental, and rhythmic expression of these monoterpene synthase genes in snapdragon flowers revealed coordinated regulation of phenylpropanoid and isoprenoid scent production.The Plant Cell 06/2003; 15(5):1227-41. · 8.99 Impact Factor
