Rapid assessment of single-copy nuclear DNA variation in diverse species

We investigated the use of PCR primers designed to conserved exons within nuclear DNA to amplify potentially variable regions such as introns or hypervariable exons from a wide range of species. We then explored various approaches to assay population-level variation in these PCR products. Primers designed to amplify regions within the histone H2AF, myoglobin , MHC DQA , and aldolase (ALD) genes gave clean amplifications in diverse mammals (DQA) , and in birds, reptiles and mammals ( aldolase, H2AF, myoglobin ). The sequenced PCR products generally, but not always, confirmed that the correct locus had been amplified. Several primer sets produced smaller size fragments consistent with preferential amplification of intronless pseudogenes; this was confirmed by sequencing seal and reptile H2AF PCR products. Digestion with randomly selected four-base recognizing enzymes detected variation in some cases but not in others. In species/gene combinations with either low (e.g. seal H2AF, ALD-A ) or high (e.g. skink ALD-1 ) nucleotide diversity it was more efficient to sequence a small number of distantly related individuals (e.g. one per geographic population) and from these data to identify informative or potentially informative restriction enzymes for 'targeted' digestion. We conclude that for studies of population-level variation, the optimal approach is to use a battery of primers for initial PCR of both mtDNA and scnDNA loci, select those that give clean amplifications, and sequence one sample from each population to (i) confirm gene identity, (ii) estimate the amount of variation and, (iii) search for diagnostic restriction sites. This will allow determination of the most efficient approach for a large-scale study.