Pitfalls in determining size homoplasy of microsatellite loci

Size homoplasy of microsatellite loci can be detected by single-strand conformation polymorphism analysis (SSCP) or sequence analysis. The present note demonstrates pitfalls of sequencing microsatellite loci. A complex chloroplast microsatellite locus was polymerase chain reaction (PCR) amplified from 33 Abies alba individuals, and the products cloned and sequenced. After 2-7 clones per individual had been sequenced and aligned, differences in repeat numbers occurred among clones of the same individual. It was concluded that the DNA polymerase slippage during PCR most probably caused this variation. A solution specific to the analysed complex locus was found by splitting it into two new loci.