Methods

The general approach to PCR mapping is simple. Two sets of primers are needed to detect differences in gene order. The first primer set consists of the "anchored" primers, which are designed within genes close to, but not moved by, a putative inversion. The second primer set consists of the "inversion" primers, designed within genes moved by the putative inversion, and as close to the inversion boundaries as possible. Figure 6.3 illustrates these primer locations. To test for gene order, each anchored primer is combined with each inversion primer. The combination of working and failing PCR reactions indicate which gene order is present. For example, in Figure 6.3, if the gene order is A1, I1, I2, A2, then the primer combinations A1 + I1 and A2 + I2 would be positive, whereas the combinations A1 + I2 and A2 + I1 would be negative. This approach has been used successfully to examine the distribution of a large inversion in moss plastid genomes (Sugiura et al., 2003).

To apply PCR mapping to the IR of fern plastid genomes we designed two sets of primers as above. The anchored primers were located in rpl2, rpl32, chlL, and chlB (Figure 6.4). These genes are outside the IR and also outside the putative region that has been reorganized. Inversion primers were designed within rrn5, rps7, rrn16, and psbA (Figure 6.4). These primers can be used to determine whether a taxon has the gene order of either Adiantum, or Angiopteris, or a structure resulting from only one of the two inferred inversions. To compensate for changes in gene orientation, both forward and reverse inversion primers were designed.

We attempted to sample a representative from each major lineage of the lep-tosporangiate ferns. We included a member of the filmy ferns (Trichomanes), not included by Raubeson and Stein (1995). We determined gene order in the IR region for Angiopteris, Osmunda, Trichomanes, Gleichenia, Lygodium, Marsilea, Dickso-nia, Pteridium, and Adiantum.

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