Those sequences that did not produce a significant hit with the nr database were compared to the PFAM database for annotation. The latter comprises a large collection of multiple sequence alignments and hidden Markov mod els covering many common protein domains. Signif icant BLAST results against TAIR database were used for functional gene ontology annotation. Transcriptome comparison, selleck chemical A. tuberculatus vs. A. hypochondriacus The raw sequence files derived from the recently reported A. tuberculatus transcriptome pyrosequencing effort were downloaded directly from the NCBI Sequence Read Archive at Traces sra sra. cgi study SRP002251. Reads were assembled after quality control, following an identical Tran script annotation for A. tuberculatus was performed by querying the UniRef 100, and Amaranthaceae ESTs databases.
Both transcriptomes were then aligned with each other using BLASTN to identify homologous con tigs. Sequence homology was defined only at E values 1 �� 10 10 and identity 90%. Homologous transcripts were quantified and classified into five different cate gories, i. e. those, i producing the same hit, ii different hits, iii and iv one hit for one species and no hit for the other, and vice versa, or v no hit, when queried against the above databases. Annotated transcripts detected only in A. hypochondriacus or A. tuberculatus were also quantified. Digital expression analysis The number of reads per gene was counted in each of the 454 sequencing outputs derived from the salt stress, water stress, insect herbivory and bacterial infection treatments and also from stem tissue.
Genes having read counts lower than 5 were eliminated. To calculate relative expression profiles in each stress treatment, Rela tive Abundance values were computed for each gene per treatment sample by dividing its 454 sequence count by the total 454 sequence count in the treatment sample. Differentially expressed genes in one or more treatments were detected by using the R and c2 test statistics using a freely available web tool. A gene was considered to be differentially expressed when at least one statistical test yielded significance values 0. 0001. A similar procedure was employed to identify transcripts that were stem speci fic or highly abundant in this tissue.
The following considerations were adopted for the organization of the digital stress related gene expression data, i a minimum or baseline control expression value for a given Anacetrapib gene was assigned to the lowest RA in the four treatment set examined. The RAs that produced an expression ratio 2 when divided by MIN were also considered as MINs, ii a gene was considered to be sig nificantly expressed by a given treatment when its RA yielded a ratio 2 when divided by MIN, and iii maximum expression levels for a given gene were assigned to the treatment having the highest SE. Treat ments were reported to produce additional MEs when their respective SEs yielded a ratio 2 when divided by ME.