Joly et al. BMC Genomics 2010, 11:422 http://www.biomedcentral.com/1471-2164/11/422 dS rates across all possible pairwise sequence compari- sons within each of the 369 HGGs using the ML method. The distribution of omega ratios (ω) was skewed towards extreme low values for non-secreted HGGs, with almost 75% of highest pairwise estimates of ω < 0.2 and only 5% > 0.8 (Figure 5). This distribution was quite different for secreted HGGs, with proportions around 40% and 20% of highest pairwise estimates of ω < 0.2 and > 0.8, respectively. For 13 HGGs, the dN value was significantly greater than dS (ω = dN/dS > 1.2) in at least one pairwise comparison (Table 3). Ten of these groups under positive selection were secreted HGGs (corresponding to 12% of the total secreted HGGs). Using a different approach based on Fixed-Effect Likeli- hood (FEL) statistics, Feau and colleagues detected posi- tive selection in seven gene groups (including five putative secretome members) from a similar dataset [68]. Two of these seven gene groups corresponded to secreted HGGs unravelled using the dN/dS approach described here (92 and 6067, corresponding to MEL_49 and MEL_55, respectively, in [68]). Interestingly, eight positively selected secreted HGGs were found to encode cysteine-rich proteins with an even number of Cys resi- dues that may be involved in disulfide bonds. An even number of Cys residues is generally indicative of the Page 9 of 16 presence of disulfide bonds, which are formed between the thiol groups of cysteines. Disulfide bonds play an important role in the folding and stability of some pro- teins, usually proteins secreted to the extracellular med- ium, and are typical features of a subset of fungal and oomycete effectors, especially those acting in the plant apoplast [11,27]. To identify additional HGGs under positive selection and detect the amino acid residues that are under posi- tive selection, we contrasted the M2A/M1A, M8/M7 and M8/M8A models with likelihood ratio tests (Addi- tional Files 5 and 6; see Methods) [64,66]. Significant evidence of positive selection was found in 4 (including 2 secreted HGGs) of the 369 HGGs. Selective pressures had previously been identified for one of these secre- tome members using FEL statistics and a population genetics approach [68,69]. This particular secreted HGG has homology with HESP-417 from M. lini, a gene known to be expressed in haustoria and encoding a secreted protein with an even number of Cys residues [26]. We assessed the position of positively selected HGGs on above SimiTri plots (indicated by arrows, Figure 4): only one non-secreted HGG (1278) had homologues in the three databases, two secreted HGGs (747 and 6067) Figure 5 Elevated dN/dS ratios among Melampsora homologous gene groups (HGGs) encoding secreted proteins. The rates of non- synonymous nucleotide substitutions per non-synonymous site (dN) and the rates of synonymous nucleotide substitutions per synonymous site (dS) were calculated across all possible pairwise comparisons within each of the 369 HGGs using the maximum likelihood method implemented in the codeml program (runmode = -2) in the PAML 4 software package. The distribution of highest calculated ω (dN/dS ratios) among all pairwise comparisons within each HGG is shown. In cases where dN > 0 and dS = 0 (i.e. ω = ∞), the second higher ratio was selected.