Metabolic Profiles, Genetic Diversity, and Genome Size of Bulgarian Population of Alkanna tinctoria
by
, , , , , , and
Milena Nikolova
1,
Ina Aneva
1,
Petar Zhelev
2,
Ivanka Semerdjieva
1,3,
Valtcho D. Zheljazkov
4
,
Vladimir Vladimirov
1,5,
Stoyan Stoyanov
1,
Strahil Berkov
1 and
Elina Yankova-Tsvetkova
1,* 1
Department of Plant and Fungal Diversity and Resources, Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
2
Department of Dendrology, University of Forestry, 1797 Sofia, Bulgaria
3
Department of Botany and Agrometeorology, Agricultural University, Mendeleev 12, 4000 Plovdiv, Bulgaria
4
Department of Crop and Soil Science, Oregon State University, 3050 SW Campus Way, 109 Crop Science, Building, Corvallis, OR 97331, USA
5
Botanical Garden, Bulgarian Academy of Sciences, 1000 Sofia, Bulgaria
*
Author to whom correspondence should be addressed.
Plants 2023, 12(1), 111; https://doi.org/10.3390/plants12010111
Submission received: 22 November 2022
/
Revised: 21 December 2022
/
Accepted: 22 December 2022
/
Published: 26 December 2022
(This article belongs to the Special Issue Metabolism, Genetics and Biodiversity in Wild Crops Relatives and Herbals)
Abstract
:Alkanna tinctoria (L.) Tausch Boraginaceae is a medicinal plant whose root is used for its antimicrobial and anti-inflammatory properties. A. tinctoria roots have been subject to numerous studies. However, the aerial parts have been explored less. The objective of the present study was to compare the chemical profile of aerial parts and roots as well as the total alkannin content in roots of 11 populations of the species from different floristic regions of Bulgaria. Methanolic extracts from 22 samples were analyzed by GC/MS. Phenolic, fatty, and organic acids, sterols, polyols, fatty alcohols, and sugars were identified. Ononitol (4-O-methyl-myo-inositol) was found as the main compound in the aerial parts. The total alkannin content in the roots was evaluated by the spectrophotometric method and compared with that of the commercial product. Populations with high alkannin content and rich in other bioactive compounds were identified. A relatively low genetic diversity in the studied populations was observed. The present study is the first comprehensive study on metabolite profiles and genetic diversity of the Bulgarian populations of A. tinctoria. The occurrence of ononitol in the aerial parts of the species is reported for the first time, as well as the phenolic acid profiles of the species in both aerial parts and roots. The results showed that aerial parts of the plant are also promising for use as a source of valuable biologically active substances.
1. Introduction
Alkanna tinctoria (L.) Tausch (alkanet, alkanna) is a medicinal plant native to Southern Europe, Northern Africa, and Southwestern Asia. The species has limited distribution in Bulgaria; its habitats are located only in three floristic regions of the country in fragmented populations. The roots of the species are used as a source of red pigments in traditional communities and as a dye in the cosmetics, food, and textile industries [1,2,3]. The roots are also used as a natural remedy to prevent and treat ulcers, wounds, fever, inflammation, aging, and herpes [1,4]. In Bulgarian folk medicine, A. tinctoria roots are applied internally in the form of a decoction as an astringent for gastrointestinal disorders, and externally, to prepare ointments for application to wounds, pustules, and burns, and poultices made from the whole plant are used to treat mumps.
As the root is the used plant part of A. tinctoria, the majority of studies have been focused on the chemical composition and biological activity of the plant roots [2,3,5,6,7,8]. Studies on the other plant parts are rarer. Phytochemical analysis on A. tinctoria leaves extracts revealed that aqueous and ethanol extracts contained biologically active compounds, representatives of main chemical groups such as alkaloids, bufadienoloides, carbohydrates, flavonoids, gallotannins, phenolics, proteins, pseudotannins, resins, saponins, steroids, tannins, triterpenoides but individual compounds have not been identified [8]. The main phytochemical components of A. tinctoria roots that are responsible for its biological activity are the naphthoquinones alkannin, shikonin, and their derivatives: acetylalkannin, angelylalkannin, 5-methoxyangenylalkannin, acetylshikonin, dimethylacryl alkannin, naphtharzin, arnebifuranone, shikalkin, alkanfuranol and alkandiol [2,3,5,6]. A number of analytical techniques have been applied for the determination of the alkannin/shikonin (A/S) content of A. tinctoria [9,10]. High-performance liquid chromatography (HPLC) has been the most frequently utilized technique for qualitative and quantitative determinations of A/S and their derivatives [11,12,13], spectrophotometric method for quantitative assay of A/S content has been also applied [14,15,16].
The genetic variation of the populations of any species is a fundamental characteristic determining its capacity to persist and adapt to environmental changes [17,18]. The investigation of genetic diversity and structure of endangered species is of main importance for genetic resource conservation and plant breeding programs [19,20]. Revealing the genetic diversity of natural populations and cultivars is a basic requirement for the development of sustainable agricultural practices [21].
The genetic diversity of Alkanna species has been studied by employing different genetic markers and for different purposes. Wolff et al. [22] applied RAPD markers in their study on A. orientalis in the Sinai Desert, Egypt, and concluded that the main drivers of gene flow are periodic flash floods. The same classes of markers were applied by Abdel-Hamid [23] to distinguish between two subspecies of A. tinctoria in Lybia. Recently, more informative codominant markers have been developed for describing the genetic diversity of Alkanna species [24]. Genetic variation of three endemic Alkanna species in Bulgaria was studied by Semerdjieva et al. [25] based on highly polymorphic ISSR markers. The authors provided important inferences about the taxonomic and evolutionary relationships of the studied species. Taxonomic markers, like rpl32-trnL (UAG) и trnH–psbA [26], were applied successfully to different systematic groups, including the species of the Boraginaceae family. Even though these markers have some limitations, they could bring additional insight into the phylogenetic relationships among the Alkanna species. The genetic structure and its determinants in A. tinctoria and A. sieberi from Greece were studied by Ahmad et al. [27].
The genome size is a highly relevant characteristic of living organisms that presents the DNA content of the non-replicated gametic chromosome set [28,29]. It is defined with the term C-value proposed by Swift [30]. This value has been considered characteristic and invariable for each species. Information on the C-value can be useful in different areas of plant science, including ecology and phytogeography [29,31,32]. A correlation between genome size and environmental conditions has been reported by Bennett and Smith [33] and Grime and Mowforth [29]. Relationships between DNA amount and latitude [33], altitude [34], temperature [35], fertilizer treatment [36,37], and seedling growth rate [38] have been found.
The citometrical studies in Alkanna species are few. The genome size of A. tinctoria and A. sieberi was previously reported by Ahmad et al. [27].
The aim of the present work was to compare GC/MS-based metabolite profiles of the aerial parts and roots of 11 populations of A. tinctoria from different floristic regions as well as total alkannin content in roots in correlation with their origin (genetic profile and genome size) in order to facilitate the selection of desirable accessions for possible cultivation. The introduction of A. tinctoria into culture would preserve its natural populations in the Bulgarian flora.
2. Results
2.1. Phytochemical Study
2.1.1. GC/MS Analysis
Twenty-four (24) metabolites were identified in the methanolic extracts from 22 samples of roots and aerial parts of A. tinctoria following GC/MS analyses (Appendix A Table A1 and Table A2). The identified compounds were phenolic, fatty, and organic acids, sterols, polyols, fatty alcohols, and sugars (Appendix A Table A1 and Table A2). Similar metabolic profiles with the quantitative variation of individual components between the studied samples of the different populations were found. Sucrose was the main metabolite in the roots. Monosaccharides were identified in large amounts also. Caffeic acid was the most abundant phenolic acid. The highest amounts of phenolic acids were found in the samples from the Danubian Plain (AT1, AT2). Eastern Rhodopes (AT11) and the River Struma Valley (AT3, AT9).
