ACTA BOT. CROAT. 81 (1), 2022 1 Acta Bot. Croat. 81 (1), 1–11, 2022 CODEN: ABCRA 25 DOI: 10.37427/botcro-2021-026 ISSN 0365-0588 eISSN 1847-8476 Analysis of Hypericum accessions by DNA fingerprinting and flow cytometry Anca Butiuc-Keul1,2,3, Ana Coste4,*, Holger Budahn1, Frank Dunemann1, Anca Farkas2,3, Dragoș Postolache5, Evelyn Klocke1 1 Institute for Breeding Research on Horticultural Crops, Julius Kühn Institute, Federal Research Centre for Cultivated Plants, Erwin-Baur-Str. 27, 06484 Quedlinburg, Germany 2 Babeş-Bolyai University, Faculty of Biology and Geology, Department of Molecular Biology and Biotechnology, M. Kogalniceanu St. 1, 400084 Cluj-Napoca, Romania 3 Babeş-Bolyai University, Centre for Systems Biology, Biodiversity and Bioresources, Clinicilor St. 5-7 400006, Cluj-Napoca, Romania 4 Institute of Biological Research Cluj-Napoca, Branch of National Institute of Research and Development for Biological Sciences, Republicii St. 48, 400015 Cluj-Napoca, Romania 5 National Institute for Research and Development in Forestry “Marin Drăcea”, Eroilor Boulevard 128, Voluntari, 077190 Ilfov County, Romania Abstract – Hypericum perforatum, H. umbellatum, H. maculatum, and H. hircinum accessions originating from bo- tanical gardens across Europe were examined by flow cytometry and molecular markers. 2C DNA content of 17 Hypericum perforatum accessions (Hp) and the H. perforatum cultivar Topaz amounted to between 1.56 pg and 1.62 pg. In four Hp accessions some individual plants were found with a DNA content corresponding to 6Cx (2.34 - 2.39 pg). All plants of accession Hp8 showed a DNA content of 6Cx (2.41 pg). In root tips of Hp plants with an average DNA amount of 1.58 pg, 32 chromosomes were detected, corresponding to 2n = 4x. This is the first ploidy and/or DNA content report for H. umbellatum, H. maculatum and H. hircinum. H. umbellatum and H. maculatum, each contained 0.76 pg DNA and 16 chromosomes were counted. The 2C DNA content of H. hircinum was 1.00 pg with the best metaphase plate revealing 32 chromosomes. Additionally, a combined marker analysis, based on inter-simple se- quence repeats (ISSR) and sequence related amplified polymorphism (SRAP), was conducted to gain a better under- standing of diversity especially within the accessions of H. perforatum. A total of 27 (11 ISSR and 16 SRAP) primer combinations were screened, showing 699 bands, of which 661 were polymorphic. UPGMA clustering revealed that accessions from the same geographic area tended to be more closely related, while H. maculatum was grouped sepa- rately from all H. perforatum accessions. Both methods have shown similar sensitivities in detecting the genetic diver- sity of the analyzed genotypes. Our results may be useful for Hypericum breeding programs and the development of effective conservation strategies. Keywords: chromosome number, DNA, genetic diversity, molecular markers, St. John’s wort Introduction Hypericum L. (Hypericaceae) is a species-rich genus that colonized the temperate regions of the northern hemisphere and underwent rapid radiation during the Pleistocene ( Scheriau et al. 2017). The genus consists of almost 500 species of shrubs, herbs and a few trees (Nürk and Blattner 2010), which were grouped into 36 sections (Robson 1981). Among Hypericum species only H. perforatum is widely used in medicine. So far, a limited number of species within the ge- nus was studied, and the chemical compounds of approxi- mately three quarters of Hypericum species have not been surveyed yet (Karioti and Bilia 2010). Hypericum perforatum extracts have multiple effects as an antidepressant, antiviral, antimicrobial and anti-inflammatory drug due to the main constituents, such as naphthodianthrones (hypericin and pseudohypericin), phloroglucinol derivatives (hyperforin), and flavonoids (quercetin, quercitrin, hyperoside and rutin). * Corresponding author e-mail: ana.coste@icbcluj.ro ACCEPTED, AHEAD OF PRINT VERSION OF THE MANUSCRIPT 81(1), 1st April 2022 Please cite this article as: Butiuc-Keul A, Coste A, Budahn H, Dunemann F, Farkas A, Postolache D, Klocke E: Analysis of Hypericum accessions by DNA fingerprinting and flow cytom- etry. Acta Botanica Croatica, DOI: 10.37427/botcro-2021-026 Running title: ANALYSIS OF HYPERICUM ACCESSIONS BUTIUC-KEUL A., COSTE A., BUDAHN H., DUNEMANN F., FARKAS A., POSTOLACHE D., KLOCKE E. 2 ACTA BOT. CROAT. 