Heteroduplex mobility assay detects DNA mutations for differentiation of closely related phytoplasma strains

We present the first use of DNA heteroduplex mobility assay (HMA) to detect the point mutations including substitutions and deletions/insertions in 16 S rDNA of aster yellows phytoplasma (AY27) and to differentiate phytoplasma s collected from field samples of clover proliferation (CP) and alfalfa wit ches'-broom (AWB). The phytoplasmal 16S rDNA fragment was amplified from AY 27 by polymerase chain reaction (PCR) and cloned into a plasmid vector. The cloned DNA fragment was subjected to in vitro mutation to produce 1- to 4- base substitutions and 1- to 3-base deletions. The mutated 16S rDNA fragmen ts were analyzed by HMA. The results showed that a single two-base substitu tion or a single-base deletion/insertion in the 529 bp DNA fragment was dir ectly detected and that a DNA divergence at a level of as low as 0.2% was d etectable by HMA. Heteroduplex mobilities were affected by the number and c omposition of the phytoplasma DNA bases in mismatches or gaps and were prop ortional to the degree of DNA divergences. Gaps caused greater retardation in heteroduplex mobility than mismatches did. HMA was highly sensitive in d etecting the mixed infections of phytoplasmas. In analyses of CP and AWE fi eld samples collected in Alberta, two CP and one AWE phytoplasma isolates w ere differentiated from others by HMA but not by restriction fragment lengt h polymorphism (RFLP). Therefore, HMA provides a simple, rapid, highly sens itive and analytical method to detect and estimate the genetic divergence o f phytoplasmas when other methods such as RFLP are not readily applicable. (C) 2000 Elsevier Science B.V. All rights reserved.