By contrast, the phylogeny based on intron 2 sequence shows that the β-like globin genes of all eulipotyphlan species examined - together with the HBD genes of human, rabbit, and armadillo - form a well-supported monophyletic group with high bootstrap support.
Starting with the genomic amplicons, the sequenced clones were almost equally divided between two clusters (arbitrarily labeled A and B on Figure 4) based on intron length, intergenic spacer size, and sequence differences in both non-coding and coding regions (Figure 3, 4, 5).
In comparison, the success rate of intron-primed exon-crossing (based on salmonid intron sequences) was considerably higher: a single clear PCR product was obtained for 21 of 22 loci (95%) and high-quality sequences were obtained for 17 of these (77%) – i.e., a success rate almost four times higher than that obtained using the EPIC approach (χ2 = 7.771, d.f. = 1, P = 0.005).
The tree based on LdhA intron sequences (Additional file 6a) is similar to the full-sequence tree but relationships within the cluster of alleles from NA D. pulicaria, which also contains alleles from all the other species, are not well resolved.
Here we present phylogenetic evidence of a vertical inheritance pattern of the Nae.S516 intron in Naegleria that includes each of the domains NaGIR1, NaGIR2, and NaHEG, and corroborates a previous study based on 5 intron sequences [ 6].
Our mitochondrial trees did not return the Metaves clade that had been proposed based on one nuclear intron sequence.
We also conducted separate phylogenetic reconstructions based on coding sequence, intron 2 sequence, and the 5′ and 3′-flanking sequences of each gene.
Polymerase chain reaction primer pairs for mRNA quantification, intending to flank at least one intron, were designed based on coding sequences obtained from the GenBank Sequence Database (http://www.ncbi.nlm.nih.gov/Genbank/index.html) (Table 1>).
