RAPID ASSESSMENT OF SINGLE-COPY NUCLEAR-DNA VARIATION IN DIVERSE SPECIES

We investigated the use of PCR primers designed to conserved exons wit hin nuclear DNA to amplify potentially variable regions such as intron s or hypervariable exons from a wide range of species. We then explore d 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 amplification s in diverse mammals (DQA), and in birds, reptiles and mammals (aldola se, H2AF, myoglobin). The sequenced PCR products generally, but not al ways, confirmed that the correct locus had been amplified. Several pri mer sets produced smaller size fragments consistent with preferential amplification of intronless pseudogenes; this was confirmed by sequenc ing seal and reptile H2AF PCR products. Digestion with randomly select ed 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 m ore efficient to sequence a small number of distantly related individu als (e.g. one per geographic population) and from these data to identi fy informative or potentially informative restriction enzymes for 'tar geted' digestion. We conclude that for studies of population-level var iation, the optimal approach is to use a battery of primers for initia l PCR of both mtDNA and scnDNA loci, select those that give clean ampl ifications, and sequence one sample from each population to (i) confir m gene identity, (ii) estimate the amount of variation and, (iii) sear ch for diagnostic restriction sites. This will allow determination of the most efficient approach for a large-scale study.