Ononitol (4-O-methyl-myo-inositol) was identified as the main component in the aerial parts of the studied samples (Table 1 and Figure 1). Ononitol identification in the extracts was made by comparing its mass spectra and RI with mass spectral databases (NIST; GOLM; Figure 2). The highest content of ononitol was found in the samples from the River Struma valley (AT3, AT10). In addition, the samples from this location had the highest content of sugars and sugar derivatives. Seven phenolic acids were identified; among them, caffeic, 4-hydroxybenzoic, and 4-hydroxycinnamic were the main ones. The highest content of phenolic acids was found in the samples from the River Struma Valley (AT10, AT5, AT3, AT9). Whole phenolic acids were more abundant in the aerial parts than in the roots. Sterols and sucrose were present in higher quantities in the roots than in aerial parts.
Generalized comparative data of established qualitative and quantitative metabolite composition of plant parts (roots and aerial parts) of studied A. tinctoria populations are presented in Table 1. The aerial parts of the species contained 4-hydroxybenzoic and caffeic acids and all identified organic acids in larger quantities than in those in the roots. However, the roots contained higher concentrations of monosaccharides, disaccharide (sucrose), sterols, and fatty alcohols. Ononitol was identified only in the aerial parts of the species.
2.1.2. Spectrophotometric Analysis of Alkannin Derivatives
The total alkannin content in the hexane extracts of the roots of the studied samples was determined spectrophotometrically (Table 2). The highest alkannin content, exceeding that of the control sample, was found in the root extract of the population from Kresna town (AT3) in the Struma Valley region. Comparable alkannin content to that of the control was found in the sample from Lebnitsa village (AT8), Ilindentsi village (AT4), Kulata village (AT10), and also from the River Struma Valley region as well as Ostrov village, Danubian Plain (AT3). The lowest alkannin content was found in the sample from Spatovo village, the Struma Valley region (AT8), and Odrintsi village, at the Eastern Rhodopes Mountains (AT11).
2.2. Study of Genetic Diversity
The percentage of polymorphic bands in the studied populations ranged from 55 to 72 (mean 61.5%) and was the highest in the northern population AT1 (Table 3). A similar percentage of polymorphic loci (64.58) was established previously in other Alkanna species from Bulgaria (A. graeca) by Semerdjieva et al. [25]. The genetic distances among populations were relatively low, they ranged from 0.01 to 0.14, and the most distinct was the population Orsoya (AT1), situated at 1–1.5 km distance from the Danube River and at an air distance of at least 200 km from the remaining populations (results not shown). The cluster dendrogram (Figure 3) demonstrates the distinct position of population AT1 while the other populations are close to each other, and the grouping does not indicate a particular trend of spatial variation.
The estimated genetic diversity level in the studied population of A. tinctoria was low (He = 0.220–0.298), and with similar values in individual populations (Table 3)
2.3. Flow Cytometry Analysis
The estimated values of the DNA content in fresh leaves of the studied specimens of A. tinctoria are presented in Table 4 and show remarkable congruence—1C = 1.326 pg in both studied populations.
The estimated values of the DNA content in the seeds of A. tinctoria are presented in Table 5.
The CV of measurement of 5000 PI-stained particles in the samples, each containing 10 mature seeds from 11 A. tinctoria populations (AT1 to AT11), with no internal standard, varied in the range 4.7–10.7%. No difference between the populations was detected in the position of the peaks on the obtained histograms, i.e., similar fluorescence mean values were estimated for the different samples under the same gain values of the flow cytometer.
3. Discussion
3.1. Phytochemical Study
3.1.1. GC/MS Analysis
Primary and secondary metabolites were identified by GC/MS analysis in the aerial parts and roots of A. tinctoria. The identified lipid compounds in the roots of the species are in accordance with previously reported data [39]. The present study provided data on phenolic acids profiles of the species both in the aerial parts and roots for the first time. Previous studies concerning the chemical composition of aerial parts of A. tinctoria reported only total phenolic content without identification of the individual compounds [1,40]. An important result of the present study was the identification of ononitol in the aerial parts of A. tinctoria. Because chemical composition studies have previously been focused on A. tinctoria roots, ononitol has not been reported in this species until now. Ononitol (4-O-methyl-myo-inositol) belongs to the group of polyols or cyclitols (Inositols). This compound, together with pinitol, are distinguished from the other methyl derivatives (such as bornesitol) and isomers of inositol by the presence of fragment ion at m/z 260 in their mass spectra [41]. Previous studies reported that ononitol exhibited important biological activities such as hepatoprotective, analgesic, and anti-inflammatory [42,43]. It has been reported that O-methyl-inositols such as ononitol are accumulated in plants in response to abiotic stresses such as drought and salts [44,45]. Ononitol has been reported mainly for Fabaceae species but also for Brassicaceae and Ericaceae [46]. In Boraginaceae, bornesitol (1-O-methyl-myo-inositol) seems to be widespread [47], but information about the distribution of ononitol in this family was not found. The occurrence of ononitol in the aerial parts of A. tinctoria becomes the first report on the presence of ononitol in the family Boraginaceae.
3.1.2. Spectrophotometric Analysis
Differences in roots alkannin content between samples collected from different floristic regions and from different localities within one region were found. The alkannin content found in this study was comparable to those reported previously on Bulgarian populations of A. tinctoria [14]. Genova et al. [14] studied roots of the species from five localities in the Struma Valley Region different from those of the present study, and found variation of alkannin content from 3.40% to 13.00%. The latter authors found the highest amount of alkannin in the corky tissue of the main and lateral roots of medium adult individuals (10.06–12.85%) as well as in the corky tissue of young roots (11.99%). Less alkannin content was found in the secondary cortex, and no alkannin was found in secondary wood [14]. Papageorgiou et al. [48] reported also that the root age was crucial for shikonin content of Lithospermum erythrorhizon Sieb. et Zucc. In the cell suspension cultures, it has been found that shikonin derivative production was strongly affected by light, temperature, and pH of the medium. Alkaline pH (7.25–9.50), temperature 25 °C, and dark were reported to be optimal conditions for increased alkannin production [16]. In the present study, a low alkannin content was found in the sample (AT11) that was collected from calcareous rock, which gives us reason to assume this rock is probably unfavorable to accumulation of alkannin
Overall, the different amounts of alkannin and other biologically active compounds synthesized in each of the studied populations of A. tinctoria could be due to a number of factors such as (1) genotype; (2) soil type; (3) exposition; (4) environmental conditions; (5) the content of the available soil macro and micronutrients [49,50,51]; (6) fungal infections [52,53,54,55]; and (7) physiological status of plants [56]. Environmental factors are often proved to have a significant effect on the concentration, with lower effect on the composition of biologically active substances. In the case of Sinopodophyllum hexandrum, climate factors were more important than the soil properties [57]; however, the majority of studies revealed that these peculiarities are species-dependent [58,59]. The studied populations in the Struma River Valley (AT6, AT7) and the Eastern Rhodopes Mountain (AT11) are influenced by a Mediterranean climate, but their habitats differ. For example, the species distributed in the Eastern Rhodopes Mountain grow in Leptosols soils or calcareous rock with low anthropogenic impact due to the area’s depopulation. In contrast, in the Struma River Valley, the species grow on the Siliceous or Calcareous stony slopes in grassy communities. The species in these populations (AT6, AT7) have been exposed to greater anthropogenic impacts such as grazing and trampling. The conditions in the populations of the Danubian Plain were different (AT1, AT2), where the species grow on Inland sandy dunes and are influenced by a temperate-continental climate (Table 6). The present results confirmed previous research that, in general, the concentrations and type of plant secondary molecules are determined by the species, genotype, developmental stage, physiology, and environmental factors during growth [56].
3.2. Study of Genetic Diversity
Studies on the distribution of genetic diversity in the populations of Alkanna species have shown that, in most cases, there were no noticeable differences in the level of intrapopulation diversity among populations. The assessment of three endemic Alkanna species in Bulgaria by ISSR markers revealed close values of the diversity parameters, such as percent of polymorphic loci, expected heterozygosity, and polymorphism index of Shannon [25]. The preliminary results of our study have shown that the level of genetic diversity in A. tinctoria was comparable with that of the other Alkanna species. It should be noted that the comparison in this study was based only on one type of marker (ISSR) with dominant inheritance. Including other types of markers, such as microsatellites, or simple sequence repeats (SSR), already developed for A. tinctoria [24] could provide valuable additional information about the level of genetic diversity within and among populations.
