Phylogenetic identification of Balkan endemic Stachys species and genomic stability during ex vitro conservation

The genus Stachys is one of the largest in the Lamiaceae family. Representatives of the genus are among the most ancient medicinal plants used in the ethnomedicine. The Balkan endemic species S. thracica, S. bulgarica and S. scardica are included in The Red Data Book of Bulgaria and due to their endangered status are scarcely studied. The aim of the present work was to examine the genetic status of these three endemic Stachys species during the process of their ex situ conservation. To gain information about their taxonomic position in the genus Stachys, we applied the DNA barcoding approach. Nuclear (ITS) and plastid (rbcL, matK and trnH‑psbA) DNA barcodes were generated and aligned with accessions available in the data base. In the constructed phylogenetic trees S. thracica was placed in a cluster together with S. alpina, S. germanica and S. cretica, while S. bulgarica and S. scardica were clustered with S. officinalis. The ex situ conservation was achieved by the initiation of in vitro shoot cultures and their subsequent adaptation in ex vitro conditions. To check the genomic stability of the plants during the acclimatisation from in vitro conditions to ex vitro, analysis by sequence-related amplified polymorphism (SRAP) markers was performed. No difference was detected between the SRAP profiles of in vitro cultivated and ex vitro adapted S. thracica and S. scardica plants. In S. bulgarica, only 0.4% fragment difference was detected. The obtained results indicated that the three Stachys species preserved their genetic stability during the process of in vitro multiplication, which is a prerequisite for conserved bioactive capacity