PCR-RFLP analysis of introns of nuclear genes in Populus and Prunus

Abstract

Many PCR primer sequences are available in the literature that amplify genes, introns and spacers from plant chloroplast and mitochondrial DNA, which are reasonably conserved over evolutionary distances. Similar attempts with nuclear DNA are hampered by greater diversity in exon sequences, the presence of gene families and pseudogenes, and often a lack of suitable sequences in the public data banks. However, within a species or a genus, using published sequences to design primers that amplify presumably polymorphic introns is more straightforward. It is often possible to find primers that amplify gene fragments from a number of angiosperm taxa, although specific PCR approaches are needed in many situations. These include using polymerases with different characteristics, and specific temperature profiles and optimization strategies. Primer sequences based on sequence data from even a broad range of genera may still fail to amplify the desired fragment from specific species, genera, or families — a finding in accordance with DNA sequence evolution theory. Amplified fragments can be analysed by restriction digestion followed by agarose electrophoresis as a simple but often effective way of detecting polymorphisms. Examples are given from such approaches including peroxidases in Populus sp., specific and broad-range primers for phenylalanine ammonia lyase (PAL) in both Populus and Prunus, the hypervariable S-RNAases in Prunus avium, and cinnamoyl alcohol dehydrogenase (CAD) in a number of angiosperm taxa. In Populus, using these primers allowed us to detect introgression of genes from hybrid clones into the native Populus nigra genepool, while in Prunus avium, we expect to gain insight into the peculiar mating system of this species.