This study demonstrated a low level of genetic diversity in the Bulgarian population of A. tinctoria that correlates with its limited distribution. Lower genetic diversity is attributed to endemic species and to species that are not widely distributed [60]. The low level of genetic diversity in their populations is due to the non-plasticity caused by the narrow distribution [61]. In turn, the reduction in genetic variation might suggest a decline in adaptation to a changing environment, leading to an increased danger of extinction and increased inbreeding [62,63]. In the studied Bulgarian populations of A. tinctoria, the established genetic diversity was low, while in the Greek populations of the species, Ahmad et al. [27] found moderate genetic diversity. This fact is consistent with the assumption of the same authors that limitation in population size and distribution is a major factor influencing population genetic diversity, which decreases with decreasing population size: the species is more common in Greece and other Mediterranean countries compared to Bulgaria, where it is considered very rare [64].
3.3. Flow Cytometry Analysis
This is the first report on the DNA content in the Bulgarian accessions of A. tinctoria. The estimated value of 1C = 1.326 pg was congruent with the data published for the species from Greek accessions (1C = 1.21–1.28 pg, [27]). Studies in the genome size in mature seeds, 10 seeds per population in a total of 11 populations (AT1 to AT11), revealed that 1C varied in the range of 1.28 to 1.49 pg (Table 4). As it was observed in the Greek populations [27], no pattern of variation of the C-values was observed speaking in favor of variation in the ploidy level within and between populations. Moreover, the observed small variation did not exhibit any geographical pattern. For example, the range of variation of the two accessions from the Danubian Plain floristic region in North Bulgaria was 1C = 1.29–1.31 pg, whereas, in the Struma River Valley in the South Bulgaria region, the variation was 1C = 1.28–1.43 pg. The analysis of the measurement histograms of the samples, each containing 10 seeds of A. tinctoria without internal standard, showed there were no different classes of the observed fluorescence mean values (i.e., only one peak per run present). This suggests there is no variation in the ploidy level between the different seeds (respectively, different individuals). This finding agrees with the established normal running of the reproductive processes leading to the formation of reduced male (sperm cells) and female (embryo sacs) gametes in A. tinctoria [65].
In general, flow cytometry is a useful screening method for estimating the reproductive development pathways using mature seeds. Matzk et al. [66] presented 10 different pathways of seed formation depending on the participation of reduced or unreduced gametes and the type of formation of the embryo and endosperm. While measuring the DNA content in the seed samples from A. tinctoria, only one peak was formed and clearly visible on the histograms. This suggests that only PI-stained nuclei of the embryos were detectable, and no endosperm was present. The latter was confirmed by the embryological studies on the species [65]. In this case, the observed pattern of peak formation on the histograms can be explained by two development pathways of seed formation; the fusion of reduced egg and sperm cells (amphimixis) or autonomous development of an unreduced egg cell (apomixis) (cf. [66], Figure 1). Thus, it can be inferred that flow cytometry alone cannot be used for estimating the seed development pathway in A. tinctoria due to the lack of endosperm in the mature seeds.
Ahmad et al. [27] established a chromosome number of 2n = 30 in the studied Greek populations of A. tinctoria. This number agrees with the karyological findings of other authors for the species [67,68,69], including for a Bulgarian population [70]. Ahmad et al. [27] consider A. tinctoria as a tetraploid species. Polyploids are known to have advantages over diploids that make them more suitable for growing as crops due to a number of gains, such as pathogen resistance, temperature stress alleviation, salinity-induced stress alleviation, and drought stress alleviation [71].
4. Materials and Methods
4.1. Plants Material
The plant material used in the study, roots, and aboveground parts were collected from plants at the flowering stage from 11 Bulgarian populations of A. tinctoria located in three different floristic regions of the country (Table 6, Figure 4). A commercial sample of A. tinctoria roots was used as a control sample (AT0).
Figure 4.
Map of the studied localities of A. tinctoria. ●/red circle/: the established localities.
Table 6.
List of the studied localities of A. tinctoria in Bulgaria.
| Floristic Region | Locality | GPS Data | Altitude | Population Size, ha | Habitat |
|---|---|---|---|---|---|
| Danibian Plain | Orsoya village, Lom district (AT1) | N 43.78639 E 23.09111 | 30 | 150 | Inland sandy dunes |
| Danibian Plain | Ostrov village, Oryahovo district (AT2) | N 43.66873 E 24.10173 | 30 | 50 | Inland sandy dunes |
| Valley of River Struma | North of Kresna town (AT3) | N 41.73182 E 23.15583 | 210 | 0.1 | Siliceous stony slope |
| Valley of River Struma | North of Ilindentsi village, Strumyani district (AT4) | N 41.65349 E 23.23062 | 380 | 1 | Calcareous stony slope |
| Valley of River Struma | North of Mikrevo village, Strumyani district (AT5) | N 41.63887 E 23.17440 | 170 | 1 | Siliceous stony slope |
| Valley of River Struma | West of Ploski village, Sandanski district (AT6) | N 41.61957 E 23.23851 | 300 | 0.1 | Grassy communities on sandy soils |
| Valley of River Struma | North of Struma village, Sandanski district (AT7) | N 41.55849 E 23.23130 | 130 | 3.5 | Grassy communities on sandy soils |
| Valley of River Struma | South of Lebnitsa village, Sandanski district (AT8) | N 41.50464 E 23.25049 | 110 | 0.5 | Siliceous stony slope |
| Valley of River Struma | West of Spatovo village, Sandanski district (AT9) | N 41.50243 E 23.31010 | 180 | 2.5 | Grassy communities on sandy soils |
| Valley of River Struma | East of Kulata village, Petrich district (AT10) | N 41.39304 E 23.37154 | 160 | 1.5 | Grassy communities on sandy soils |
| Eastern Rhodopes Mountain | East of Odrintsi village, Ivaylovgrad district (AT11) | N 41.43415 E 26.14828 | 90 | 0.5 | Grassy communities on calcareous rock and Leptosols soils |
4.2. Methods
4.2.1. Phytochemical Study
Extraction Procedure
One hundred (100 mg) powdered roots of each A. tinctoria sample were extracted with 1 mL of MeOH for 24 h at room temperature. A 50 μL (1 mg/mL) of 3,5 dichloro-4-hidroxy benzoic acid was added at the beginning of the extraction as an internal standard.
GC/MS Analysis
Dried methanolic extracts of the samples were dissolved in 50 μL of pyridine. Then, 50 μL of N.O-bis-(trimethylsilyl)trifluoroacetamide was added, and the samples were heated at 70 °C for 2 h. After cooling, the samples were diluted with 300 μL of chloroform and analyzed using GC-MS. The GC–MS spectra were recorded on a Thermo Scientific Focus GC coupled with Thermo Scientific DSQ mass detector (Austin, TX, USA) operating in EI mode at 70 eV. The chromatographic conditions were previously described by Berkov et al. [72]. The measured mass spectra were deconvoluted using AMDIS 2.64 software before comparison with the databases. Retention Indices (RI) of the compounds were measured with a standard n-alkane hydrocarbons calibration mixture (C9–C36) (Restek. Cat No. 31614, supplied by Teknokroma, Barcelona, Spain). The compounds were identified by comparing their mass spectra and retention indices (RI) with those of authentic standards and the National Institute of Standards and Technology (NIST) spectra library. The response ratios were calculated for each metabolite relative to the internal standard using the calculated areas for both components.
Spectrophotometric Analysis
The powdered roots of A. tinctoria samples (0.250 g) were extracted with n–hexane (50 mL × 1) under reflux for 20 min, and after filtration, the rest plant material was extracted with 20 mL × 2 for 15 min each extraction. The combined filtrates were poured into a 100 mL measuring flask, and it was filled to the measure with n-hexane. The absorption of the resulting solutions was recorded in quartz cuvettes at a length of wave λ = 520 nm against pure n–hexane using a spectrophotometer (Jenway 6320D). Alkannin derivatives were quantified by a standard curve, which was made by using a standard of alkannin in hexane in the concentration range of 0.02–0.7 mg/mL. Quantification was done according to Genova et al. [14]. A commercial product of A. tinctoria roots was used as a control sample.
