A total of 221729 SSRs with a motif length between 1 and 10 bp and minimum three repeats were found in the entire R. solanacearum genome. Of those, 139993 (63.14%) were located on the chromosome (Table 1) and 81736 (36.86%) were located on the megaplasmid (Table 2). This corresponds wel with the size distribution between both replicons (63.96% of all bases are in the chromosome, 36.04% are in the megaplasmid). The SSRs were evenly distributed both over the chromosome as over the megaplasmid (Fig. 1). The total number of repeats is lower than expected by chance; especially the number of mononucleotide repeats is significantly lower than expected (Tables 1 and 2). Trinucleotide repeats occur more than expected by chance alone, both in the chromosome and the megaplasmid (Tables 1 and 2). Mononucleotide repeats of length = 3 bp and dinucleotide repeats are distributed over coding and non-coding regions as expected, both in the chromosome and the megaplasmid. As mononucleotide repeats become larger, there is more and more deviation from the expected distribution; these larger mononucleotide repeats are almost exclusively located in non-coding regions. Our data also show that trinucleotides are overrepresented in protein-coding regions of both replicons (Table 3). The nucleotide composition of the SSR tracts in the R. solanacearum chromosome and megaplasmid are shown in Tables 4 and 5, respectively. Our data show that (i) the G+C composition of mononucleotide repeats in both replicons is significantly lower than the overall composition, but this difference can exclusively be attributed to non-coding regions; (ii) G and C mononucleotide repeats are underrepresented in coding and non-coding regions of both replicons and (iii) CG and GC dinucleotide repeats are vastly overrepresented both in coding and non-coding regions of both replicons, while other dinucleotide repeats are underrepresented.

Dinucleotide relative abundances are shown in Table 6. The dinucleotides TA and AT are strongly underrepresented in both replicons while GC is moderately overrepresented in both replicons. CC and GG are moderately underrepresented in the chromosome. The average absolute dinucleotide relative abundance difference (δ*) between both replicons is 9.78. To assess the variability of dinucleotide relative abundances within a replicon, both replicons were divided into 12 and 7 (for the chromosome and the megaplasmid, respectively) equally-sized, nonoverlapping fragments and ρ*_XY values were calculated for each fragment. δ*(f,g) values within replicons ranged from 6.63 to 31.77 (mean ± standard deviation: 14.49 ± 5.35) (for the chromosome) and from 4.83 to 20.63 (13.11 ± 4.55) (for the megaplasmid). These differences are not significantly smaller than the between-replicon differences (data not shown). Significantly over- or underrepresented tetranucleotides are shown in Table 7. CTAG, AATT, CATG, GATA and TATA are underrepresented in both replicons. GTAG and TTAA are overrepresented in both replicons.

Our data clearly show that the R. solanacearum genome contains numerous SSRs with a motif length between 1 and 10 bp, although not as many as expected by chance alone. Mutations in SSRs are thought to be the result of slipped strand mispairing during DNA replication; slipped strand mispairing can occur because the tertiary structure of SSRs allows mismatching and repeats can be inserted or excised during DNA duplication 414243. The observation of upper limits for SSR length in Escherichia coli suggested that the tendency for repeat length to arise via mutation is counteracted by selection 36. We observed similar upper limits: the upper limit for total length of mononucleotide SSRs is 13 bp and 11 bp for the chromosome and megaplasmid, respectively and, in addition, very few other SSRs with a total length >15 bp (for the chromosome) or >18 bp (for the megaplasmid) are observed. Both strand separation and slippage are more likely for mononculeotide SSRs, explaining why mononucleotide SSRs are more likely to undergo slipped strand mispairing; longer SSRs with a lower repeat number have less opportunity to undergo slipped strand mispairing and there will be less mutability in their repeat number 36. This may explain why larger mononucleotide SSRs are overrepresented in non-coding regions of the R. solanacearum genome as selection has ample opportunity to operate against these larger repeats that cause frameshift and nonsense mutations in coding regions. This hypothesis is supported by the fact that poly(A) and poly(T) SSRs are overrepresented, especially in the non-coding regions, in both replicons (Tables 4 and 5): strand separation for these poly(A) and poly(T) tracts is considerably easier than for poly(G) or poly(C) tracts, increasing the possibility of slipped strand mispairing.

