PCR-RFLP image analysis: A practical method for estimating isolate-specific allele frequency in a population consisting of two different strains of the pinewood nematode, Bursaphelenchus xylophilus (Aphelenchida: Aphelencoididae)

Abstract

A practical method for estimating the allele frequency in a population of the pinewood nematode, Bursaphelenchus xylophilus, was developed by employing the technique of PCR-RFLP image analysis, in which a relative quantity of DNA templates is determined. At first, nematode individuals of two isolates, a virulent (S10) and an avirulent isolate (C14-5), were mixed at various proportions, and then PCR-RFLP patterns of the heat-shock protein 70A (hsp70A) gene fragment were obtained from each mixture. Regression analysis between logarithmic values of relative intensity of isolate-specific bands and those of a relative number of individuals of the two strains showed a strong linear relationship. These regression lines were applied to pursue temporal changes in the frequency of the hsp70A alleles in several experimental populations. The changes observed in allele frequencies were well explained with an overdominance model.