RAPD-derived, PCR-based mitochondrial markers for Larix species and their usefulness in phylogeny Vladimir L. Semerikov 1,3, *, Giovanni G. Vendramin 2 , Federico Sebastiani 4 & Martin Lascoux 3 1 Institute of Plant and Animal Ecology, 620144, Ekaterinburg, Russia; 2 Plant Genetics Institute, CNR, Via Madonna del Piano, 50019, Sesto Fiorentino (Firenze), Italy; 3 Evolutionary Biology Centre, Program in Evolutionary Functional Genomics, Uppsala University, Norbyva ¨gen 18D, 75236, Uppsala, Sweden; 4 Department of Plant Biotechnology, Genexpress, University of Florence, Via della Lastruccia 14, 50019, Sesto Fiorentino (Firenze), Italy (*Corresponding author: Phone: +7-343-2103858; Fax: +7-343-2606500; E-mail: semerikov@ipae.uran.ru) Received 26 April 2005; accepted 30 August 2005 Key words: Larix, minisatellite, mitochondrial DNA Abstract Random amplified polymorphic DNA (RAPDs) from plants contains numerous fragments of mitochon- drial origin. In the present study, the association between RAPD bands and previously recognized mito- chondrial polymorphism in a Larix population was used to identify fragments of mitochondrial origin and to develop PCR-based mitochondrial DNA markers useful to study phylogeny in larches, Larix sp. (Pinaceae). The management of genetic resources in forest tree species demand knowledge of the genetic structure of populations that might be revealed using genetic markers associated with all three plant genomes – nuclear, chloroplast and mito- chondrial. In the present study, we chose to focus on mitochondrial DNA as it is maternally inher- ited in conifers and consequently only dispersed through seeds that have a much lower mobility than pollen. To develop PCR-based mitochondrial markers for larches we took advantage of the fact that RAPD reactions in plants can yield numerous fragments of mitochondrial origin (Aagard et al. 1995). We used the correlation between RAPD phenotypes and already characterized mitochon- drial genotypes obtained with eight RFLPs (six probe–enzyme combinations and two PCR- RFLPs) in our previous study of Eurasian Larix phylogeography (Semerikov and Lascoux 2003). In a hybrid population, mitochondrial haplotypes characteristic of either L. sibirica or L. gmelinii were found in equal proportions, whereas both nuclear and chloroplast DNA were typical of L. gmelinii. This information was used here to find RAPD bands associated with species-specific mitochondrial haplotype. RAPDs were first used to screen two samples of four individuals each, one bearing L. sibirica haplotypes and the second dis- playing L. gmelinii haplotypes, respectively. A few hundred RAPD primers were screened. Bands that were present in the PCR product of one sam- ple and absent in the other were selected. Selected RAPDs were tested on 12 individuals with L. sibirica mtDNA haplotypes and 12 individuals with L. gmelinii haplotypes. A band present in at least half of the individuals carrying L. sibirica haplotypes and completely absent in individuals carrying the L. gmelinii haplotypes or vice versa, was supposed to be of mitochondrial origin. Four Conservation Genetics (2006) 7:621–625 Ó Springer 2005 DOI 10.1007/s10592-005-9059-8