Barvkar et al. BMC Genomics 2012, 13:175 http://www.biomedcentral.com/1471-2164/13/175 amplicons were analyzed on 1.0% agarose gels and eluted using GenElute gel extraction kit (Sigma-Aldrich, USA) followed by cloning into pGEM-T Easy vector (Promega, USA). Plasmid DNA was isolated using GenElute plasmid extraction kit (Sigma-Aldrich, USA) and sequenced using MegaBACE 500 (GE Healthcare, UK) DNA analysis system. Sequence alignment and phylogenetic analysis The predicted amino acid sequences of the UGT genes were initially aligned using ClustalW with default gap penalties [54]. These alignments were visually inspected for indels and to minimize insertion/deletion events in unalignable regions. Trees were constructed from 409 alignable amino acid positions (60.41%) for all the sequences. Distance as well as Parsimony analyses were performed using MEGA5 [55]. Only the regions of unam- biguous alignments were used in the phylogenetic analyses with Dayhoff substitution matrix (PAM250) and trees were constructed by neighbour-joining algorithm [56] with bootstrapping (1000 replicates). Eighteen Arabidopsis UGT sequences, one from each UGT family and one sesame sequence (UGT94D1) were also included in the analyses (Additional file 9). Intron mapping and organization A flax UGT intron map was constructed by determining the intron splice sites, phases and positions. The introns were serially numbered relative to their positions in the amino acid sequence produced by aligning all the flax UGTs. Intron phases were determined as follows: introns positioned between two codons as phase 0, introns positioned after the first base in the codon as phase 1, and introns positioned after the second base in the codon as phase 2. Detection of orthologs of flax UGTs in four sequenced dicots Blast2Go [57] was used to search the orthologs for flax UGTs in four sequenced dicots, Ricinus communis (Euphorbiaceae), Populus trichocarpa (Salicaceae), Vitis vinifera (Vitaceae) and Arabidopsis thaliana (Brassicaceae), using default parameters except for E value cut off of < e−100. These four dicots were selected based on the genome homologies with flax as reported by Ragupathy et al. [58]. Digital expression analysis The putative UGT coding sequences were BLAST searched against the Linum usitatissimum NCBI-EST dataset (dated: June, 2011; 2,86,895 sequences; http://www.ncbi.nlm.nih. gov/nucest?term=Linum%20usitasimum) to identify tran- scriptional evidence for individual UGT genes and to esti- mate the number of ESTs expressed per tissue type and Page 11 of 13 gene model. These tissue types include flower (FL), globular embryo (GE), heart embryo (HE), torpedo embryo (TE), bent embryo (BE), mature embryo (ME), seed coat at globular stage (GC), seed coat at torpedo stage (TC), pooled endosperm (EN), etiolated seedling (ES), stem (ST), leaf (LE), peeled stem (PS) [59], 12 days DAF bolls and outer fibrous stem tissue. Additionally, microarray expression data for 48,021 flax unigenes (http:// www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE21868) were also used. RMA - normalized, averaged gene-level signal intensity (log2) values for the unigenes exhibiting specified sequence similarity were used from all the biological as well as technical replicates and averaged further. A heat map for digital expression analysis was con- structed with these values using TIGR MultiExperiment Viewer (MeV, http://www.tm4.org/mev.html). Reverse transcription quantitative real time PCR Total RNA from mature leaves (ML), stem (ST), root (RT), etiolated seedling (ES), flower (FL) and seed develop- mental stages (4, 8, 12, 16, 22, 30, 48 DAF) of flax variety NL260 was isolated as described earlier. DNaseI treated total RNA was reverse transcribed using oligo(dT) primer and MultiScribeTM reverse transcriptase (Applied Biosystems, USA). Gene specific primers for 10 glycosyl- transferase genes (Additional file 8) were designed using Primer3 [60]. PCR conditions were optimized for anneal- ing temperature and primer concentration. Primers used for real-time PCR are listed in Additional file 8. Real-time PCR was carried out in 7900HT Fast real-time PCR system (Applied Biosystems, USA) using FastStart universal SYBR green master mix (Roche, USA). Each 10 μL real-time PCR cocktail contained 0.125-0.4 μM concentrations of both forward and reverse gene-specific primers (Additional file 8), 4 μL of 1:16 diluted first strand cDNA, 1× SYBR green master mix and sterile milliQ water to make up the reac- tion volume. Real-time PCR amplification reactions were performed with following conditions: 95°C denaturation for 10 min, followed by 40 cycles of 95°C for 3 s, with primer annealing and extension at 60°C for 30 s. Following amplifi- cation, a melting dissociation curve was generated using a 62–95°C ramp with 0.4°C increment per cycle in order to monitor the specificity of each primer pair. Eukaryotic translation initiation factor 5A (ETIF5A) gene from flax was used as a housekeeping or reference gene for all the real-time PCR reactions [17]. Housekeeping gene was selected after confirming the stability of this gene across all the tissue type used in the study. For each biological replicate, two independent technical replications were performed and averaged for further calculations. PCR conditions were optimized such that PCR efficiencies of housekeeping gene and the gene of interest were similar and closer to 2.0. PCR efficiencies were calculated using LinRegPCR [61]. Relative transcript abundance