Chromosoma (2003) 111:518-524 DOI 10.1007/s00412-002-0228-y Eddy Chukwura Agbo • Birgitta Duim • Phelix A. O. Majiwa • Philippe Biischer • Eric Claassen • Marinus F. W. te Pas Multiplex-endonuclease genotyping approach (MEGA): a tool for the fine-scale detection of unlinked polymorphic DNA markers Received: 7 November 2002 / Revised: 30 November 2002 ! Accepted: 30 November 2002 / Published online: 22 March 2003 © Springer-Verlag 2003 Abstract Restriction enzyme-detectable polymorphisms have been used for assessing genetic differences and generating informative genetic markers. The most de- tailed fingerprinting analyses have been obtained using the AFLP (amplified fragment length polymorphism) technique, which accesses subsets of polymorphisms at one or two restriction sites. To combine increased discriminatory power with the stringency of polymerase chain reaction amplification, it would be beneficial to access additional independent restriction sites per analy- sis, and to amplify subsets of DNA restriction fragments with only one pair of oligonucleotide primers. We have now developed a unique approach that permits the simultaneous use of four or more endonucleases in combination with one pair of adapters/primers, and applied it to genotype 21 trypanosome populations to subspecific level. The approach takes advantage of the fact that some endonucleases create cohesive ends that are compatible with the overhang sites created by other endonucleases. We demonstrate the greater resolution of identifiable polymorphic fragments over the conventional Edited by: W. Hennig E. C. Agbo (~) • M. F. W. te Pas Division of Animal Sciences, Institute for Animal Science and Health, Edelhertweg 15, 8200 AB Lelystad, The Netherlands e-mail: e.e.c.agbo @id.wag-ur.nl B. Duim Department of Medical Microbiology,University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands P. A. O. Majiwa International Livestock Research Institute, P.O. Box 30709, Nairobi, Kenya P. Btischer Department of Parasitology, Institute of Tropical Medicine, Nationalestraat 155, 2000 Antwerp, Belgium E. Claassen Department of Immunology, Erasmus University Rotterdam, Dr Molewaterplein 50, 3015 GE Rotterdam, The Netherlands ligation-mediated restriction analysis method ~. d~,cuss the value of the approach as a tool ¢,,t ~ine genetic mapping of Trypanosoma brucei. Finally. we propose use of the method for fine characterisation and for identifying co-dominant genetic markers in a variety of other taxa. Introduction A variety of fingerprinting methods have been developed for accurate assessment of genetic diversity, and to address questions regarding population structure and genetic relatedness among prokaryotic and eukaryotic organisms (for review, see Mueller and Wolfenbarger 1999). The ideal genomic fingerprinting method should provide an unbiased estimate of the total genomic variance in the population and must be sufficiently sensitive to detect minimal genetic differences and minimise errors due to sampling variance. This is particularly relevant for the comparative analysis of genomes of closely related organisms where the relatively limited genetic variation between subspecies can only be revealed by the enhanced resolution power of the technique employed. Trypanosome species are unicellular, protozoan para- sites that cause debilitating disease, collectively called trypanosomosis (Kassai 1988), with fatal outcome in man and animals. Trypanosoma brucei consists of three subspecies that are indistinguishable by conventional morphological and antigenic criteria, but differ in their geographical distribution and host specificity (Gibson et al. 1980). For epidemiological identification and tracking of the subspecific strains, T. b. garnbiense can be distinguished from T. b. brucei and T. b. rhodesiense (Hide et al. 1998; Agbo et al. 2002). However, the distinction between T. b. brucei and T. b. rhodesiense has always been more difficult to determine (Godfrey et al. 1990; Hide 1999; Agbo et al. 2002). Resistance to normal human serum (equivalent to human infectivity) is an important trait that distinguishes T. b. gambiense and T. b.