Supplementary File A

Supplementary File A

The design of PCR primers for amplification and sequencing of mitochondrial DNA (mtDNA) can be plagued by several common problems. For instance, primers often co-amplify nuclear pseudogenes along with the intended mitochondrial fragment (Sorenson and Quinn 1998). A second complication in mitochondrial primer design is the presence multiple copies of a genetic region within the same mitochondrial genome, such as occurs after tandem duplication events (e.g., Gibb et al. 2007). Large duplicated regions appear to be particularly common in the mitochondrial genomes of seabird species, including several species that are closely related to Abbott’s boobies (Papasula abbotti) and to frigatebirds (e.g., Abbott et al. 2005; Eda et al. 2010; Morris-Pocock et al. 2010a,b; Rains et al. 2011). Therefore, we took particular care to characterize any potential mitochondrial duplication in Abbott’s boobies, Christmas Island frigatebirds (Fregata andrewsi), and great frigatebirds (F. minor) before designing any primers for final amplification of the mitochondrial cytochrome b gene and the non-coding control region.

In previous work, we found that the mitochondrial genomes of brown (Sula leucogaster), red-footed (S. sula), and blue-footed boobies (S. nebouxii) each possessed large, duplicated regions that included partial copies of cytochrome b and full copies of tRNAThr, tRNAPro, ND6, tRNAGlu and the control region. Full details of this duplication can be found in the original publication (Morris-Pocock et al. 2010b). Briefly, the gene order of the duplication in all three species was ND5 - cytochrome b - tRNAThr (copy 1) - tRNAPro (copy 1) - ND6 (copy 1) - tRNAGlu (copy 1) - Control Region 1 - partial copy of cytochrome b - tRNAThr (copy 2) - tRNAPro (copy 2) - ND6 (copy 2) - tRNAGlu (copy 2) - Control Region 2 – tRNAPhe – 12S (see Figure 1 in Morris-Pocock et al. 2010b). To determine whether similar duplications exist in Abbott’s boobies, Christmas Island frigatebirds, and great frigatebirds, we used followed an approach that is similar to that used by Morris-Pocock et al. (2010b). We briefly outline this approach in four steps, below:

1.  In one individual of each species, we attempted to amplify the region between the two putative copies of the control region using an unconventional primer pairing. The primers were oriented such that they would only form a PCR product if a duplication was present. For Abbott’s boobies we used the primer pair SlMCR-L740/b6, and for both frigatebird species we used the primer pair SlMCR-L980/b6 (see Table S1 for all primer sequences). Each of these amplifications yielded products which were sequenced in both directions using the amplification primers. This sequencing effort revealed the presence of a partially duplicated copy of cytochrome b in all three species. As found in other booby species, the partial cytochrome b copy was located to the 3’ end of the first control region (Figure 1 in Morris-Pocock et al. 2010b).

2.  We took advantage of the fact that the 5’ end of cytochrome b was not duplicated to design PCR primers that only amplified the full (and presumably functional) cytochrome b copy. The primer pairs that we used for this amplification were b3/b6 for Abbott’s boobies and b3-fb/b6 for both frigatebird species. Primers b3 and b6 were previously used in other booby species and our sequencing results from the current study indicated that they would only amplify the full copy of cytochrome b under standard PCR conditions. Due to a mutation in the priming sites of b3 in both frigatebird species, we designed an alternate but analogous primer, b3-fb, for frigatebirds.

3.  Using the sequence obtained in Step 1 as a guide, we PCR amplified and sequenced as much of the area surrounding the duplicated control regions as possible. We accomplished this by using (i) primers that were developed based on the sequence obtained in Step 1, and (ii) primers that we developed in our previous study of booby control region variation (Morris-Pocock et al. 2010b; Table S1). This sequencing effort revealed that Abbott’s boobies, Christmas Island frigatebirds, and great frigatebirds all have the same gene order that was identified previously in brown, red-footed, and blue-footed boobies (Morris-Pocock et al. 2010a). As in our previous work, we found that the two control region copies within the mitochondrial genomes of each species were identical, with the exception of small region (<30 base pairs) at the 5’end of the control region.

4.  We took advantage of sequence differences at the 5’ end of duplicated control regions to design primers for each species that only amplified the second control region copy (CR2). We chose this copy for amplification as it is surrounded the “typical” gene arrangement (i.e., tRNAGlu and tRNAPhe) and because we previously amplified this copy in other sulid species (Morris-Pocock et al. 2010a, Taylor et al. 2011a,b). The primer pairs that we used to amplify CR2 were: SaMCR-L99a/SdMCR-H750 (Abbott’s booby), FmMCR-L123A/FmMCR-H750 (great frigatebirds), and FaMCR-L168/FmMCR-H750 (Christmas Island frigatebirds).

We performed all PCRs in 25 μL reaction volumes [~5 ng DNA, 1x Multiplex Mix (Qiagen), 0.7 mM each forward and reverse primer]. Reaction mixtures were denatured at 95°C for 15min, followed by 35 cycles of 94°C for 30 sec, annealing for 30 sec and 72°C for 30 sec, and a final extension at 72°C for 3min. Annealing temperatures were 50°C for all reactions except for those describe in Step 4, which were 60°C. We sequenced PCR products using forward and reverse primers on a 3730XL DNA Analyzer (Applied Biosystems, Foster City, CA) at the Genome Quebec Innovation Centre (McGill University, Montreal, Quebec).

Literature cited

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