4.2.2. Study of Genetic Diversity
Six samples from the studied natural populations of A. tinctoria were included in the genetic analysis. Most of them were from Southwest Bulgaria, and one was from the Danubian plain (Table 3). A total of 2 to 12 individuals per population was studied. The samples collected were dried in silica gel before the analysis. The extraction of DNA was done using two approaches: (1) by CTAB protocol [73] and (2) by using Invisorb® Spin Plant Mini Kit (Invitek Molecular GmbH, Berlin, Germany), following the instructions of the producer. The quality of the extracted DNA was tested by spectrophotometer NanodropTM Lite (Thermo Fisher Scientific, Waltham, MA USA).
Seven Inter Simple Sequence Repeats (ISSR) markers were used to characterize the level of genetic diversity within and among populations of the species. The markers used and other details of the methods applied are presented in Table 7. The electrophoresis and other procedures were performed according to Semerdjieva et al. [25] and Petrova et al. [74].
Data analysis considered the specific patterns of ISSR markers variation. Analysis of dominant marker data, incl. ISSRs is more difficult than that of codominant data and not as straightforward. In the latter, when studying diploid organisms, heterozygotes can be scored directly from the electropherograms, while dominant markers require a different approach [75,76,77].
A binary matrix was constructed using the presence (1) and absence (0) of a particular band. Expected heterozygosity and percent of polymorphic bands were used to characterize the within-population diversity, while Nei’s [78] genetic distances among the population pairs were calculated to reveal the inter-population variation. Genetic distances were used as raw data for applying cluster analysis to reveal the grouping among the studied populations. Software DendroUPGMA (version 2002) [79] was used to construct the dendrogram reflecting the grouping of genetically similar populations.
4.2.3. Flow Cytometry Analysis
Nuclear DNA content was measured by CyFlow SL Green flow cytometer (PARTEC, Germany), equipped with a green (532 nm) solid-state laser. Two types of plant material were used for the study. For precise measurement of the DNA content, fresh leaves collected in the field and then cultivated in the vegetation house of IBER—BAS plants were used for the study. Pisum sativum ‘Kleine Rheinländerin’ (1C = 4.38 pg, [80]) was applied as an internal standard. The plant material was treated with the extraction and staining kit ‘CyStain® PI Absolute P’ (SYSMEX) following the protocol prescribed by the manufacturer. Altogether 5000 PI-stained particles were measured per run, with 5 runs per preparation. Runs with a CV above 5% for the Alkanna plants or the standard were discarded. For detecting any deviations in the ploidy level of the Alkanna individuals, mature seeds were used. Ten seeds of Alkanna and a piece of fresh leaf of Pisum sativum ‘Kleine Rheinländerin’ were co-chopped with a razor-blade and treated simultaneously with the extraction and staining kit ‘CyStain® PI Absolute P’ (SYSMEX) following the protocol prescribed by the manufacturer. Only one run per preparation was measured, with 5000 PI-stained particles per run. Additionally, the same treatment of 10 seeds of Alkanna per preparation was done without an internal standard in order to estimate any significant deviations in the DNA content without influence from the internal standard.
5. Conclusions
The phytochemical analysis revealed that Bulgarian plants of Alkanna tinctoria (roots and aerial parts) are rich in valuable bioactive compounds such as alkannin and ononitol, especially those from Kresna (AT3), North of Ilindentsi village (AT4), South of Lebnitsa village (AT8), in the Struma valley, Ostrov village (AT2), in the Danubian Plain. Plant material from these locations was recommended as a promising source for cultivation as a crop in order to provide a raw material for the established market needs and to preserve the natural populations of the species. However, further agronomic studies would need to be conducted to identify the optimal soil, water, nutrient requirements, harvest, and post-harvest handling of Alkanna tinctoria as an agronomic crop, which may take a significant amount of time.
