Genetic Diversity

The incitant of stem canker / blackleg disease of oilseed rape can be differentiated (as described before) in two main groups (A- and B-type). With the development of DNA-approaches polymorphism between these groups were also early reported.
The prescence of a linear plasmid within the genome of A-type isolates was published. Also chromosomal polymorphisms and restriction fragment length polymorphisms were observed between representative isolates of both groups. With the development of the polymerase chain reaction -PCR-, which early becomes a routine method, polymorphism between randomly amplified DNAs (RAPDs) were reported. Later larger repetitive, GC rich elements were used for studies on diversity resulting in more reliable results due to less miss priming risks by the use of higher annealing temperatures.
In our own studies we used repetetive elements, which seem to be distributed over a huge number of organisms. We successfully used ERIC- and VNTR- primers. The first two figures display genetic variation found within an international isolate collection of the International Blackleg of Crucifers Network (IBCN), comprising 91 isolates of different geographical and host origin. Two gels showing amplified and separated DNA fragments resulting from either ERIC- or VNTR PCR analysis are presented. On both gels you see sirodesmin producing isolates (A-types) on the left and non-producing reference isolates (B-types) on the right side flanked and separated by a molecular size standard. Among the 91 isolates 9 genotypes were found. Results show that A-types are a very homogenous group of isolates displaying only very little variation. In contrast B-type isolates are very polymorphic. They can be further clustered in 6-7 sub-groups. All so far characterised B-type isolates with German origin belong to the NA1-group firstly described by Koch et al. (1991). This seem to be also the case for isolates on the European scale. However, these were only isolated from oilseed rape, wheras higher diversity was found for North American isolates originated from cruciferous weeds like Lepidium sp. Erysimum sp., Sysimbrium sp. and Thlaspi sp.. 6 of the 7 genotypes were found among isolates originated from these cruciferous weeds. Another genotype was charcterised originated from canola grown in Australia (NA-AUS). Our own results are equivalent to the results published by Mendes-Pereira et al. (2004). Until recently, the NA-Aus isolate completed the so far known variabilty within Phoma lingam. New own investigations (Islam et al. 2004) on 21 Canadian isolates originated from Thlaspi arvense displayed two new genoptypes not reported so far. Results are consistant with length polymorphisms of the "internal transcribed spacer" region of the rDNA. Results of ITS-PCR using prímers ITS4 and ITS5 are shown in the last figure. IBCN reference isolates are shown on the left between two marker lanes. Wheras Thlaspi ITS-products are shown at the right. The first lane at the right shows the IBCN Thlaspi isolate. The other 6 lanes represent 3 isolates each of the two new Thlaspi genotypes found.