81 (1), 2022 Because the substances are present only in small amounts but of great commercial interest, there have been strong ef- forts to enhance their production by biotechnological meth- ods (Coste et al. 2011, Franklin et al. 2016). For this purpose, molecular characterization of native plants and/or acces- sions is necessary in order to identify the genotypes and to develop molecular markers associated with valuable traits. In addition, the knowledge of the full range of ploidy variation is valuable for a proper management of genetic re- sources of Hypericum ssp. Extensive cytological investiga- tions of Hypericum perforatum have made this species a model for examining aposporous apomixis (Barcaccia et al. 2007, Mártonfi and Mártonfiová 2011). This species has a basic chromosome number of 8, but mostly it is tetraploid (2n = 4x = 32), although diploids (2n = 2x = 16) and hexa- ploids (2n = 6x = 48) occur in natural populations (Matzk et al. 2001). Apomictic (polyploid) individuals can faculta- tively produce both sexual and variable apomictic seeds (Matzk et al. 2001, 2003, Barcaccia et al. 2007, Galla et al. 2011). Despite comprehensive studies about its modes of re- production in relation to different ploidies, there is little knowledge about the distribution of different genome sizes and ploidies of individual plants within accessions, this dis- tribution being an important feature especially for manage- ment of genetic resources. Furthermore, aneuploid individ- uals have been identified in Australian populations (2n-1 = 31) (Mayo and Langridge 2003). H. perforatum is hypothe- sized to hybridize easily with its sister H. maculatum, with mainly diploid populations (Brutovská et al. 2000; Barcaccia et al. 2007). Up to date, there have been reports of two dip- loid subspecies for H. maculatum (subsp. maculatum and subsp. immaculatum) and one tetraploid subspecies H. mac- ulatum subsp. obtusiusculum (Koch et al. 2013). Regarding H. hircinum, this species was first reported as a possible pen- taploid cytotype, with chromosome number varying be- tween 40 (Loon and Jong 1978, Robson 1981) and 32 (Matzk et al. 2003, Castro and Rosselló 2006). To our knowledge, no data regarding H. umbellatum ploidy level and DNA con- tent have been reported. In the present paper, flow cytometry (FCM) was con- ducted to estimate the 2C DNA content of single plants of 17 accessions of H. perforatum (Hp) from botanical gardens all over Europe and the cultivar Topaz as well as Romanian accessions of wild species H. maculatum Crantz (Hm), H.  umbellatum A. Kern. (Hu) and H. hircinum L. (Hh). The occurrence of various ploidies within the accessions was noted. For confirmation of the ploidy, chromosome counting was performed. Molecular characterization of Hypericum germplasm was accomplished by ISSR and SRAP markers. Materials and methods Plant material Within this study, different accessions (populations) be- longing to the genus Hypericum and covering four species namely Hypericum perforatum (Hp), Hypericum maculatum (Hm), H. umbellatum (Hu) and H. hircinum (Hh) from different botanical gardens of Europe were analyzed. In most of the accessions, seeds were from plants cultivated in botanical gardens (Hp1-8, Hp11-13, Hp15, Hm23, Hh24) but some of them were collected from plants from natural pop- ulations (Hp9-10, Hp14, Hp16-17, Hu21, Hm22) according to Tab. 1. Seeds were germinated in soil and plants grown in the greenhouse (at least 30 seeds/accession). Flow cytometry analysis Estimation of DNA content and ploidy by FCM was per- formed using fresh leaf material from young plants. Small amounts of leaf tissue of a sample and the internal standard were chopped together in 0.5 mL of Galbraith’s buffer ( Galbraith et al. 1983), supplemented with 0.5 mL Partec CyStain propidium iodide solution containing DNase-free RNase following the manufacturer’s instructions (Partec) and filtered through a cell-strainer cap (BD FalconTM) with 35 µm pore size. Analyses were performed with a flow cytometer BD FACS Calibur (USA). For each sample, no fewer than 5000 particles were registered by 488 nm laser beam. For estimation of the nuclear DNA content, Raphanus sativus L. was used as an internal standard (2C = 1.11 pg; Doležel et al. 1992). Analyses were carried out for at least Tab. 1. Accessions of Hypericum spp. studied and their origin (spe- cies code are given in parenthesis, * – seeds collected from plants cultivated in a botanical garden, ** – seeds collected from natural populations). Accession name Origin Seeds / DNA source H. perforatum (Hp) 1 Germany Botanical Garden Ulm Hp2* Germany Botanical Garden Frankfurt Hp3* Germany Botanical Garden Regensburg Hp4* Germany Humboldt University Berlin Hp5* Germany Botanical Garden Constance Hp6* Germany Botanical Garden Hamburg Hp7* Switzerland Botanical Garden St. Gallen Hp8* Austria Botanical Garden Salzburg Hp9** Austria Botanical Garden Graz Hp10** France Botanical Garden Nancy Hp11* France Botanical Garden Ville de Renne Hp12* France Botanical Garden Talence Hp13* Poland Botanical Garden Wrocław Hp14** Norway Botanical Garden Oslo Hp15* Italy Botanical Garden Trento Hp16** Italy Botanical Garden Siena Hp17** Estonia Läänemaa, Hort Bot Tartu University Hp cultivar ’Topaz’ Germany Seed provider, Fürstenwalde H. umbellatum (Hu21) ** Romania Gilău H. maculatum (Hm22) ** Romania Piatra Craiului H. maculatum (Hm23) * Romania Botanical Garden Cluj-Napoca H. hircinum (Hh24) * Romania Botanical Garden Cluj-Napoca ANALYSIS OF HYPERICUM ACCESSIONS ACTA BOT. CROAT. 