The present study demonstrated that aerial parts of the plant are also promising as a source of valuable biologically active substances such as ononitol, which has shown pronounced hepatoprotective, analgesic, and anti-inflammatory activity. This study reported ononitol as a newly discovered inositol for the genus Alkanna and the Boraginaceae family.
Author Contributions
Conceptualization, M.N. and E.Y.-T.; methodology, M.N., S.B., V.V., I.A. and P.Z.; validation, M.N. and S.B.; formal analysis, M.N., V.V., I.A. and P.Z.; investigation, M.N., V.V., I.A. and P.Z.; resources, S.S., I.S. and V.D.Z.; writing—original draft preparation, M.N. and E.Y.-T.; writing—review and editing, M.N., V.V., I.A., P.Z., I.S., V.D.Z. and E.Y.-T.; visualization, S.B., I.A., P.Z. and S.S.; supervision, E.Y.-T.; project administration, E.Y.-T.; funding acquisition, E.Y.-T. All authors have read and agreed to the published version of the manuscript.
Funding
This research was funded by the Bulgarian National Science Fund, grant number KП-06-H26/6/13.12.2018.
Data Availability Statement
Not applicable.
Acknowledgments
The authors are grateful for the financial support provided by the Bulgarian National Science Fund (Grant KП-06-H26/6/13.12.2018) and for the technical support provided by the Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences.
Conflicts of Interest
The authors declare no conflict of interest.
Appendix A
Table A1.
Identified metabolites in the roots of A. tinctoria by GC/MS *.
| Compounds | RI | Studied Samples | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| AT0 | AT1 | AT2 | AT3 | AT4 | AT5 | AT6 | AT7 | AT8 | AT9 | AT10 | AT11 | ||
| Phenolic acids | |||||||||||||
| 4-Hydroxybenzoic | 1634 | 1.8 ± 0.8 | 0.9 ± 0.4 | 2.3 ± 0.9 | 0.6 ± 0.3 | 0.7 ± 0.2 | 2.4 ± 1.9 | 1.2 ± 0.7 | 0.5 ± 0.1 | 2.6 ± 1.8 | 0.8 ± 0.3 | 2.2 ± 1.3 | 1.1 ± 0.3 |
| Vanilic | 1776 | 1.0 ± 0.5 | 0.1 ± 0 | 0.3 ± 0.1 | 0.3 ± 0.1 | 0.2 ± 0.1 | 0.4 ± 0.2 | 0.3 ± 0.1 | 0.01 ± 0 | 0.1 ± 0 | 0.1 ± 0 | 0.5 ± 0.2 | 1.5 ± 0.1 |
| Protocatechuic | 1813 | 0.9 ± 0.6 | 0.2 ± 0.1 | 2.3 ± 0.0 | 1.0 ± 0.6 | 0.3 ± 0.1 | 0.4 ± 0.2 | 0.6 ± 0.3 | 0.5 ± 0.3 | 0.5 ± 0.3 | 0.3 ± 0.1 | 1.8 ± 0.9 | 2.9 ± 1.2 |
| Ferulic | 2086 | 0.6 ± 0.3 | 1.4 ± 0.8 | 0.1 ± 0 | 1.0 ± 0.5 | 0.1 ± 0 | 0.2 ± 0.1 | 1.1 ± 0.7 | 0.1 ± 0 | 0.1 ± 0 | 0.3 ± 0.1 | 0.2 ± 0.1 | 0.2 ± 0.1 |
| Caffeic | 2141 | 16.8 ± 4.4 | 24.8 ± 11 | 10.4 ± 2.3 | 28.4 ± 13 | 1.4 ± 0.6 | 5.0 ± 2.8 | 11.2 ± 1.7 | 5.3 ± 1.4 | 4.7 ± 2.1 | 14.9 ± 5.6 | 1.8 ± 1.1 | 13.7 ± 3.1 |
| Organic acids | |||||||||||||
| Succinic acid | 1310 | 11.8 ± 4.4 | 6.1 ± 3.3 | 6.5 ± 3.1 | 5.1 ± 3.1 | 11.5 ± 4.4 | 1.8 ± 0.7 | 15.1 ± 5.2 | 3.9 ± 1.1 | 17.1 ± 3.8 | 3.5 ± 1.8 | 6.0 ± 2.3 | 41.9 ± 19 |
| Malic acid | 1488 | 1.6 ± 1.0 | 2.4 ± 1.7 | 0.8 ± 0.3 | 24.2 ± 4.6 | 9.2 ± 3.1 | 1.3 ± 0.5 | 9.3 ± 2.7 | 1.2 ± 0.5 | 14.5 ± 1.6 | 1.6 ± 0.9 | 12.1 ± 2.6 | 17.5 ± 8.1 |
| Sugars and polyols | |||||||||||||
| Fructose 1 | 1855 | 111.6 ± 33 | 179.9 ± 47 | 155.7 ± 37 | 522.3 ± 98 | 12.7 ± 3.8 | 24.0 ± 5.8 | 514.3 ± 29 | 23.9 ± 5.5 | 284.7 ± 89 | 50.6 ± 23 | 11.2 ± 2.2 | 75.5 ± 25 |
| Fructose 2 | 1864 | 421.8 ± 41 | 129.7 ± 27 | 224.4 ± 51 | 779.1 ± 112 | 96.1 ± 28 | 39.7 ± 4.4 | 645.6 ± 143 | 8.1 ± 2.4 | 303.7 ± 153 | 22.4 ± 12 | 37.2 ± 3.9 | 14.2 ± 1.7 |
| Glucose | 1887 | 419.6 ± 55 | 124.6 ± 38 | 238.1 ± 38 | 403.2 ± 104 | 74.0 ± 22 | 40.8 ± 5.3 | 948.0 ± 63.8 | 78.5 ± 14 | 593.0 ± 196 | 94.6 ± 34 | 112.4 ± 33 | 247.4 ± 53 |
| Myo-Inositol | 2090 | 177.1 ± 48 | 1.7 ± 0.9 | 23.9 ± 5.6 | 113.9 ± 34 | 14.3 ± 6.5 | 79.6 ± 35 | 26.3 ± 8.9 | 15.1 ± 6.1 | 166.4 ± 67 | 32.5 ± 16 | 58.5 ± 18 | 19.4 ± 2.3 |
| Sucrose | 2628 | 535.3 ± 76 | 2813.9 ± 393 | 2197.8 ± 259 | 4215.4 ± 531 | 1431.3 ± 198 | 826.4 ± 106 | 5914.2 ± 448 | 534.5 ± 82 | 4298.9 ± 394 | 598.3 ± 69 | 1777.6 ± 58 | 2121.1 ± 293 |
| Lipids | |||||||||||||
| Hexadecanoic acid | 1925 | 40.7 ± 13 | 75.1 ± 10 | 86.3 ± 15 | 76.4 ± 24 | 11.7 ± 3.1 | 5.5 ± 2.1 | 52.0 ± 3.3 | 9.9 ± 4.2 | 36.2 ± 14 | 12.5 ± 5.5 | 74.1 ± 27 | 61.2 ± 23 |
| Octadecanoic acid | 2120 | 9.5 ± 3.4 | 18.5 ± 0.8 | 9.4 ± 2.2 | 21.3 ± 15 | 12.8 ± 2.8 | 3.4 ± 0.7 | 15.3 ± 1.9 | 0.1 ± 0 | 8.3 ± 3.1 | 8.9 ± 2.1 | 28.9 ± 10 | 12.6 ± 4.3 |
| Teracosanol | 2740 | 1.2 ± 0.8 | 5.1 ± 1.1 | 3.8 ± 1.4 | 2.1 ± 0.3 | 5.5 ± 2.2 | 5.2 ± 0.7 | 5.2 ± 1.6 | 0.1 ± 0 | 3.7 ± 1.1 | 1.4 ± 0.7 | 5.0 ± 2.3 | 21.1 ± 5.5 |
| Hexacosanol | 2938 | 0.1 ± 0 | 7.0 ± 2.8 | 8.5 ± 1.7 | 1.2 ± 0.4 | 1.6 ± 0.9 | 11.3 ± 3.4 | 3.6 ± 1.9 | 1.5 ± 0.3 | 0.1 ± 0 | 1.7 ± 0.8 | 4.7 ± 2.0 | 3.0 ± 1.2 |
| Campestrol | 3239 | 4.7 ± 1.4 | 32.3 ± 14 | 15.2 ± 2.8 | 6.2 ± 0.9 | 4.2 ± 1.1 | 5.2 ± 1.3 | 0.1 ± 0 | 2.6 ± 1.1 | 4.9 ± 2.1 | 6.4 ± 2.2 | 15.8 ± 6.7 | 0.1 ± 0 |
