Wang et al. BMC Genomics 2012, 13:529 http://www.biomedcentral.com/1471-2164/13/529 Page 6 of 13 MEAM1 57,741 sequences ASIA II 3 52,535 sequences MED 168,900 sequences Reciprocal best match E<1e-5 20,929 reciprocal best matched sequences 22,415 reciprocal best matched sequences Swissprot BLASTx hit to the same gene 3,308 pairs of orthologous gene 2,819 pairs of orthologous gene 210 pairs of 5’UTR 2,966 pairs of CDS 337 pairs of 3’UTR 102 pairs of 5’UTR 2,529 pairs of CDS 102 pairs of 3’UTR Figure 3 Identification of the orthologous gene pairs between Asia II 3 & MEAM1 and between Asia II 3 & MED. With Megablast, homologous genes were identified using the bidirectional best-hit method. Putatively orthologous gene pairs were further filtered by Blastx against all known proteins in Swissprot database using a threshold of 1×10-5. Coding sequences (CDS) of the orthologous genes were determined based on the annotation of Swissprot Blast hits. After removing the untranslated regions (UTRs), CDS with unexpected codons or shorter than 150 bp were removed. The numbers of gene pairs obtained in each step are shown in the boxes. Left: Asia II 3 & MEAM1; Right: Asia II 3 & MED. between MEAM1 and MED (0.83%) (Table 4) [30]. Gen- ome sequences are very useful for constructing phylo- genetic trees with high resolution and accuracy. Using all the orthologous genes among the three species, a neighbor-joining tree was reconstructed (Figure 4). The phylogenetic distance between MEAM1 and MED is less than that of the invasive whiteflies to the native Asia II 3. This is not surprising as both MEAM1 and MED ori- ginated from the Middle East Asia Minor and Mediter- ranean Basin regions whereas Asia II 3 is a native species in China [14,34]. The sequence divergence between Asia II 3 and MEAM1 To reveal the molecular variation between Asia II 3 and MEAM1, the sequence divergence was analyzed between the orthologous genes of their transcriptomes (Table 5). The average divergence between the 5’UTRs of Asia II 3 and the MEAM1 orthologous genes is 3.35% which is Table 4 Sequence divergence of MEAM1/Asia II 3 and MED/Asia II 3 Total ortholog pairs: Total aligned length (kb): Mean aligned length (bp): Longest aligned length (bp): Mean homology (%): Lowest homology (%): Highest homology (%): Standard deviation: MEAM1/Asia II 3 2,966 1,434.4 483.6 3,081 98.27% 70.43% 100% 0.00024 MED/Asia II 3 2,529 1,072.1 423.9 2,919 98.16% 79.68% 100% 0.00029 almost twice as much as between the same region of MEAM1 and MED. Similar to previous reports, the di- vergence rate at the CpG sites in the 5’UTR (8.28%) is much higher than that of non-CpG sites (3.05%). For the 3’UTR, the overall difference between Asia II 3 and MEAM1 is 2.91%. CpG and non-CpG sites differ at 12.42% and 2.52%, respectively (Table 5). Hence, in the 3’UTR, CpG sites contain 4.93 times of differences com- pared to non-CpG sites. Among the 2,966 orthologous gene pairs, the overall divergence in CDS is 1.73%. In non-CpG sites, the divergence is slightly lower (1.29%), whereas the CpG site divergence (7.77%) is about 6.0 times as high as the non-CpG site divergence (Table 5). Nucleotides in CDS can further be classified as non- degenerative (nd) sites (any nucleotide change cause amino acid replacement) and four fold degenerate (4d) sites (no substitution cause amino acid change). At nd sites the overall divergence is 0.47%, whereas the overall MEAM 1 MED Asia II 3 0.005 Figure 4 Phylogeny of Asia II 3, MEAM1 and MED. Aligned fragments were extracted for phylogenetic analysis using MEGA 5 and the phylogenetic tree was constructed by the neighbor-joining method.