81 (1), 2022 3 five randomly selected individuals per accession and three repetitions for each individual. For accessions with differ- ing cytotypes up to 30 individual plants were tested (Tab. 2). Data evaluation was accomplished with BD software CellQuestTMPro. Nuclear DNA content was calculated using the linear relationship between the ratio of the 2C peak positions of the target species/internal standard on the area histogram of fluorescence intensities. The statistics were carried out using the Real Statistics Resource Pack Version 4.9 for Microsoft Excel. Squash preparations of root tip cells Root tips were harvested from greenhouse plants and pretreated with 8-hydroxyquinoline for 2.5 hours at room temperature and fixed overnight in ethanol: glacial acetic acid (3:1 v/v) at 4 °C. The fixed root tips were transferred to 70% ethanol and stored at 4 °C. For further investigation fixed root tips were washed in distilled water for 10 min and then incubated at 37 °C for 45 min in an enzyme mix con- sisting of 4% celullase, 1% pectolyase and 45% acetic acid, pH 4.0. After being washed with distilled water, the tips were squashed on the slide in 45% acetic acid. If under a bright microscope in phase contrast metaphase chromo- somes were observed, the slide was frozen for at least 15 min at -80 °C and the cover slip was blown off. After an air dry- ing for 10 min or longer 15 µl DAPI VECTASHIELDÒ anti- fade mounting medium with DAPI (4’, 6-diamidino-2-phe- nylindol, Vector Laboratories) was added. The chromosomes were detected and photographed in fluorescent light (mi- croscope Axioimager Z1 with CCD-camera Axiocam, Zeiss). The image analysis was carried out with the software program Isis (MetaSystems, Germany). ISSR and SRAP markers analysis Marker analysis was conducted with 90 individuals (5 individuals/available accession), a representative sub-set of accessions and individuals screened by flow cytometry. Ge- nomic DNA was isolated from young leaves of plants grown Tab. 2. DNA content of accessions of Hypericum perforatum (Hp), H. umbellatum (Hu), H. maculatum (Hm), H. hircinum (Hh) assessed with internal standard Raphanus sativus (2C = 1.11 pg). -1, -2: plants belonging to the same accession but with different DNA content. Accession № of plants investigated № of measure- ments Mean DNA content (pg) Standard Deviation Ploidy 1Cx Content (pg) Hp1 5 15 1.58 0.03 4x 0.40 Hp2 15 35 1.57 0.04 4x 0.39 Hp3-1 15 33 1.58 0.03 4x 0.40 Hp3-2 1 3 1.99 0.02 ? ? Hp4 5 15 1.56 0.01 4x 0.39 Hp5 5 15 1.59 0.02 4x 0.40 Hp6-1 24 30 1.57 0.03 4x 0.39 Hp6-2 2 7 2.38 0.05 6x 0.40 Hp7-1 30 38 1.56 0.02 4x 0.39 Hp7-2 5 10 2.34 0.02 6x 0.39 Hp8 5 12 2.41 0.07 6x 0.40 Hp9-1 25 33 1.58 0.03 4x 0.40 Hp9-2 1 3 2.37 0.03 6x 0.40 Hp10-1 23 44 1.57 0.02 4x 0.39 Hp10-2 2 5 2.39 0.05 6x 0.40 Hp11 5 15 1.59 0.03 4x 0.40 Hp12 5 14 1.62 0.01 4x 0.40 Hp13 5 15 1.58 0.01 4x 0.40 Hp14 5 15 1.57 0.02 4x 0.39 Hp15 5 15 1.59 0.02 4x 0.40 Hp16 5 15 1.59 0.01 4x 0.40 Hp17 5 15 1.59 0.02 4x 0.40 Hp ’Topaz’ 6 7 1.58 0.01 4x 0.40 Hp (average for 4x) 188 369 1.58 0.01 0.40 Hp (average for 6x) 15 37 2.38 0.02 0.40 Other species Hu21 5 13 0.76 0.02 2x 0.38 Hm22 10 14 0.76 0.03 2x 0.38 Hm23 5 14 0.76 0.01 2x 0.38 Hh24 11 14 1.00 0.04 4x 0.25 BUTIUC-KEUL A., COSTE A., BUDAHN H., DUNEMANN F., FARKAS A., POSTOLACHE D., KLOCKE E. 4 ACTA BOT. CROAT. 