Chloroplast DNA Variation in Some Representatives of the Asian, North American and Mediterranean Firs (Abies spp)

Using PCR-RFLP analysis, a comparative study on the restriction site polymorphism within 8 genes and regions of the Abies chloroplast DNA has been conducted covering 15 Asian, 6 North American and 7 Mediterranean species. A variable degree of divergence was observed among individual species of a given region as well as between geographical groups. A group of the Mediterranean firs, consisting of closely related species, differed profoundly from both Asian and North American representatives. Although a higher level of restriction site variants was detected among the Asian firs, two thirds of them were allocated to the difference between A. mariesii and the other Asian firs. The North American species exhibited the highest level of polymorphism resulting in several subgroups on a cladogram. At the individual species level, the Asian species A. mariesii and the North American species A. lasiocarpa diverged conspicuously from their counterparts in their respective regions. The results of restriction site polymorphism analysis are discussed with ragard to crossability and taxonomic status of individual species.

Conidia as a Substrate for Internal Transcribed Spacer-Based PCR Identification of Members of the Leptosphaeria maculans Species Complex

The blackleg disease of oilseed rape is caused by an ascomycete species complex termed Leptosphaeria maculans (anamorph Phoma lingam). L. maculans isolates collected worldwide were gathered in the International Blackleg of Crucifers Network (IBCN) collection. Representative IBCN isolates, along with one P. nigrificans isolate, were further analyzed using polymerase chain reaction (PCR) amplification of the internal transcribed spacer (ITS) region. ITS size polymorphism discriminated three groups: (i) P. nigrificans, (ii) Tox+ and ‘Lepidium’ isolates, and (iii) NA1, NA2, NA3, ‘Thlaspi’, and ‘Erysimum’ isolates. Digestion of the ITS region with 19 selected endonucleases showed restriction site polymorphism between the different subgroups: digestion with RsaI could discriminate Tox+ from ‘Lepidium’ isolates, whereas digestion with four enzymes, i.e., HaeIII, EcoRII, RsaI, and AluI, was needed to discriminate between NA1, NA2, NA3, ‘Thlaspi’, and ‘Erysimum’ isolates. No restriction site polymorphism was observed between isolates within the ‘Thlaspi’, Tox+, NA1, and NA2 subgroups. Direct amplification of the ITS region could be achieved using intact conidia, collected either in axenic cultures or on leaf lesions, with only a 4-min 95°C denaturation step prior to PCR reaction. A routine identification protocol requiring no DNA extraction and a sequential use of a few restriction enzymes following PCR has been used successfully for large-scale identification of French field isolates.