Separation of chromosomes of Phytophthora species using CHEF gel electrophoresis

Abstract

A broadly applicable technique for separation of chromosomes of Phytophthora species is desirable as a genetic tool for estimating genome size, determining chromosome number, and localizing specific genes on chromosomes. Chromosomal DNAs from seven different Phytophthora species were electrophoresed using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Ramped switching intervals of 3 to 45 min at 50 V for 7 days (14°C) allowed resolution of 13 bands for Phytophthora megasperma isolate 63, in agreement with the chromosome number reported from cytological studies. At 45 V, at least six chromosomes were resolved for P. cactorum, four to five for P. boehmeriae, seven to nine for P. megasperma isolate 77, and three to four for P. capsici. Some separation but poor resolution was observed for chromosomes of P. palmivora and P. parasitica. Chromosomes of P. infestans failed to separate, migrating as a single high molecular weight band. Substantial variation in band intensity was observed within isolates of all species. Specific hybridization to chromosomes on CHEF gels was obtained with the seven species using a ribosomal DNA probe from Pythium paroecandrum.