Barvkar et al. BMC Genomics 2012, 13:175 http://www.biomedcentral.com/1471-2164/13/175 present analysis of flax UGT genes might help to narrow down the substrate choice of a specific gene. Detection of orthologs and functional divergence of unique flax UGTs Detection of orthologs is critically important for accurate functional annotation and has been widely used to facili- tate the studies on comparative and evolutionary genomics [39]. Several methods such as the BlastP [40], inparanoid [41] and reciprocal smallest distance [42] have been reported to detect orthologs. In the present study, we used BlastP to identify the orthologs for flax UGTs from four sequenced dicots (Ricinus communis, Populus trichocarpa, Vitis vinifera and Arabidopsis thaliana). Of the 137 flax UGTs, 130 UGTs had orthologs from the four dicots and seven flax-diverged UGTs were detected. Based on the microarray and EST data, 95 of these 130 orthologs (73%) showed expression evidence; while, five of the seven flax diverged UGTs revealed expression evidence, suggesting their functional divergence. Thus, the flax diverged UGTs, with significantly different primary sequences than those of other surveyed dicots, might have evolved inde- pendently since the last common ancestor between flax and these dicots. As the number of flax diverged UGTs identified in our analysis is small, other methods such as inparanoid search need to be conducted to identify more flax diverged UGTs that the present analysis might have missed. However, we could not perform this analysis, as the flax scaffold sequences are not yet publicly available for conducting the inparanoid search. Intron mapping to understand the evolution of UGT family To understand the evolution of a gene family within phylogenetic groups, introns, more specifically their position, phase, loss and gain, can serve as an important tool [43]. Therefore, we conducted intron mapping in the 137 flax UGTs among which 40.14% sequences were intron less. This percentage is less than that observed in Arabidopsis, wherein >50% genes were intron less [12]. In flax UGTs, a total of seven intron positions were identified with the number of introns per family in the range of one to four. Most families showed the presence of conserved introns 3 (53.65%) and 4 (32.92%), which could probably be considered as the oldest among the seven introns iden- tified. Intron 3 was present in almost all members of the groups F-J and N; while intron 4 was dominant in groups L and K. Interestingly, in these groups wherever intron 3 was present, intron 4 was absent and vice versa except in case of LuUGT709E3, where both the introns were present; while in case of LuUGT87J2, both were absent. In other groups, the introns 3 and 4 were absent in some members of groups A, D, M and E. This suggests that either of these introns was gained prior to diversification Page 9 of 13 of flax UGTs. This is also supported by the observation that most of the conserved introns were in the same phase. It is a commonly held view that the majority of conserved introns are ancient elements and their phases usually remain unchanged [44]. In fact, it has been further suggested that the intron sliding or shifts of intron-exon boundary over a few nucleotides causing change of intron phase are rare events and introns retain their phase for a long evolutionary time [45]. Furthermore, the introns other than the conserved introns were found only within a single restricted group of closely related sequences or in only a single gene, suggesting a general pattern of intron gain during evolution of the flax UGT gene family. A clear case of loss of a conserved intron and gain of intron 5 was seen in the subfamily of closely related genes LuUGTB17-LuUGTB19 from group A. Similarly, in case of LuUGT73B12 and LuUGT73B13, loss of con- served introns and gain of intron 2 was also observed. Thus, analysis of the evolution of the flax UGT multigene family provides evidence for both intron gain and loss and thereby strongly supports the “intron-late” theory of intron evolution [46]. Expressed flax UGTs: identified by digital expression analysis and supported by RT-qPCR Functional divergence among duplicated genes is one of the most important sources of evolutionary innovation in complex organisms. Interestingly, among the 22 duplicated genes, five pairs of genes LuUGT94G3 and LuUGT94G4, LuUGT73B12 and LuUGT73B13, LuUGT712B1 and LuUGT712B5, LuUGT86A8 and LuUGT86A9 and LuUGT74S5 and LuUGT74S6, showed evidence of differential expression. For example, LuUGT74S5 showed seed coat specific expression, while its duplicated counterpart, LuUGT74S6, remained unex- pressed. Evidence for differential expression was also provided by the duplicated gene pair LuUGT86A8 and LuUGT86A9. This suggests that after duplication, the genes acquired either differential or tissue specific expression patterns. In an earlier study, Haberer et al. [47] estimated that about two thirds of duplicate gene pairs had divergent expression in Arabidopsis. To predict and understand the roles of these UGT genes in various tissue types, gene expression pattern analysis is very helpful to infer which gene family members are expected to perform distinct or similar roles. With this aim, we performed expression analysis of flax UGTs using EST libraries, microarray data and RT-qPCR. About 62% flax UGTs showed expression evidence based on the EST data and one or more ESTs were detected per tissue type, providing strong evidence that most of the flax UGT genes were expressed in varied tissue types. The expression