| Stigmasterol | 3319 | 0.1 ± 0 | 19.5 ± 0.9 | 0.6 ± 0.2 | 0.1 ± 0 | 1.3 ± 0.8 | 2.8 ± 0.5 | 6.1 ± 1.3 | 1.3 ± 0.4 | 0.1 ± 0 | 2.6 ± 0.9 | 2.9 ± 1.1 | 0.1 ± 0 |
| β-Sitosterol | 3335 | 23.8 ± 3.9 | 112.5 ± 41 | 126.0 ± 20 | 32.8 ± 9.5 | 13.6 ± 4.4 | 15.4 ± 5.9 | 32.6 ± 7.4 | 20.9 ± 7.1 | 30.1 ± 12 | 45.9 ± 19 | 28.7 ± 17 | 37.0 ± 16 |
* The amounts of metabolites are represented as response ratio that represents peak area ratios using 3,5 dichloro-4-hidroxybenzoic acid (50 μg) as quantitative internal standard.
Table A2.
Identified metabolites of the aerial parts of A. tinctoria by GC/MS *.
| Compounds | RI | Studied Samples | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| AT1 | AT2 | AT3 | AT4 | AT5 | AT6 | AT7 | AT8 | AT9 | AT10 | AT11 | ||
| Phenolic acids | ||||||||||||
| 4-Hydroxybenzoic | 1635 | 7.5 ± 4.1 | 6.4 ± 3.2 | 7.9 ± 3.4 | 2.0 ± 0.8 | 5.2 ± 2.2 | 2.4 ± 0.9 | 5.8 ± 2.3 | 4.1 ± 1.7 | 2.8 ± 0.7 | 5.6 ± 2.5 | 2.3 ± 0.6 |
| Vanilic | 1776 | 0.4 ± 0.2 | 0.3 ± 0.2 | 0.4 ± 0.2 | 0.1 ± 0 | 0.3 ± 0.1 | 0.2 ± 0 | 0.4 ± 0.2 | 0.2 ± 0.1 | 0.3 ± 0.1 | 0.5 ± 0.2 | 0.3 ± 0.1 |
| 4-Hydroxycinnamic cis | 1783 | 0.6 ± 0.3 | 0.3 ± 0.1 | 0.1 ± 0 | 0.2 ± 0.1 | 0.5 ± 0.1 | 0.1 ± 0 | 0.9 ± 0.3 | 0.1 ± 0 | 0.3 ± 0.2 | 0.6 ± 0.3 | 0.1 ± 0 |
| Protocatechuic | 1813 | 2.9 ± 0.8 | 1.0 ± 0 | 3.2 ± 0.6 | 0.8 ± 0.4 | 1.4 ± 0.8 | 0.5 ± 0.2 | 1.0± | 0.5 ± 0.1 | 0.7 ± 0.2 | 2.0 ± 0.9 | 0.5 ± 0.3 |
| 4-Hydroxycinnamic trans | 1948 | 3.8 ± 1.1 | 3.3 ± 0.8 | 7.4 ± 2.4 | 2.1 ± 1.0 | 3.9 ± 2.2 | 0.8 ± 0.1 | 3.1 ± 0.5 | 0.4 ± 0.2 | 1.3 ± 0.9 | 4.7 ± 0.3 | 1.9 ± 0.8 |
| Ferulic | 2086 | 0.9 ± 0.2 | 1.7 ± 0.6 | 3.1 ± 0.6 | 0.4 ± 0.1 | 0.8 ± 0.1 | 0.6 ± 0.2 | 0.3 ± 0.1 | 2.6 ± 1.1 | 1.1 ± 0.8 | 1.0 ± 0.3 | 0.1 ± 0 |
| Caffeic | 2141 | 21.5 ± 5.7 | 19.8 ± 5.3 | 26.9 ± 17 | 41.3 ± 12 | 80.1 ± 24 | 30.1 ± 0.9 | 33.0 ± 12 | 11.2 ± 4.9 | 40.6 ± 18 | 93.2 ± 34 | 21.8 ± 4.8 |
| Organic acids | ||||||||||||
| Succinic acid | 1310 | 112.9 ± 38 | 113.1 ± 42 | 88.3 ± 32 | 21.8 ± 6.7 | 55.3 ± 18 | 25.0 ± 9.7 | 74.6 ± 25 | 39.0 ± 10 | 30.5 ± 12 | 38.4 ± 13 | 122.4 ± 36 |
| Glyceric acid | 1340 | 7.2 ± 2.3 | 8.8 ± 2.7 | 5.9 ± 2.1 | 2.3 ± 0.5 | 11.6 ± 0.9 | 1.2 ± 0.6 | 9.0 ± 3.4 | 3.8 ± 1.2 | 4.8 ± 2.3 | 4.5 ± 1.7 | 6.4 ± 2.3 |
| Malic acid | 1488 | 11.5 ± 4.4 | 19.8 ± 3.1 | 21.3 ± 10 | 11.9 ± 0.6 | 8.5 ± 1.7 | 13.8 ± 2.0 | 10.8 ± 0.4 | 15.1 ± 3.4 | 6.7 ± 2.2 | 11.2 ± 2.1 | 11.3 ± 4.4 |
| Sugars and polyols | ||||||||||||
| Fructose 1 | 1855 | 272.8 ± 72 | 308.1 ± 28 | 62.4 ± 14 | 27.8 ± 1.1 | 48.9 ± 19 | 117.9 ± 38 | 24.4 ± 11 | 97.2 ± 28 | 24.6 ± 2.4 | 107.7 ± 28 | 82.2 ± 12 |
| Fructose 2 | 1864 | 33.0 ± 11 | 102.6 ± 43 | 46.2 ± 12 | 20.9 ± 3.2 | 24.7 ± 11 | 123.7 ± 19 | 40.3 ± 15 | 62.8 ± 19 | 45.6 ± 0.8 | 64.8 ± 12 | 55.0 ± 6.7 |
| Glucose | 1887 | 236 ± 96 | 483.7 ± 87 | 115.5 ± 44 | 47.8 ± 15 | 131.0 ± 25 | 99.5 ± 21 | 29.2 ± 3.3 | 142.6 ± 44 | 47.2 ± 14 | 190.7.6 ± 34 | 43.8 ± 2.2 |
| Ononitol | 1986 | 2326.2 ± 341 | 2198.8 ± 238 | 3989.9 ± 301 | 1098.8 ± 105 | 1230.1 ± 175 | 2092.5 ± 285 | 2502.3 ± 202 | 1739.4 ± 182 | 2237.8 ± 287 | 3255.7 ± 358 | 2773.1 ± 342 |
| Myo-Inositol | 2080 | 72.7 ± 22 | 37.3 ± 16 | 36.0 ± 9.8 | 7.3 ± 1.9 | 27.4 ± 17 | 18.5 ± 2.8 | 15.0 ± 0.8 | 26.2 ± 9.2 | 19.2 ± 9.3 | 18.7 ± 8.2 | 27.9 ± 9.3 |
| Sucrose | 2628 | 1575.7 ± 121 | 2138.3 ± 332 | 2465.1 ± 326 | 626.0 ± 152 | 836.3 ± 113 | 401.7 ± 92 | 503.9 ± 96 | 841.5 ± 104 | 569.9 ± 31.8 | 2291.9 ± 165 | 1291.5 ± 89 |
| Lipids | ||||||||||||
| Hexadecanoic acid | 1925 | 140.4 ± 32 | 99.5 ± 24 | 148.1 ± 67 | 58.5 ± 18 | 86.0 ± 27 | 25.7 ± 9.3 | 67.0 ± 24 | 47.9 ± 13 | 31.2 ± 19 | 36.2 ± 10 | 33.7 ± 8.6 |
| Octadecanoic acid | 2132 | 20.0 ± 5.4 | 19.3 ± 5.1 | 57.1 ± 32 | 11.2 ± 3.6 | 4.2 ± 1.9 | 13.6 ± 4.2 | 31.9 ± 18 | 28.6 ± 3.7 | 6.4 ± 2.3 | 0.0 | 8.2 ± 2.4 |
| Teracosanol | 2740 | 7.3 ± 2.8 | 2.2 ± 0.9 | 2.4 ± 1.1 | 1.4 ± 0.3 | 0.8 ± 0.3 | 0.3 ± 0.1 | 3.2 ± 1.2 | 2.6 ± 0.9 | 0.4 ± 0.1 | 1.2 ± 0.2 | 0.1 ± 0 |
| Campestrol | 3239 | 9.3 ± 3.1 | 3.2 ± 1.7 | 10.0 ± 4.5 | 1.5 ± 0.5 | 3.9 ± 1.2 | 1.9 ± 0.4 | 5.9 ± 2.2 | 2.0 ± 1.1 | 6.8 ± 4.7 | 6.7 ± 2.4 | 0.1 ± 0 |
| β-Sitosterol | 3335 | 39.5 ± 4.6 | 27.4 ± 3.5 | 31.4 ± 13 | 14.5 ± 4.6 | 26.1 ± 8.6 | 11.4 ± 3.6 | 28.4 ± 16 | 12.2 ± 1.3 | 21.8 ± 5.7 | 49.3 ± 8.5 | 12.7 ± 6.3 |
* The amounts of metabolites are represented as response ratio that represents peak area ratios using 3,5 dichloro-4-hidroxybenzoic acid (50 μg) as quantitative internal standard.
