Development of mitochondrial SNP markers in different Populus species

KEY MESSAGE : We aim to develop, for the first time, poplar species-specific mitochondrial SNP markers which can be employed as diagnostic tool, e.g., to identify mitochondria belonging to different poplar species used in protoplast fusion experiments and for population genetics approaches. In most higher plant species, the inheritance of chloroplasts and mitochondria is maternal. In contrast to chloroplast DNA, however, the mitochondrial genome, especially for woody plants including poplar, has seldom been the object of molecular biological research. Published studies on mitochondrial DNA variation in poplar have dealt with genetic relationships using RFLP analyses for population genetic purposes, but so far not for species identification. In this study, we for the first time aim to develop poplar species-specific mitochondrial SNP markers which can be employed as a diagnostic tool, e.g., to identify mitochondria belonging to different poplar species used in protoplast fusion experiments. In total, seven mitochondrial regions were PCR amplified, sequenced and screened for SNPs differentiating clones belonging to six different poplar species from three sections. Two regions (Sc438, contig42) revealed to be monomorphic in the six Populus species. For the five other regions, SNPs could be identified that are suitable for distinguishing between Populus species, either by a SNP-related CAPS (cleaved amplified polymorphic site) marker to be employed in robust PCR–RFLPs, or simply on the basis of the sequence to be screened by PCR and subsequent Sanger sequencing. As a proof of concept, the developed mitochondrial SNP markers confirmed results obtained with chloroplast SNP markers in interspecific poplar hybrids, either with respect to the direction of the cross or the reclassification of putative pure lines as hybrid lines.