81 (1), 2022 under standard greenhouse conditions, by using the CTAB method described by Doyle and Doyle (1987). DNA concen- tration was estimated using a UV-Vis spectral photometer Nanodrop 8000. ISSR (Inter Simple Sequence Repeats) amplification was performed with 11 primers (Rostami- -Ahmadvandi et al. 2013). For SRAP (Sequence Related Amplified Polymorphism) analysis, sixteen primer combi- nations (Li and Quiros 2001) were used. Primer sequences are given in Tab. 2. PCR amplification was performed in a 0.2 mL tube containing 12.5 μL 2x DreamTaq Green PCR master mix (Thermo Fisher Scientific, USA), 10.25 μL nuclease-free water (Lonza, Switzerland), 25 pmol of each primer (Eurogentec, Belgium) and 5 ng of genomic DNA in a final volume of 25 μL. For ISSR analysis the GeneAmp PCR system 9700 (Applied Biosystems, Forster City, USA) was programmed as follows: 94 °C for 4 min, 35 cycles of 94 °C for 30 sec, 46 °C for 30 sec and 72 °C for 55 sec followed by a final elongation step at 72 °C for 5 min. For SRAP analysis, the following program was used: 94 °C for 4 min, 5 cycles of 94 °C for 30 sec, 35 °C for 30 sec, and 72 °C for 55 sec followed by 30 cycles of 94 °C for 30 sec, 50 °C for 30 sec, 72 °C for 55 sec, and a final elongation at 72 °C for 5 min. Amplification products were separated in 1.5% w/v agarose (Cleaver Scientific, United Kingdom) gel in 1×TBE buffer (Lonza, Switzerland) and stained with 0.5 μg/mL ethidium bromide (Thermo Fisher Scientific, USA). ISSR and SRAP patterns were assessed as dominant markers. Band patterns for both marker systems were re- corded in 1/0 matrices to determine the level of genetic sim- ilarity between the different accessions on the basis of Jac- card’s coefficient (Jaccard 1908). The resulting matrix of similarity was analyzed by the unweighted pairgroup meth- od with arithmetic mean (UPGMA) and the dendrogram was obtained using MultiVariate Statistical Package 3.21 (Kovach 2007). Shannon’s information index (I) (Shannon and Weaver 1949) and expected heterozygosity (He) were calculated, using GenAlEx software version 6.5 (Peakall and Smouse 2012). The polymorphism information content (PIC) value of each individual locus was calculated accord- ing to Sehgal et al. (2009) as: PICj = 1–Ʃpi2n=1 Where i is the ith allele of the jth marker, n is the number of alleles at the jth marker and p is the allele frequency. Resolving power (Rp) for the individual marker system was bases on the distribution of detected bands within the sampled clones and was calculated based on the formula described by Prevost and Wilkinson (1999): Rp = ΣIb Where Ib (informativeness) is 1- [2 x |0.5-p|] and p is the ratio of present bands among the analyzed accessions. Results Estimation of nuclear DNA content and determination of ploidy The flow cytometric measurements consistently showed a high reproducibility in repeated measurements of the same plant despite the presence of numerous secondary metabo- Fig. 1. Flow cytometric histograms (1: 2C Peak of internal standard Raphanus sativus, 1.11 pg). a – Hypericum perforatum Hp7-1: 2: 2C = 4Cx peak, 1.55 pg, b – Hp7-2: 2: 2C = 6Cx peak, 2.34 pg, c – H. maculatum Hm23: 2: 2C = 2Cx peak, 0.76 pg. ANALYSIS OF HYPERICUM ACCESSIONS ACTA BOT. CROAT. 81 (1), 2022 5 lites, which often influence the peak performance. The CV (coefficient of variation) of histogram peaks was below 5.0 in each case. Raphanus sativus served as an internal standard for genome size estimation. Both 2C peaks from internal standard and Hypericum sample were well separated from one another (Fig. 1). The genome size of 17 Hp accessions and the cultivar Topaz of the tetraploid genotypes amounted to between 1.56 pg and 1.62 pg (Tab. 2, Fig. 1a) with an aver- age genome size of 2C = 4Cx = 1.58 pg. The Hypericum ac- cessions were obtained from various European botanical gardens and from Romanian regions (Tab. 1). The accession Hp8 from the Salzburg botanical garden, Austria, revealed for five tested plants a hexaploid cytotype with a DNA con- tent of 6Cx = 2.41 pg (Tab. 2). Besides tetraploid plants we found in Hp6, Hp7, Hp9 and Hp10 at least one plant which showed a genome size corresponding to the hexaploid chro- mosome level (Fig. 1a, b), taking it into account that the 1Cx DNA content for all Hp accessions amounted to 0.40 pg. However, one exception was noticed. In the Hp3 from the botanical garden in Regensburg, Germany, one plant with a genome size of 1.99 pg was observed. Only speculation could be offered about the nature of this cytotype. For both Romanian H. maculatum accessions as well as for H. umbellatum a small genome size with 0.76 pg was es- timated (Fig. 1c). The genome size of H. hircinum was 1.00 pg (Tab. 2). Thus, the 1Cx value of H. hircinum differed con- siderably from the others and was only 0.25 pg. Chromosome number For correct assignment of genome size to ploidy, the numbers of metaphase chromosomes were counted in stained root tips. The chromosomes of Hypericum are very small and morphologically similar, making chromosome counting difficult. In addition, the plant tissue digestion posed problems with regard to achieving well-spread chro- mosome plates. The unsatisfactory quality in connection Tab. 3. SRAP (sequence related amplified