References
- Jaradat, N.A.; Zaid, A.N.; Hussen, F.; Issa, L.; Altamimi, M.; Fuqaha, B.; Nawahda, A.; Assadi, M. Phytoconstituents, antioxidant, sun protection and skin anti-wrinkle effects using four solvents fractions of the root bark of the traditional plant Alkanna tinctoria (L.). Eur. J. Integr. Med. 2018, 21, 88–93. [Google Scholar] [CrossRef]
- Tung, N.H.; Du, G.J.; Yuan, C.-S.; Shoyama, Y.; Wang, C.Z. Isolation and chemopreventive evaluation of novel naphthoquinone compounds from Alkanna tinctoria. Anti-Cancer Drugs 2013, 24, 1058–1068. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Tung, N.H.; Du, G.J.; Wang, C.Z.; Yuan, C.S.; Shoyama, Y. Naphthoquinone components from Alkanna tinctoria (L.) Tausch show significant antiproliferative effects on human colorectal cancer cells. Phytother. Res. 2013, 27, 66–70. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Abdel-Gelil, O.E.; Atwa, N.A.; Moustafa, A.R.A.; Mansour, S.R. Alkanna Species: A Promising Herbal Medicine and its Uses. J. Food Nutr. Res. 2019, 2, 309–315. [Google Scholar] [CrossRef]
- Assimopoulou, A.; Dimitrios, B.D.; Boskou, V.P. Antioxidant activities of alkannin, shikonin and Alkanna tinctoria root extracts in oil substrates. Food Chem. 2004, 87, 433–438. [Google Scholar] [CrossRef]
- Assimopoulou, A.N.; Papageorgiou, V.P. Radical scavenging activity of Alkanna tinctoria root extracts and their main constituents, hydroxynaphthoquinones. Phytother. Res. 2005, 19, 141–147. [Google Scholar] [CrossRef]
- Ozer, M.; Sarikurkcu, C.; Tepe, B. Essential oil composition and antioxidant activities of alkanet (Alkanna tinctoria subsp. tinctoria). Food Sci. Biotechnol. 2010, 19, 1177–1183. [Google Scholar] [CrossRef]
- Khan, U.A.; Rahman, H.; Qasim, M.; Hussain, A.; Azizllah, A.; Murad, W.; Khan, Z.; Anees, M.; Adnan, M. Alkanna tinctoria leaves extracts: A prospective remedy against multidrug resistant human pathogenic bacteria BMC Complement. Altern. Med. 2015, 15, 127. [Google Scholar] [CrossRef] [Green Version]
- Papageorgiou, V.P.; Assimopoulou, A.N.; Samanidou, V.F.; Papadoyannis, I.N. Recent Advances in Chemistry, Biology and Biotechnology of Alkannins and Shikonins. Curr. Org. Chem. 2006, 10, 2123–2142. [Google Scholar] [CrossRef]
- Papageorgiou, V.P.; Assimopoulou, A.N.; Ballis, A.C. Alkannins and Shikonins: A New Class of Wound Healing Agents. Curr. Med. Chem. 2008, 15, 3248–3267. [Google Scholar] [CrossRef]
- Assimopoulou, A.N.; Karapanagiotis, I.; Vasiliou, A.; Kokkini, S.; Papageorgiou, V.P. Analysis of alkannin derivatives from Alkanna species by high-performance liquid chromatography/photodiode array/mass spectrometry. Biomed. Chromatogr. 2006, 20, 1359–1374. [Google Scholar] [CrossRef] [PubMed]
- Assimopoulou, A.N.; Sturm, S.; Stuppner, H.; Papageorgiou, V.P. Preparative isolation of Alkannin/Shikonin derivatives by High-Speed Counter-Current Chromatography. Planta Med. 2008, 74, PC51. [Google Scholar] [CrossRef]
- Tappeiner, J.; Vasiliou, A.; Ganzera, M.; Fessas, D.; Stuppner, H.; Papageorgiou, V.P.; Assimopoulou, A.N. Quantitative determination of alkannins and shikonins in endemic Mediterranean Alkanna species. Biomed. Chromatogr. 2013, 28, 923–933. [Google Scholar] [CrossRef] [PubMed]
- Genova, E.; Kitanov, G.; Stefanova, I. Study on the alkannin (shikonin) content in the roots of dyer’s alkanet (Alkanna tinctoria (L.). Pharmacia 1995, 3, 15–17. (In Bulgarian) [Google Scholar]
- Ozgen, U.; Demirkaya Miloglu, F.; Bulut, G. Quantitative determination of shikonin derivatives with UV-Vis spectrophotometric methods in the roots of Onosma nigricaule. Rev. Anal. Chem. 2011, 30, 59–63. [Google Scholar] [CrossRef]
- Malik, S.; Bhushan, S.; Sharma, M.; Ahuja, P.S. Physico-chemicalfactors influencing the shikonin derivatives production in cellsuspension cultures of Arnebia euchroma (Royle) Johnston, amedicinally important plant species. Cell Biol. Int. 2011, 35, 152–157. [Google Scholar] [CrossRef]
- Hughes, A.R.; Inouye, B.D.; Johnson, M.T.J.; Underwood, N.; Vellend, M. Ecological consequences of genetic diversity. Ecol. Lett. 2008, 11, 609–623. [Google Scholar] [CrossRef]
- Ondaa, Y.; Mochida, K. Exploring Genetic Diversity in Plants Using High-Throughput Sequencing Techniques. Curr. Genom. 2016, 17, 358–367. [Google Scholar] [CrossRef] [Green Version]
- Weising, K.; Nybom, H.; Wolff, K.; Meyer, W. Applications of DNA Fingerprinting in Plants and Fungi DNA Fingerprinting in Plants and Fungi; CRC Press: Boca Raton, FL, USA, 1995. [Google Scholar]
- Baird, V.; Abbott, A.; Ballard, R.; Sosinski, B.; Rajapakse, S. DNA diagnostics in horticulture. In Current Topics in Plant Molecular Biology: Technology Transfer of Plant Biotechnology; Gresshoff, P., Ed.; CRC Press: Boca Raton, FL, USA, 1997; pp. 111–130. [Google Scholar]
- Esquinas-Alcázar, J. Protecting crop genetic diversity for food security: Political, ethical and technical challenges. Nat. Rev. Genet. 2005, 6, 946–953. [Google Scholar] [CrossRef]
- Wolff, K.; El-Akkad, S.; Abbott, R.J. Population substructure of Alkanna orientalis (Boraginaceae) in the Sinai Desert, in relation to its pollination behavior. Mol. Ecol. 1997, 6, 365–372. [Google Scholar] [CrossRef] [Green Version]
- Abdel-Hamid, A.M.E. DNA fingerprint of Alkanna tinctoria subspecies in Misurata, Libya. Middle East. J. Sci. Res. 2011, 7, 555–560. Available online: http://www.idosi.org/mejsr/mejsr7%284%2911/20.pdf (accessed on 20 September 2022).
- Ahmad, M.; Lazic, D.; Hansel-Hohl, K.; Lexer, C.; Sehr, E.M. Development of novel microsatellite markers for Alkanna tinctoria by comparative transcriptomics. Appl. Plant Sci. 2019, 7, e11296. [Google Scholar] [CrossRef] [PubMed]
- Semerdjieva, I.; Petrova, G.; Yankova-Tsvetkova, E.; Doncheva, T.; Kostova, N.; Nikolova, R.; Zheljazkov, V.D. Genetic diversity, reproductive capacity and alkaloids content in three endemic Alkanna species. PLoS ONE 2020, 15, e0233516. [Google Scholar] [CrossRef] [PubMed]
- Nasrollahi, F.; Kazempour-Osaloo, S.; Saadati, N.; Mozaffarian, V.; Zare-Maivan, H. Molecular phylogeny and divergence times of Onosma (Boraginaceae s.s.) based on nrDNA ITS and plastid rpl32-trnL(UAG) and trnH–psbA sequences. Nord. J. Bot. 2019, 37, e02060. [Google Scholar] [CrossRef] [Green Version]
- Ahmad, M.; Leroy, T.; Krigas, N.; Temsch, E.M.; Weiss-Schneeweiss, H.; Lexer, C.; Sehr, E.M.; Paun, O. Spatial and ecological drivers of genetic structure in Greek populations of Alkanna tinctoria (Boraginaceae), a polyploid medicinal herb. Front. Plant Sci. 2021, 12, 706574. [Google Scholar] [CrossRef]
- Bennett, M.; Leitch, I. Genome Size Evolution in Plants; Ryan Gregory, T., Ed.; The Evolution of the Genome Academic Press; Elsevier Inc.: London, UK, 2005; pp. 89–162. [Google Scholar] [CrossRef]
- Pustahija, F.; Brown, S.C.; Bogunić, F.; Bašić, N.; Muratović, E.; Ollier, S.; Hidalgo, O.; Bourge, M.; Stevanović, V.; Siljak-Yakovlev, S. Small genomes dominate in plants growing on serpentine soils in West Balkans, an exhaustive study of 8 habitats covering 308 taxa. Plant Soil 2013, 373, 427–453. [Google Scholar] [CrossRef]
- Swift, H. The constancy of desoxyribose nucleic acid in plant nuclei. Proc. Nat. Acad. Sci. USA 1950, 36, 643–654. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Grime, J.P.; Mowforth, M.A. Variation in genome size—An ecological interpretation. Nature 1982, 299, 151–153. Available online: https://www.nature.com/articles/299151a0 (accessed on 20 September 2022). [CrossRef]
