Abstract
BackgroundExpression profiling has been proposed as a means for screening non-model organisms in their natural environments to identify genes potentially important in adaptive diversification. Tag profiling using high throughput sequencing is a relatively low cost means of expression profiling with deep coverage. However the extent to which very short cDNA sequences can be effectively used in screening for candidate genes is unclear. Here we investigate this question using an evolutionarily distant as well as a closely related transcriptome for referencing tags. We do this by comparing differentially expressed genes and processes between two closely related allopolyploid species of Pachycladon which have distinct altitudinal preferences in the New Zealand Southern Alps. We validate biological inferences against earlier microarray analyses.ResultsStatistical and gene annotation enrichment analyses of tag profiles identified more differentially expressed genes of potential adaptive significance than previous analyses of array-based expression profiles. These include genes involved in glucosinolate metabolism, flowering time, and response to cold, desiccation, fungi and oxidation. In addition, despite the short length of 20mer tags, we were able to infer patterns of homeologous gene expression for 700 genes in our reference library of 7,128 full-length Pachycladon ESTs. We also demonstrate that there is significant information loss when mapping tags to the non-conspecific reference transcriptome of A. thaliana as opposed to P. fastigiatum ESTs but also describe mapping strategies by which the larger collection of A. thaliana ESTs can be used as a reference.ConclusionWhen coupled with a reference transcriptome generated using RNA-seq, tag sequencing offers a promising approach for screening natural populations and identifying candidate genes of potential adaptive significance. We identify computational issues important for the successful application of tag profiling in a non-model allopolyploid plant species.
Highlights
Expression profiling has been proposed as a means for screening non-model organisms in their natural environments to identify genes potentially important in adaptive diversification
A. thaliana as opposed to P. fastigiatum ESTs and describe mapping strategies by which the larger collection of A. thaliana ESTs can be used as a reference
Screening individuals that are naturally occurring across environmental and altitudinal gradients for differential gene expression is one approach proposed for the preliminary identification of candidate genes important in adaptive diversification and plastic responses [1,2,3]
Summary
Expression profiling has been proposed as a means for screening non-model organisms in their natural environments to identify genes potentially important in adaptive diversification. The extent to which very short cDNA sequences can be effectively used in screening for candidate genes is unclear We investigate this question using an evolutionarily distant as well as a closely related transcriptome for referencing tags. Screening individuals that are naturally occurring across environmental and altitudinal gradients for differential gene expression is one approach proposed for the preliminary identification of candidate genes important in adaptive diversification and plastic responses [1,2,3]. Implementing this approach may involve large numbers of comparisons and requires a cost effective means of expression profiling. Studies have already demonstrated that sequencing tags (typically 20–36 bases of cDNA) produces more robust results and detects more differentially expressed genes than several different microarray platforms, when using a con-specific reference genome/
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