Genetic DNA Marker for A2 mating type in Phytophthora infestans

The Phytophthora infestans requires two mating types for sexual reproduction. Amplified fragment length polymorphism (AFLP) was used to specifically detect different mating types of P. infestans. The AFLP primers E+AA (5'-GACTGCGTACCAATTCAA-3') and M+CAA (5'-GATGAGTCCTGAG-TAAC AA-3') detected a fragment that is specific in the A2 mating type of P. infestans. This fragment was cloned and sequenced. Based on the sequence data, PHYB-1 and PHYB-2 primer were designed to detect the A2 mating type of P. infestans. A single 347 bp segment was observed in the A2 mating type of P. infestans, but not in the A1 mating type of P. infestans or other Phytophthora spp. Identification of mating type was performed with phenotype (sexual reproduction) and genotype (CAPs marker) methods. Two factors, the annealing temperature and template DNA quantity, were investigated to determine the optimal conditions. Using mating type-specific primers, a unique band was obtained within annealing temperatures of 57$^{\circ}C$-62$^{\circ}C$ and DNA levels of 10pg-100 ng (data not shown).