polymorphism) and ISSR (inter-simple sequence repeats) primers used for amplification with their respective codes and nucleotide sequences. Primer Sequence Primer Sequence SRAP ISSR SRP2 Fw 5'-TGAGTCCAAACCGGAGC-3' Rv 5'-GACTGCGTACGAATTAAT-3' A (GACA)3RT SRP5 Fw 5'-TGAGTCCAAACCGGAAG-3' Rv 5'-GACTGCGTACGAATTAAT-3' C (GACAC)2 SRP6 Fw 5'-TGAGTCCAAACCGGATA-3' Rv 5'-GACTGCGTACGAATTTGC-3' UBC808 (AG)8C SRP11 Fw 5'-TGAGTCCAAACCGGATA-3' Rv 5'-GACTGCGTACGAATTGAC-3' UBC809 (AG)8G SRP12 Fw 5'-TGAGTCCAAACCGGAGC-3' Rv 5'-GACTGCGTACGAATTGAC-3' UBC811 (GA)8C SRP13 Fw 5'-TGAGTCCAAACCGGAAT-3' Rv 5'-GACTGCGTACGAATTGAC-3' UBC112 (GACA)4 SRP14 Fw 5'-TGAGTCCAAACCGGACC-3' Rv 5'-GACTGCGTACGAATTGAC-3' UBC818 (CA)8G SRP15 Fw 5'-TGAGTCCAAACCGGAAG-3' Rv 5'-GACTGCGTACGAATTGAC-3' UBC855 (AC)8YT SRP17 Fw 5'-TGAGTCCAAACCGGAGC-3' Rv 5'-GACTGCGTACGAATTTGA-3' UBC856 (ACAC)4YG SRP20 Fw 5'-TGAGTCCAAACCGGAAG-3' Rv 5'-GACTGCGTACGAATTTGA-3' UBC857 (AC)8T SRP25 Fw 5'-TGAGTCCAAACCGGAAG-3' Rv 5'-GACTGCGTACGAATTAAC-3' UBC873 (ATG)6 SRP26 Fw 5'-TGAGTCCAAACCGGATA-3' Rv 5'-GACTGCGTACGAATTGCA-3' SRP27 Fw 5'-TGAGTCCAAACCGGAGC-3' Rv 5'-GACTGCGTACGAATTGCA-3' SRP28 Fw 5'-TGAGTCCAAACCGGAAT-3' Rv 5'-GACTGCGTACGAATTGCA-3' SRP29 Fw 5'-TGAGTCCAAACCGGACC-3' Rv 5'-GACTGCGTACGAATTGCA-3' SRP30 Fw 5'-TGAGTCCAAACCGGAAG-3' Rv 5'-GACTGCGTACGAATTGCA-3' BUTIUC-KEUL A., COSTE A., BUDAHN H., DUNEMANN F., FARKAS A., POSTOLACHE D., KLOCKE E. 6 ACTA BOT. CROAT. 81 (1), 2022 with low mitotic index resulted in a fluctuating number of chromosomes being counted. In the accessions Hp3, Hp4, Hp7, and Hp9 we noticed 30 – 34 chromosomes, mainly 32 (Fig. 2a, Tab. 4). In H. umbellatum and H. maculatum 16 chromosomes were detected (Fig. 2b, Tab. 4). In squash preparations of H. hircinum 30 – 40 chromosomes were found, in the best preparations 32 chromosomes (Fig. 2c, Tab. 4). Especially in H. hircinum, chromosomes often so stuck together that even at different focal levels an unam- biguous assessment of the chromosomes was problematic. ISSR and SRAP marker analysis In all, 699 amplified DNA bands were scored using 11 ISSR primers (298 markers) and 16 SRAP primers (401 markers) for the 17 H. perforatum accessions and one H. maculatum accession (Hm22). The overall number of detected individual bands per primer ranged from 9 (SRP17) to 53 (SRP30), with an aver- age of 27.1/25.1 (ISSR/SRAP) (Tab. 5). The combined analy- sis of ISSR and SRAP markers revealed a total of 661 (94.6%) polymorphic bands. The marker performance was estimat- ed by two parameters: PIC value and resolving power (RP). ISSR primers and SRAP primer combinations showed the same mean PIC value (0.38). The highest PIC value was de- termined for primers UBC808, UBC809, UBC857, UBC873 and SRP26 (0.49). The resolving power (RP) of primers test- ed varied between 0.44 (UBC818/ SRP29) and 1.69 (UBC112). The mean values for Shannon’s information in- dex (I) and expected heterozygosity (He) were 0.39/0.25 for ISSR primers, and 0.44/0.29 for SRAP markers (Tab. 5). Combined ISSR-SRAP UPGMA revealed two major clusters (Fig. 3): Cluster I comprising all H. perforatum ac- cessions and cluster II represented by H. maculatum (Hm22) as a clearly separated outgroup. Within cluster I we ob- served 6 subclusters: Hp1 and Hp2 from Germany; Hp7 (Switzerland) and Hp9 (Austria); Hp13 (Poland) and Hp14 (Norway); Hp5 and Hp6 from Germany. French Hp10 and Hp11 and Italian Hp16 and Hp15 grouped with Hp17 from Estonia. Four H. perforatum accessions, Hp3 and Hp4 from Germany as well as Hp8 from Austria and Hp12 from France were separated in individual branches from the oth- er genotypes. H. maculatum is clearly distinguished from all H. perforatum accessions. Discussion Nuclear DNA content and ploidy The key to a successful breeding program is a better un- derstanding of the extent and nature of genetic diversity pres- ent in wild, conserved and/or actively utilized germplasm of various species. There is a broad interest in gaining a better understanding of diversity within Hypericum species, espe- cially in H. perforatum, due to its pharmaceutical importance but also for its remarkable evolutionary and adaptive capaci- ties. Therefore, we employed FCM for analysis of ploidy and genome size as well as two types of molecular markers (ISSR and SRAP) to reveal the genetic diversity and relationships among several Hypericum accessions. FCM is a powerful tool for genome size estimation and ploidy determination. With all the advantages and possi- Fig. 2. Chromosomes in root tips stained with DAPI. a – Hypericum perforatum Hp7, 32 chromosomes, b – H. maculatum Hm22, 16 chromosomes, c – H. hircinum Hh24, 32 chromosomes. Scale bar: 5 µm. Tab. 4. Chromosome number in root tips of different Hypericum accessions (Hp - H. perforatum, Hu – H. umbellatum, Hm – H. maculatum, Hh – H. hircinum). Accession № of chromosome counts Counted chromosomes Hp3 3 31 - 32 Hp4 2 30 - 31 Hp7 23 28 - 34, of which 10 times 32 chromosomes Hp9 1 32 - 34 Hu21 11 14 - 16 Hm22 9 13 - 16 Hh24 13 30 - 40, of which 5 times 32 chromosomes ANALYSIS OF HYPERICUM ACCESSIONS ACTA BOT. CROAT. 