The dinucleotide TA is underrepresented in both replicons. TA is underrepresented in almost all prokaryotic genomes; this could be due to the fact that (i) TA forms the thermodynamically least stable DNA (allowing unwinding of the helix), (ii) RNases preferentially degrade UA dinucleotides in mRNA, and/or (iii) TA is part of many regulatory sequences 38. AT is significantly underrepresented in the R. solanacearum genome but is overrepresented in the genome of most α-Proteobacteria and in the genomes of the β-proteobacterial species R. eutropha and Bordetella pertussis 39. CC and GG are slightly underrepresented in the chromosome but not in the megaplasmid, although the differences in relative abundances are small (Table 6). The dincucleotide GC is overrepresented in both replicons; this is also the case in most other β-Proteobacteria and γ-Proteobacteria 39. In general, within species δ*(f,g)-differences among nonoverlapping 50 kb contigs of bacteria are in the range 18–43 39 and genome signatures of chromosomes and plasmids from the same host are at least weakly similar to each other [δ*(f,g) < 115] 4445. δ*(f,g) values reported for the multiple chromosomes of A. tumefaciens, Deinococcus radiodurans, V. cholerae and B. melitensis were between 27.0 and 30.8 45. A comparison of both R. solanacearum replicons based on dinucleotide relative abundances indicates that they are very similar with δ*(f,g) = 9.78. A comparison of δ*(f,g) values within and between replicons revealed that the variability in δ*(f,g) within a replicon is not significantly smaller than the difference in δ*(f,g) between both replicons. CTAG is significantly underrepresented in the R. solanacearum genome as it is in most proteobacterial organisms. Possible reasons for the underrepresentation of this tetranucleotide include structural defects or special functional roles associated with CTAG 38. AATT, CATG, GATA and TATA are underrepresented in both replicons while GTAG and TTAA are overrepresented. ATTG, CATC and TTGG occur slightly less than expected in the megaplasmid but their relative abundance in the chromosome is in the normal range. The general mechanisms underlying tetranucleotide extremes are unclear but besides the above-mentioned structural defects or functional roles associated with specific tetranucleotides, it has been suggested that restriction avoidance may play an important role in the maintenance of tetranucleotide extremes 39).

The sequences of the chromosome (AL646052) and the megaplasmid (AL646053) of R. solanacearum strain GMI1000 were downloaded from the GenBank database.

We used the software developed by Gur-Arie et al. 36 to screen the entire genome of R. solanacearum for SSRs withg a motif length between 1 and 10 bp and a minimal number of three repeats. This software can be downloaded from ftp://ftp.technion.ac.il/supported/biotech/ssr.exe and reports motif, motif length, repeat number and genomic location of all SSRs. To determine whether the observed SSR frequencies of a given motif length and repeat number occurred as expected by chance, they were compared with the mean frequencies observed in three randomly shuffled genomes. Randomised sequences were generated with shuffleseq (part of the EMBOSS package, http://www.hgmp.mrc.ac.uk/software/EMBOSS). Statistical significance was tested with two-tailed t-tests using SPSS 11.0.1 (SPSS). To determine the distribution of SSRs between coding and non-coding regions of the genome, all coding regions were extracted from the sequence using Artemis 4.0 46 and parsed into a new sequence file using seqret (EMBOSS).

We determined the compositional bias in di- and tetra-nucleotides in the chromosome and megaplasmid of R. solanacearum GMI1000. Both sequences were concatenated with their inverted complementary sequence using revseq, yank and union (EMBOSS). Mononucleotide frequencies were calculated using Artemis 4.0 46, di-, tri- and tetra-nucleotide frequencies were calculated using compseq (EMBOSS). Dinucleotide relative abundances ρ*_XY were calculated using the equation ρ*_XY = f_XY/f_Xf_Ywhere f_XY denotes the frequency of dinucleotide XY and f_X and f_Y denote the frequencies of X and Y, respectively 38. Similarly, the corresponding fourth-order oligonucleotide measures (which factor out all lower-order biases) is given by τ*_XYZW = (f* _XYZWf* _XYf* _XNZf* _XN1N2Wf* _YZf* _YNWf* _ZW)/(f* _XYZf* _XYNWf* _YZWf* _Xf* _Yf* _Zf* _W) were N is any nucleotide and X, Y, Z and W are each one of A, C, G and T 38. Statistical theory and data from previous studies 3839 indicate that the normal range of ρ*_XY, is between 0.78 and 1.23. In this study we used the refined criteria of discrimination proposed by Karlin et al. 38. Overrepresentation is indicated by + (1.23 = ρ* < 1.30), ++ (1.30 = ρ*< 1.50) and +++ (ρ* ≥ 1.50), while underrepresentation is indicated by - (0.70 < ρ* = 0.78), -- (0.50 < ρ* = 0.70) and --- (ρ* = 0.50). The dissimilarities in relative abundance of dinucleotides between both sequences were calculated using the equation described by Karlin et al. 38: δ*(f,g) = 1/16Σ |ρ*_XY(f)-ρ*_XY(g)| (multiplied by 1000 for convenience), were the sum extends over all dinucleotides. To assess the variability of dinucleotide relative abundances within a replicon, both replicons were divided into 12 and 7 (for the chromosome and the megaplasmid, respectively) non-overlapping fragments and ρ*_XY values were calculated for each fragment. The average δ*(f,g) within each replicon was also calculated.