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The following files are available with the online version of this article: the e-values of the best matches for the initial BLASTX searches against bacteria, C. elegans and Drosophila for the WMi dataset (Additional data file 1), together with a mapping file (Additional data file 2) that gives the MI contig number associated with each filename; the best match and e-value for the BLASTX search of the WMj dataset against bacteria (Additional data file 3), C. elegans (Additional data file 4) and Drosophila (Additional data file 5); the e-value for the best match to bacteria, C. elegans and Drosophila resulting from a TBLASTX search for the NMi dataset (Additional data file 6), the NMj dataset (Additional data file 7), the NMh dataset (Additional data file 8), where a value of 100 indicates no match found; a text file with details of the data given in each of these dataset files (Additional data file 9); the alignment in Phylip format used to calculate the NodL tree (Additional data file 10); the alignment in Phylip format of the original sequences, before any manual adjustments were made (Additional data file 11); and a key giving the gi number listed in the alignment for each species (Additional data file 12).
To provide experimental evidence that the Meloidogyne nodL sequences represent nematode loci, we cloned and sequenced a full-length transcript from M. incognita ( Mi-NodL ). Identification of the SL1 trans -splice leader at the 5' end of the message [ 41 ] , and a poly(A) tail at the 3' end, confirmed that this is a bona fide nematode gene (Figure 3).
However, four of these genes do map to the same M. loti linear chromosome (Figure 4), including nodL and the glutamine synthetase gene, both of which are involved in nodulation/nitrogen fixation in rhizobia.
Because the absolute value of the test statistic calculated from the simulated datasets exceeded 0.082 only 62 of 10,000 times, we reject the null hypothesis of identical expected codon usage in the M. incognita and R. leguminosarum nodL genes and conclude that codon usage in these genes is significantly different between the species.
To examine further the relationship between putative nodL candidates found in M. incognita and M. javanica with the cognate genes in rhizobia, we undertook a phylogenetic analysis and found that the two nematode genes fall squarely within the rhizobial nodL clade (Figure 2).
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