Genome size and ISSR markers for Mentha L. (Lamiaceae) genetic diversity assessment and species identification

The systematics of the Mentha genus is very complicated and still unclear. This is mainly due to the existence of variation in basic chromosome number, frequent interspecific hybridization, cytomixis, polymorphism in morphology and essential oil composition under different environmental conditions, colonial mutant propagation, as well as the occurrence of polyploidy, aneuploidy and nothomorphs. Morphological traits, chemical composition, and even caryological analysis failed in discrimination of Mentha species. Therefore, the aim of this study was to combine genome size estimation with ISSR markers for quick and precise species identification, determination the inter- and intraspecific variation within this genus, as well as description of relationships between genotypes. Genome size estimation revealed, that investigated Mentha species possessed very small and small genomes. Additionally, for 13 Mentha species, this is the first report on genome size. Within one species different values of genome size occurred, which indicated polyploidy and aneuploidy. The smallest genome (0.63 pg/2C) was detected in M. gattefossei, while the largest in M. × verticillata (4.15 pg/2C). Flow cytometric measurements of genome size allowed to distinguish some species and cytotypes, and thus it can be used as a first step in species identification. More precise and detailed characterisation were provided by ISSR markers. All the primers used in ISSR-PCR, revealed to be useful in species identification, while primers (GACA)4, (GTG)6T, (CTC)4RC, (GA)6CC enabled to distinguish M. aquatica, M. canadensis, M. longifolia, M. pulegium and M. spicata cytotypes, and primers (GACA)4, (GA)6CC and (GA)8YC to identify M. × gracilis and M. × piperita cultivars. The phylogenetic analysis revealed the existence of seven main clusters and two species (M. requienii and M. × smithiana) being not clustered to any of the created groups within the Mentha genus. This analysis mostly confirms the latest taxonomical classification of the Mentha genus, however some exceptions are discussed. The identification system of combined genome size estimation, and ISSR markers proved to be useful for species in Mentha genus.