Transcriptome sequencing and microarray design for functional genomics in the extremophile Arabidopsis relative Thellungiella salsuginea (Eutrema salsugineum)

Abstract

BackgroundMost molecular studies of plant stress tolerance have been performed with Arabidopsis thaliana, although it is not particularly stress tolerant and may lack protective mechanisms required to survive extreme environmental conditions. Thellungiella salsuginea has attracted interest as an alternative plant model species with high tolerance of various abiotic stresses. While the T. salsuginea genome has recently been sequenced, its annotation is still incomplete and transcriptomic information is scarce. In addition, functional genomics investigations in this species are severely hampered by a lack of affordable tools for genome-wide gene expression studies.ResultsHere, we report the results of Thellungiella de novo transcriptome assembly and annotation based on 454 pyrosequencing and development and validation of a T. salsuginea microarray. ESTs were generated from a non-normalized and a normalized library synthesized from RNA pooled from samples covering different tissues and abiotic stress conditions. Both libraries yielded partially unique sequences, indicating their necessity to obtain comprehensive transcriptome coverage. More than 1 million sequence reads were assembled into 42,810 unigenes, approximately 50% of which could be functionally annotated. These unigenes were compared to all available Thellungiella genome sequence information. In addition, the groups of Late Embryogenesis Abundant (LEA) proteins, Mitogen Activated Protein (MAP) kinases and protein phosphatases were annotated in detail. We also predicted the target genes for 384 putative miRNAs. From the sequence information, we constructed a 44 k Agilent oligonucleotide microarray. Comparison of same-species and cross-species hybridization results showed superior performance of the newly designed array for T. salsuginea samples. The developed microarrays were used to investigate transcriptional responses of T. salsuginea and Arabidopsis during cold acclimation using the MapMan software.ConclusionsThis study provides the first comprehensive transcriptome information for the extremophile Arabidopsis relative T. salsuginea. The data constitute a more than three-fold increase in the number of publicly available unigene sequences and will greatly facilitate genome annotation. In addition, we have designed and validated the first genome-wide microarray for T. salsuginea, which will be commercially available. Together with the publicly available MapMan software this will become an important tool for functional genomics of plant stress tolerance.

Dark period transcriptomic and metabolic profiling of two diverse Eutrema salsugineum accessions

“…More than 80% of the expressed genes identified in this study were detected in both SH and YK (Table 2). This indicates a conserved transcriptome between these two accessions and is consistent with previous E. salsugineum transcriptome comparisons (Champigny et al, 2013; Lee et al, 2013). PAVs, as scored by read count in the transcript profiling experiment, accounted for 19 and 12% of all genes in this experiment and Champigny et al (2013), respectively.…”

Identification of genes involved in the drought adaptation and recovery in Portulaca oleracea by differential display

“…This is not surprising given that in species like A. thaliana which genome has been completed sequenced, about the 30e34% of its genome is composed by 'unknown' genes, based on different GO aspects (Lamesch et al, 2012). In addition, comparisons of the functional categories with the most differentially expressed genes in other extremophiles subjected to water deprivation; revealed that a large proportion of the Thellungiella salsuginea (Eutrema salsugineum) (48.9%; Lee et al, 2013) and Populus euphratica (20.8%; Tang et al, 2013) unigenes corresponded to genes without a clear classification or unknown genes. Similarly, in the Physcomitrella patens, a moss that highly tolerates desiccation, a 29.6% of the differentially proteins during dehydration were classified under the unclear on unknown function category (Wang et al, 2009).…”

Extremophyte adaptations to salt and water deficit stress

“…Metabolic profiling of the Yukon accession indicates both pre-adaptation to salt stress and a greater metabolic response to salt compared with Arabidopsis. To complement the metabolic profiling, the transcriptome response of the Yukon accession to salt stress is analysed using a 44k Eutrema salsugineum Agilent microarray platform developed by the Hincha group (Lee et al 2013). The results indicate that the numbers of genes that respond to salt in the Yukon accession are similar to Arabidopsis but occur at a higher salt concentration.…”