81 (1), 2022 7 bilities of FCM, however, it must be taken into account that the results are always expressed in relation to a known stan- dard. For genome size estimation (pg value) a plant with an already defined DNA content as internal standard should be measured together with the sample plant in the staining solution. For ploidy estimation, plants with a cytologically verified chromosome number within the same species serve as standard, providing that different ploidy degrees are in linear dependency. For 17 H. perforatum accessions from different botani- cal gardens across Europe and the cultivar Topaz, the ge- nome size was on average 1.58 pg ranging from 1.56 to 1.62 Tab. 5. Estimation of the genetic diversity of 18 Hypericum accessions. TNB – total number of bands, NPB – number of polymorphic bands, RP – resolving power-average, PIC – polymorphic information content, I – Shannon’s Information Index, He – expected heterozy- gosity, ISSR – inter-simple sequence repeats, SRAP – sequence related amplified polymorphism. Primer Detected amplification products % of polymor- phic loci RP PIC I He TNB NPB ISSR A 23 21 91.3 0.99 0.45 0.43 0.28 C 13 13 100.0 0.50 0.25 0.33 0.20 UBC808 24 20 83.3 1.38 0.49 0.49 0.33 UBC809 21 18 85.7 1.25 0.49 0.44 0.29 UBC811 46 46 100.0 0.58 0.27 0.41 0.25 UBC112 20 8 40.0 1.69 0.35 0.23 0.16 UBC818 31 31 100.0 0.44 0.21 0.33 0.20 UBC855 42 42 100.0 0.51 0.25 0.33 0.20 UBC856 24 22 91.7 0.98 0.45 0.42 0.27 UBC857 23 21 91.3 1.21 0.49 0.47 0.31 UBC873 31 27 87.1 1.27 0.49 0.45 0.30 Total 298 269 90.3 - - - - Average 27.1 24.5 - 0.98 0.38 0.39 0.25 SRAP SRP2 22 22 100.0 0.92 0.42 0.47 0.31 SRP5 22 22 100.0 0.76 0.35 0.45 0.29 SRP6 25 23 92.0 0.81 0.39 0.42 0.27 SRP11 21 21 100.0 0.78 0.37 0.43 0.28 SRP12 18 18 100.0 0.97 0.42 0.54 0.37 SRP13 26 26 100.0 0.88 0.41 0.46 0.30 SRP14 26 26 100.0 0.90 0.41 0.47 0.31 SRP15 16 16 100.0 1.08 0.45 0.55 0.38 SRP17 9 9 100.0 0.68 0.36 0.31 0.19 SRP20 21 20 95.2 0.89 0.41 0.47 0.31 SRP25 25 25 100.0 1.09 0.46 0.52 0.35 SRP26 22 18 81.8 1.37 0.49 0.48 0.33 SRP27 30 29 96.7 1.01 0.44 0.52 0.35 SRP28 30 30 100.0 0.46 0.22 0.33 0.20 SRP29 35 35 100.0 0.44 0.21 0.32 0.19 SRP30 53 52 98.1 0.58 0.29 0.34 0.21 Total 401 392 97.8 - - - - Average 25.1 24.5 - 0.85 0.38 0.44 0.29 ISSR + SRAP Total 699 661 94.6 - - - - Average 25.9 24.5 - 0.90 0.38 0.42 0.28 BUTIUC-KEUL A., COSTE A., BUDAHN H., DUNEMANN F., FARKAS A., POSTOLACHE D., KLOCKE E. 8 ACTA BOT. CROAT. 81 (1), 2022 pg in the different tetraploid accessions. Chromosome counting in root tips of different Hypericum plants with the corresponding genome size of 1.58 pg revealed 2n = 4x = 32 chromosomes. These results are in agreement with Temsch et al. (2010) (2C = 1.59 pg), and Alan et al. (2015) (2C = 1.50 pg). The slight differences result from employment of dif- ferent standards, extraction / staining buffers and flow cy- tometers (Doležel and Bartoš 2005) but the use of various accessions and the physiological state of the plant material also have an influence on the measurement. Matzk et al. (2003) developed the flow cytometric seed screen (FCSS), boosted the investigation of apomixes of Hypericum and broaden the knowledge about apomictic pathways in general. However, reports about the germina- tion capacity of the differently developed seeds and their contribution to plant populations are very limited. Thought, such information would have an impact on germplasm management and preservation strategies of rare natural populations. Mixed cytotypes in populations could provide novel traits for crop development and therefore the seed samples in gene banks should be adapted to that situation. Hypericum perforatum is predominately tetraploid (Matzk et al. 2003, Galla et al. 2011). Barcaccia et al. (2007) describe wild populations of H. perforatum in more detail as popu- lations with diploid and polyploid (mainly tetraploid) plants. Savaş Tuna et al. (2017) analyzed three seedlings from 39 Hp accessions each from different regions of Turkey and revealed a nuclear DNA content between 0.8 – 2.57 pg. Of the 39 accessions, one was diploid, 5 hexaploid and 33 tetraploid but no ploidy variation was noticed inside the accessions. Perhaps