Establishment And Application Of PCR Method For Mitochondrial DNA Haplotypes Of Phytophthora Infestans

Based on polymorphisic analysis of four mitochondrial genomes （Ia，Ib，IIa andIIb）and systemic analysis of the existed three methods for mitochondrial haplotypesidentifying, the theoretical basis of a novel PCR method for identifying mt-DNAhaplotypes was established. Genome sequencing of the four haplotypes (Ia, Ib, IIa,and IIb) allowed us to thoroughly examine mt-DNA polymorphisms and weindentified two hypervariable regions (HVRs) named HVRi and HVRii. The HVRilength polymorphism caused by a1885kb insertion/deletion was utilized to identifymt-DNA types I and types II, while another length polymorphism in the HVRii regionis caused by a variable number of tandemrepeats (n=1,2or3) of a36bp sized DNAstretch and was further used to determine mt-DNA sub-types, which were describedas n=1,2or3. Finally, the P. infestans mt-DNA haplotypes were re-defined as I R1orII R2according to PCR derived HVRi and HVRii length polymorphisms.Twenty-three isolates were chosen to verify the feasibility of our new approach foridentifying mt-DNA haplotypes and a total of five haplotypes (I R1, I R2, I R3, II R2and II R3) were identified. Additionally, we found that six isolates determined as typeI by our method were mistakenly identified as type II by the PCR–RFLP technique. Inconclusion, we propose a simple and rapid PCR method for identification of mt-DNAhaplotypes based on sequence analyses of the mitochondrial P. infestans genome.Mt-DNA haplotypes of459isolates for P. infestans were identfied by this simple PCRmethod. Of434isolates from potato late blight, five haplotypes were detected andonly haplotype II R1was not detected. Of25isolates from tomato late blight, onlyhaplotype I R3was detected. This established PCR method in this project is a simple,rapid, accurate, which could be used to study population structure, dynamics andevolution. In addition, mt-DNA type I could be originated from type II by the severaldifferent types of large fragment deletion, so we inferred that mt-DNA type II is olderthan type I.