Three major chlorotype lineages in chamomile (Matricaria chamomilla L., Asteraceae)

Genet Resour Crop Evol https://doi.org/10.1007/s10722-023-01625-5 RESEARCH ARTICLE Three major chlorotype lineages in chamomile (Matricaria chamomilla L., Asteraceae) Joana Ruzicka · Gerald Baumschlager · Dijana Jovanovic · Johannes Novak Received: 17 March 2023 / Accepted: 30 May 2023 © The Author(s) 2023 Abstract Chamomile (Matricaria chamomilla L., Asteraceae) is one of the most important medicinal plants with antiphlogistic, spasmolytic, carminative, antibacterial and antimycotic properties. Thirty-one chloroplast markers were developed, optimised for high-resolution melting analysis. Subsequently, 23 M. chamomilla accessions (247 individuals) complemented with 2 individuals of the closely related species M. discoidea were analysed with this marker set. The marker set can practically be reduced to 20 markers without information loss due to the linkage of 16 markers. In total, 20 chlorotypes (multilocus genotypes) were identified, organised in three evolutionary main lineages. Only 8 accessions were monomorphic, the other 15 accessions had between 2 and 6 chlorotypes per accession. The high number of polymorphic accessions and the high number of chlorotypes within many accessions indicate already a high degree of variability within accessions, confirmed by the 66% variation within by AMOVA. Gene diversity of the polymorphic accessions ranged between 0.069 and Supplementary Information The online version contains supplementary material available at https://doi. org/10.1007/s10722-023-01625-5. J. Ruzicka · G. Baumschlager · D. Jovanovic · J. Novak (*) Institute of Animal Nutrition and Functional Plant Compounds, University of Veterinary Medicine Vienna, Veterinärplatz 1, 1210 Vienna, Austria e-mail: 0.261. Since many cultivars had their origin in wild populations of the countries where breeding took place, a first hypothetical indication of the geographic origin of chamomile could point to Albania and adjacent countries. The ‘ancient chlorotype’ was identified by comparing published plastome sequences of Artemisia annua and Lactuca sativa. The ‘ancient chlorotype’ was present only in the closely related M. discoidea but not in any of the M. chamomilla accessions. Two commercially important tetraploid cultivars proofed to be unrelated to their presumed diploid parents. Keywords Chloroplast markers · High resolution melting (HRM) · Single nucleotide polymorphism (SNP) · Asteraceae · Matricaria chamomilla · Chamomile Introduction Matricaria L. is a small genus of 25 species in the family of Asteraceae, of which Matricaria chamomilla L. (German chamomile) (WFO) is an important medicinal plant with a trade volume of about 8,000t per year (Franke and Hannig 2012). The species origins are suggested to be in Europe and West Asia. Today, German chamomile is present in many temperate regions worldwide (Franke and Schilcher 2007). German chamomile is an annual, herbaceous plant of 10 to 90 cm height and a natively diploid Vol.: (0123456789) 13 Genet Resour Crop Evol species (2n = 2x = 18) (Albrecht and Otto 2020). Combined pharmacological and biochemical effects of several chamomile compounds, mainly from the essential oil and flavonoids, are responsible for the therapeutic effectiveness of chamomile products, which possess antiphlogistic, spasmolytic, carminative, antibacterial and antimycotic properties (Schilcher et al. 2005). Breeding activities started approximately 50 years ago from natural European populations in former Czechoslovakia, Poland, Hungary and Germany and led to the development of diploid and autotetraploid cultivars (Albrecht and Otto 2020; Franke and Schilcher 2007). Breeding focussed on the improvements of crop yield (flower heads), essential oil content and composition (correct chemotype), height adapted to harvesting and disease resistance (Albrecht and Otto 2020). Autopolyploidisation of chamomile resulted in higher weight of developing chamomile anthodia and ligulate flowers (Glücknerova et al. 1965) and in a higher cuticular flavonoid content (Repčák et al. 1999). Therefore, tetraploid varieties are important in commercial chamomile production. Several molecular marker studies were performed in German chamomile to study genetic variability (Ahmadi et al. 2014; Mehdikhani et al. 2014; Mežaka et al. 2020; Okoń et al. 2013; Otto et al. 2017; Pirkhezri et al. 2010; Ruzicka et al. 2021; Ruzicka and Novak 2020; Solouki et al. 2008; Wagner et al. 2005). Only a few of them (Otto et al. 2017; Ruzicka et al. 2021; Ruzicka and Novak 2020) were using SNP markers and only two (Ruzicka et al. 2021; Ruzicka and Novak 2020) focused on extranuclear (mitochondrial) DNA markers. Extranuclear or cytoplasmic DNA (cpDNA), located outside the cell nucleus in mitochondria and plastids, are essential tools in molecular systematics (Timmis and Ayliffe 1990), but also helpful for certain scientific intraspecific problems of e.g. phylogeography (Avise et al. 1987; Shaal et al. 1998). Extranuclear DNA is inherited only maternally (in rare cases paternally) without recombination. Therefore, cpDNA markers are usually of a lower variability than nuclear DNA markers. In case of hypervariability of nuclear markers, chloroplast markers can be more informative and provide sufficient variability (Osterberger et al. 2021). HRM (high resolution melting) is a ‘post-PCR’ method able to detect SNPs by differences of the Vol:. (1234567890) 13 melting curves of double stranded DNA recorded at high resolution (Chatzidimopoulos et al. 2019). The method allows a high throughput at low costs per reaction and is widely used for mutation scanning and SNP detection in plant breeding and genotyping (Borna et al. 2017, Sorkheh et al. 2017, Kim and Kim 2019). In this research, chloroplast markers were used to study genetic relationships between and variability within different accessions of chamomile. This study complements the mitochondrial marker previous research by Ruzicka and Novak (2020) and Ruzicka et al. (2021). Material and methods Plant material Chamomile were grown in the greenhouse and young leaves of 249 individuals from 24 accessions were harvested, dried and stored on silica until DNA extraction (Table 1). DNA extraction Approximately 1 cm2 of dried leaf material was grounded with glass beads in a swing mill (Mixer Mill MM301, Retsch GmbH, Germany) and total genomic DNA isolated using a modified CTAB extraction protocol (Schmiderer et al. 2013). Quantity and quality of the DNA were determined on a spectrophotometer (NanoDrop™ 2000, Thermo Fisher Scientific Inc., USA) and via gel electrophoresis on a 1.4% agarose gel stained with peqGREEN (VWR, Austria). The DNA was dissolved in TE buffer and stored at − 20 °C until further usage. SNP detection and primer design NGS data from a previous study (Submission ID: SUB5046906/BioProject ID: PRJNA515664) were de novo assembled to source chloroplast SNP markers. Some chloroplast consensus sequences were blasted and Artemisia annua was id (...truncated)