the test of only three seedlings per accession is not sufficient to provide reliable findings. Here we present flow cytometric DNA size determination of plants of 17 belonging to Hp accessions and the cultivar Topaz. Tetraploidy was revealed with only one exception: all plants from Hp8 from Salzburg have shown a DNA size of 2.41 pg, corresponding to the hexaploid level. This uni- formity could be explained by the origin of seeds in plants cultivated in botanical garden, most probably the collection being not very diverse. In four accessions (Hp6-7 and Hp9-10), there were mixed cytotypes (4x + 6x). This is an interesting situation, as Hp 6-7 seeds were collected from plants culti- vated in botanical gardens and Hp 9-10 belong to plants from natural populations. Similar results were found in three H. perforatum accessions (Qu et al. 2010). Despite the diverse embryo and endosperm ratios observed in seeds, Qu et al. (2010) found only tetraploids and hexaploids in seedling populations with tetraploids constituting 87 - 97% but a complete hexaploid population was not detected. Among the accessions presented here the accession Hp8 shows only hexaploid plants indicating that such accessions at least with a high proportion of hexaploid plants could exist and depend on the seed source. Moreover, in accession Hp3 a single plant was found with 1.99 pg. Since it does not fit the 1C content of 0.40 pg, one can assume that it is an aneuploid plant reflecting additionally the high plasticity of the H. perforatum genome, even if Hp3 comes from plants that are cultivated in a botanical garden. To our knowledge we report for the first time the ge- nome size of H. maculatum (two accessions, one from a natural habitat and one cultivated in a botanical garden), H. umbellatum (from natural habitat) (0.76 pg each) and H. Fig. 3. UPGMA dendrogram generated by Jaccard’s similarity coefficients showing relationships among 18 Hypericum accessions based on combined data from ISSR-SRAP (inter-simple sequence repeats analysis – sequence related amplified polymorphism). The samples are labeled with the codes listed in Tab. 1. ANALYSIS OF HYPERICUM ACCESSIONS ACTA BOT. CROAT. 81 (1), 2022 9 hircinum (from botanical garden) (1.00 pg). The first three accessions are native to Romania. Hence, the DNA size of H. hircinum of 1.0 pg is between the estimated DNA size for the diploid species H. maculatum und H. umbellatum (0.76 pg) and the tetraploid H. perforatum (1.58 pg). The genome size for H. maculatum and H. umbellatum corresponds to the chromosome number 2n = 2x = 16. For H. hircinum Loon and Jong (1978) published a chromosome number 2n = 40. They explained the high chromosome number of 40 as a possible pentaploid cytotype. Castro and Rosselló (2006) found 2n = 32 in H. hircinum subsp. cambessedesii, an endemic plant from the Balearic Islands. The H. hircinium accession investigated in the present paper was provided by the Alexandru Borza Botanical Garden from Cluj-Napoca, Romania. In this accession, we observed 30 - 40 chromo- somes; in the best metaphase plates 32, which is in agree- ment with the findings of Castro and Rosselló (2006). The result was surprising because H. hircinum has a much lower DNA size than H. perforatum (1.58 pg) although it also has 32 chromosomes. This fact underlines the high variability of the genus Hypericum. Genetic polymorphism Characterization of Hypericum species by different mo- lecular marker types has been performed over the years (Corral et al. 2011, He and Wang 2013). ISSR and SRAP markers were chosen due their advantages: cost efficiency, informativeness, versatility and reproducibility. We em- ployed a set of 11 ISSR primers and 16 SRAP primer com- binations to assess the genetic diversity among one Hm and 17 Hp accessions. ISSR and SRAP markers have proved to be highly polymorphic. The average number of polymor- phic bands per primer was the same (24) for both types of markers. Several studies report that when several marker types are used, they can be complementary tools for genet- ic diversity analysis, because they can be used to amplify different regions of the genome (Chen et al. 2013). Our study revealed a significantly higher rate of polymorphism in the analyzed Hypericum germplasm for both SRAP (97.8%) and ISSR (90.2%) markers than previously reported (He and Wang 2013). This might be explained by a larger sampling area with very different Hp accessions from botanical gar- dens in Europe and native wild accessions from Romania. The two marker systems used in our study revealed close degrees of resolution (Tab. 5). Species cluster analyses based