Barvkar et al. BMC Genomics 2012, 13:175 http://www.biomedcentral.com/1471-2164/13/175 patterns analysed using RT-qPCR very well correlated with the digital expression analysis. The frequency of ESTs per UGT gene ranged from 1–54 among the UGTs, suggesting varied expression levels. Among the different tissue types, seed and stem tissues showed the highest number of expressed UGTs. It is known that flax seeds and stem contain a large number of secondary metabolites and hence could explain the abundance of UGTs in these tissues [48,49]. However, this could also be due to a large number of EST libraries avail- able for these tissue types (seed: 9 EST libraries, 2,20,724 ESTs and stem: 3 EST libraries, 32,184 ESTs). This study also identified two genes, LuUGT85Q2 and LuUGT74S1, belonging to groups G and L respectively, which showed high expression in flower and seed coat from the torpedo stage. The members of these groups are predicted to glycosylate terpenoids, flavanoids and benzoates classes [37]; and hence, they can be considered as potential targets for screening against these predicted classes to identify their substrates. Compared to the sequence based expression analysis method, microarray provides a high-throughput tool for simultaneous analysis of expression at the whole transcriptome level. As per the microarray data, 44% flax UGTs showed expression evidence in various tissue types (Figure 3). Three genes from seed stage and one gene from leaf showed high expression, suggesting possible involvement of these genes in seed and leaf secondary metabolite glycosylation. Microarray data from two contrasting flax varieties, Drakkar and Belinka were also analyzed. Drakkar produces better quality fibres than Belinka, and is more resistant to the fungal pathogen Fusarium [14]. However, we could not detect any UGT having variety specific expression pattern. Although, plant UGTs have been reported to be involved in defence mechanism [50], the available microarray data were not generated by exposing the varieties to any pathogen. The difference in expression of the UGTs between the EST and microarray datasets might have resulted from the dif- ferences in the number of tissue types, size of each dataset and varieties used for data generation. The EST dataset was larger compared to the microarray dataset, therefore we might have obtained expression evidence for more genes using the EST dataset. Moreover, the long sequence reads of ESTs provide fairly unambiguous evidence of gene ex- pression, compared with the hybridization based micro- array data and hence EST profiling could be considered as a more reliable method for transcriptomic analysis as also suggested by Geisler-Lee et al. [13] and Moreau et al. [51]. Regarding the 37 unexpressed flax UGTs, it is possible that some or most of these genes may express at very low levels in particular tissue type or express only under specific conditions such as biotic or abiotic stresses. Hence, they might have not been represented in the EST Page 10 of 13 and microarray data as the data were generated from unchallenged libraries. Even in the large Arabidopsis EST collection gathered over several years, only 64.5% of the genes had corresponding ESTs [52]. Absence of an EST for a corresponding gene implies that it is either inactive or expressed at undetectable level in the tissues sampled or that it is a non-functional gene per se. Conclusions We identified a large number of UGT genes in the Linum usitatissimum genome. These genes were clustered into 14 distinct evolutionary groups based on the phylogenetic analysis. Two new UGT family members not observed in Arabidopsis were identified in the flax genome. Most of the identified genes were expressed in various tissue types and seven of them were flax diverged. Results of the digital expression analysis were confirmed by RT-qPCR. Two conserved introns were observed, indicating evolu- tion of flax UGTs from two lineages. The phylogenetic tree can be useful for understanding the structure- function relatedness of the UGT family members and might further facilitate their functional analysis. Methods Probing the flax genome for UGT genes The presently available draft genome sequence of flax (http://linum.ca) represents 85% genome coverage, which is derived from the low-copy fraction of the genome. This coverage is consistent with the length of the entire low-copy fraction previously estimated by reassociation kinetics [53]. We used the predicted protein database available at http://linum.ca to identify flax UGT genes. The 44 amino acid conserved sequence of the PSPG box that characterizes plant UGTs was used as a query against the 47,912 predicted flax gene models. The resulting scaffolds were analyzed to identify the genes, ORFs, intron positions and sizes using the GBrowse tool available on the same website. PCR amplification, cloning and sequencing Genomic DNA from a flax variety, NL260, was extracted using CTAB method. Total RNA from developing seeds was extracted using Spectrum Plant Total RNA kit (Sigma-Aldrich, USA) and treated with DNaseI (Promega, USA), followed by first strand cDNA synthesis using AMV Reverse Transcriptase (Promega, USA). To confirm the reading frames, primers were designed to amplify full length genes including the start and stop codons (Additional file 8). For intron-less genes, 50 ng genomic DNA, and for intron containing genes, 1.5 μl pooled cDNA from developing seeds was used as template for PCR amplification using AccuPrimeTM Pfx DNA Polymerase (Invitrogen, USA). PCR was performed using the annealing temperatures mentioned in Additional file 8. The PCR