Inter-simple sequence repeat-restriction fragment length polymorphisms for DNA fingerprinting.

As part of this laboratory’s honeybee genetics and breeding program, DNA markers are needed for uses such as the identification of populations and species and marker-assisted breeding. In particular, DNA markers are needed to discriminate Varroa-mite-resistant Russian honey bees, imported from fareastern Russia, and their offspring (1), from the other honey bees found in the USA. I tried RAPDs, but they were often unrepeatable. AFLPs require an undesirably large amount of preparation effort for each sample. Microsatellites have been useful for other studies in honey bees (2,3), but for this purpose there is inadequate variation with the primer pairs available. Microsatelliteprimed PCR involves the amplification of DNA using a single primer composed of a microsatellite sequence (simple sequence repeat; SSR) with 1–3 selective, often degenerate, nucleotides at the end. The amplified fragments are termed inter-simple sequence repeats (4). The first report of ISSRs by Zietkiewicz et al. (5) discussed several mammals, other vertebrates, plants, and E. coli. Since then, ISSRs have been reported mostly from studies in plants, particularly where inadequate variation or reproducibility were available with other methods [e.g., primer pairs in Ipomoea (4) and in Fragaria linkage analysis (6), cocoa germplasm characterization (7), intersimple sequence repeat (ISSR) inheritance in citrus (8), and classifying rice germplasm (9)]. However, ISSRs have also been used to study variation in aphids, a mosquito, and a rotifer (10); a rice pest insect (11); the silkworm (12); and corals (13). I also successfully amplified ISSR fragments from Varroa mites and Hawaiian Drosophila (data not reported). This indicates that ISSRs and ISSR-RFLPs should be detectable in any organism where DNA markers can be detected. Here I describe a new method to increase the amount of detectable DNA variation by digesting ISSRs with restriction enzymes to produce ISSR-RFLPs. The digested amplification products are detected using ethidium bromide after electrophoresis. This is a novel combination of procedures and is an improvement over just ISSRs because restriction enzyme digestion of ISSR fragments allows the detection of significant amounts of additional variation using a more repeatable and reproducible procedure. This would be particularly useful in cases where inadequate amounts of detectable genetic variation are available using ISSRs or other methods or where more variation is desired from a limited number of ISSR primers. Detection of RFLPs in PCR-amplified DNA using ethidium bromide eliminates the requirements for Southern transfers, unique probes for detection, and labeled probes or primers. This simplifies detection and reduces costs. Genomic DNA from the thorax of a single worker honey bee, Apis mellifera L., from a colony, with each colony from a different source, was extracted with 400 μL 10% Chelex® resin (14). SSR primers were purchased from the Oligonucleotide Synthesis Laboratory, NAPS Unit, University of British Columbia (Vancouver, BC, Canada). For initial evaluations of primers and DNA templates, the PCR procedure of Huang and Sun (4) was followed, but unlabeled primers were used and PCR products were detected by ethidium bromide staining after electrophoresis. Amplification was carried out in a total volume of 5 μL containing 0.35 μL Chelex extract template DNA, 0.21 μM primer, 0.2 mM dNTPs, 1.5 mM MgCl2, 0.5 U Taq DNA polymerase Benchmarks