on combined ISSR-SRAP data (Fig. 3) indicate that H. maculatum is closely related to some H. perforatum accessions. These taxa, belonging to section Hypericum “core Hypericum” (Nürk 2011), were proven to be in close contact and apparently hybridize fre- quently, which might explain the sympatric occurrence of morphologically similar taxa (Robson 2003, Koch et al. 2013). Thus, according to Brutovská et al. (2000), H. perforatum probably originates from autopolyploidization of an ancestor closely related to diploid H. maculatum, while Robson (2003) regards H. perforatum as an allopolyploid, derived from a cross between H. maculatum subsp. immaculatum and H. attenuatum. This close relationship between the two species was also supported by later studies based on different marker types, such as RFLP and cpDNA markers, as well as phylogenetic studies using nuclear internal transcribed spacer (ITS) sequences (Pilepić et al. 2011). However, it was recently implied that H. perforatum is not of hybrid origin (Koch et al. 2013). The authors suggest that H. perforatum has a single evolutionary origin arising from independent and recurrent polyploidization of two different ancestral gene pools along with occurrence of substantial gene flow within and between H. perforatum and H. maculatum. Regarding the cluster analysis of H. perforatum acces- sions, we have noticed a partially regional-based relation- ship. Some accessions from the same regions shared similar genotypes and ploidy levels (Hp1 and Hp2 from Germany; HP15 and HP16 from Italy), and the same was noticed for accessions from different geographical locations (HP9 from Austria and HP7 from Switzerland; HP13 from Poland and HP14 from Norway) (Tab. 1, 3; Fig. 3). Moreover, mixed ploidy accessions (4x and 6x) (Hp5 and Hp6; Hp9 and Hp7; Hp10 and Hp11) shared similar genotypes, while the com- plete hexaploid accession HP8 from Austria is clearly of dif- ferent genetic origin from that of the tetraploid Hp9 acces- sion from the same country (Tab. 1, 3; Fig. 3). This endorses the high plasticity in ploidy and reproductive system of H. perforatum, regardless of geographic origin (Koch et al. 2013). Differences among H. perforatum genotypes were re- ported in different H. perforatum wild populations and landraces as confirmed by molecular, morphometric and cytogenetic analyses (He and Wang 2013, Morshedloo et al. 2014). The high genetic diversity exhibited by our analysis might be explained by the diverse mating systems of H. perforatum from sexual cross to apomixis. Conclusions We report the 2C DNA content of 17 H. perforatum ac- cessions from different botanical gardens in Europe. 2C DNA content of H. perforatum found in our study was 1.58 pg. The tetraploid degree of the plants was confirmed by chromosome counting. FCM is a fast and reliable method for screening the variability inside a Hypericum accession concerning ploidy distribution. Besides the tetraploids, few hexaploid plants and one putative aneuploid plant were found in the H. perforatum accessions, independent of the origin of the seed. Mixed cytotypes (4x+6x) were identified in accessions from natural populations and cultivated in botanical gardens as well. One H. perforatum accession was characterized as hexaploid. The genome size of H. maculatum, H. umbellatum and H. hircinum was not previously reported and is 0.76 pg DNA for H. maculatum and for H. umbellatum, whereas H. hircinum has 1.00 pg DNA. This study demonstrated that both ISSR and SRAP markers were highly polymorphic in Hypericum, showing the prevalence of a wide range of diversity among the stud- ied accessions. The relative performance of ISSR and SRAP BUTIUC-KEUL A., COSTE A., BUDAHN H., DUNEMANN F., FARKAS A., POSTOLACHE D., KLOCKE E. 10 ACTA BOT. CROAT. 81 (1), 2022 markers was quite close, indicating that these markers are suitable for the determination of genetic diversity with high resolution among the Hypericum genotypes tested. Overall, marker analysis ensures information for potential applica- tions of the SRAP and ISSR marker systems in molecular breeding of Hypericum species. Complementary analysis of ploidy level and molecular markers of different accessions of Hypericum species could provide information for the se- lection of valuable accessions producing high level of natu- ral compounds useful for biotechnological applications. Acknowledgments This work was supported by DAAD scholarship (Re- search Stays for University Academics and Scientists, 2016, ID-57210259), BIOSERV 25N/2019 (core program PN2019- 2022 BIODIVERS 3) and 22PFE/2018. 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