- Vekemans, X.; Lefebvre, C.; Coulaud, J.; Blaise, S.; Gruber, W.; Siljak-Yakovlev, S.; Brown, S.C. Variation in Nuclear DNA Content at the Species Level in Armeria maritima. Hereditas 1996, 124, 237–242. [Google Scholar] [CrossRef]
- Bennett, M.D.; Smith, J.B. Nuclear DNA amounts in angiosperms. Philos. Trans. R. Soc. Lond. B Biol. Sci. 1976, 274, 227–274. [Google Scholar] [CrossRef]
- Rayburn, A.L.; Auger, J.A. Genome size variation in Zea mays ssp. mays adapted to different altitudes. Theor. Appl. Genet. 1990, 79, 470–474. [Google Scholar] [CrossRef] [PubMed]
- Ceccarelli, M.; Minelli, S.; Falcinelli, M.; Cionini, P.G. Genome size and plant development in hexaploid Festuca arundinacea. Heredity 1993, 71, 555–560. [Google Scholar] [CrossRef] [Green Version]
- Cullis, C.A. DNA differences in flax genotrophs. Nature 1973, 243, 515–516. [Google Scholar] [CrossRef] [PubMed]
- Cullis, C.A. The generation of somalic and heritable variation in rcsponsc to stress. Am. Nat. 1987, 130, 62–73. [Google Scholar] [CrossRef]
- Mowforth, M.A.; Grime, J.P. Intra-population variation in nuclear DNA amount, cell size and growth rate in Poa annua L. Funct. Ecol. 1989, 3, 289–295. [Google Scholar] [CrossRef]
- Papageorgiou, V.P.; Assimopoulou, A.N. Lipids of the hexane extract from the roots of medicinal boraginaceous species. Phytochem. Anal. 2003, 14, 251–258. [Google Scholar] [CrossRef]
- Guemmaz, T.; Arrar, L.; Baghiani, A. Total Phenolic Contents and Antioxidant Properties of Algerian Alkanna tinctoria aerial part Extracts. J. Drug Deliv. Ther. 2020, 10, 39–44. [Google Scholar] [CrossRef]
- McDonald, L.W.; Goheen, S.C.; Donald, P.A.; Campbell, J.A. Identification and quantitation of various inositols and o-methylinositols present in plant roots related to soybean cyst nematode host status. Nematropica 2012, 42, 1–8. [Google Scholar]
- Dhanasekaran, M.; Ignacimuthu, S.; Agastian, P. Potential hepatoprotective activity of ononitol monohydrate isolated from Cassia tora L. on carbon tetrachloride induced hepatotoxicity in wistar rats. Phytomedicine 2009, 16, 891–895. [Google Scholar] [CrossRef]
- Antonisamya, P.; Dhanasekaran, M.; Ha-RimKim, H.R.; Sung-GangJo, S.G.; Agastian, P.; Kwon, K.B. Anti-inflammatory and analgesic activity of ononitol monohydrate isolated from Cassia tora L. in animal models. Saudi J. Biol. Sci. 2017, 24, 1933–1938. [Google Scholar] [CrossRef]
- Wanek, W. Mechanisms of Accumulation of D-Ononitol in Vigna Umbellata in Response to Drought Stress. Ph.D. Thesis, Universitaet Wien, Vienna, Austria, 1996. [Google Scholar]
- Sheveleva, E.; Chmara, W.; Bohnert, H.J.; Jensen, R.G. Increased Salt and Drought Tolerance by D-Ononitol Production in Transgenic Nicotiana tabacum L. Plant Physiol. 1997, 115, 1211–1219. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Siracusa, L.; Napoli, E.; Ruberto, G. Novel Chemical and Biological Insights of Inositol Derivatives in Mediterranean Plants. Molecules 2022, 27, 1525. [Google Scholar] [CrossRef] [PubMed]
- Weigend, M.; Selvi, F.; Thomas, D.C.; Hilger, H.H. Boraginaceae. In Flowering Plants. Eudicots, the Families and Genera of Vascular Plants 14; Kadereit, J.W., Bittrich, V., Eds.; Springer International Publishing: Cham, Switzerland, 2016. [Google Scholar] [CrossRef]
- Papageorgiou, V.P.; Assimopoulou, A.N.; Couladouros, E.A.; Hepworth, D.; Nicolaou, K.C. The chemistry and biology of alkannin, shikonin, and related naphthazarin natural products. Angew. Chem. Int. Ed. Engl. 1999, 38, 271–300. [Google Scholar] [CrossRef]
- Akula, R.; Ravishankar, G. Influence of abiotic stress signals on secondary metabolites in plants. Plant Sign. Behav. 2011, 6, 1720–1731. [Google Scholar] [CrossRef] [PubMed]
- Pavarini, D.P.; Pavarini, S.P.; Niehues, M.; Lopes, N.P. Exogenous influences on plant secondary metabolite levels. Anim. Feed Sci. Technol. 2012, 176, 5–16. [Google Scholar] [CrossRef]
- Lim, T.; Lim, Y.; Yule, C. Distribution and caracterisation of phenolic compounds in Macaranga pruinosa and associated soils in a tropical peat swamp forest. J. Trop. For. Sci. 2017, 29, 509–518. [Google Scholar] [CrossRef]
- Churngchow, N.; Rattarasarn, M. Biosynthesis of scopoletin in Hevea brasiliensis leaves inoculated with Phytophthora palmivora. J. Plant Physiol. 2001, 158, 875–882. [Google Scholar] [CrossRef]
- Verma, N.; Shukla, S. Impact of various factors responsible for fluctuation in plant secondary metabolites. J. Appl. Res. Med. Aromat. Plants 2015, 2, 105–113. [Google Scholar] [CrossRef]
- Sampaio, B.; Edrada-Ebel, R.; Da Costa, F. Effect of the environment on the secondary metabolic profile of Tithonia diversifolia: A model for environmental metabolomics of plants. Sci. Rep. 2016, 6, 29265. [Google Scholar] [CrossRef] [Green Version]
- Borges, C.; Minatel, I.; Gomez-Gomez, H.; Pereira, L. Medicinal plants: Influence of environmental factors on the content of secondary metabolites. In Medicinal Plants and Environmental Challenges; Springer: Cham, Switzerland, 2017. [Google Scholar] [CrossRef]
- Isah, T. Stress and defense responses in plant secondary metabolites production. Biol. Res. 2019, 52, 39. [Google Scholar] [CrossRef]
- Liu, W.; Liu, J.; Yin, D.; Zhao, X. Influence of ecological factors on the production of active substances in the anti-cancer plant Sinopodophyllum hexandrum (Royle) T.S. Ying. PLoS ONE 2015, 10, e0122981. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Dong, J.E.; Ma, X.H.; Wei, Q.; Peng, S.B.; Zhang, S.C. Effects of growing location on the contents of secondary metabolites in the leaves of four selected superior clones of Eucommia ulmoides. Ind. Crops Prod. 2011, 34, 1607–1614. [Google Scholar] [CrossRef]
- Liu, W.; Yin, D.; Li, N.; Hou, X.; Wang, D.; Li, D.; Liu, J. Influence of environmental factors on the active substance production and antioxidant activity in Potentilla fruticosa L. and its quality assessment. Sci. Rep. 2016, 6, 28591. [Google Scholar] [CrossRef] [PubMed]
- Huh, M.; Huh, H.W. Patterns of genetic diversity and population structure of the clonal herb, Potentilla fragarioides var. sprengeliana (Rosaceae) in Korea. Acta Bot. Sin. 1999, 42, 64–70. [Google Scholar]
- Wang, S.Q. Genetic diversity and population structure of the endangered species Paeonia decomposita endemic to China and implications for its conservation. BMC Plant Biol. 2020, 20, 510. [Google Scholar] [CrossRef]
- Frankham, R.; Ballou, J.D.; Briscoe, D.A. Introduction to Conservation Cenetics; Cambridge University Press: Cambridge, UK, 2002. [Google Scholar]
- Tansley, S.A.; Brown, C.R. RAPD variation in the rare and endangered Leucadendron elimense (Proteaceae): Implications for their conservation. Biol. Conserv. 2000, 95, 39–48. [Google Scholar] [CrossRef]
- Petrova, A.; Vladimirov, V. Red List of Bulgarian vascular plants. Phytol. Balc. 2009, 15, 63–94. [Google Scholar]
- Yankova, E.; Grancharova, P.; Nikolova, R. On the Reproductive Biology of Alkanna tinctoria (Boraginaceae). C. R. Acad. Bulg. Sci. 2022, 75, 1000–1008. [Google Scholar] [CrossRef]
- Matzk, F.; Meistner, A.; Schubert, I. An efficient screen for reproductive pathways using mature seeds of monocots and dicots. Plant J. 2000, 21, 97–108. [Google Scholar] [CrossRef]
- Grau, J. Cytologische Untersuchungen an Boraginaceen. Mitt. Bot. Staatssamml. Münch. 1968, 7, 277–294. [Google Scholar]
- Kamari, G.; Papatsou, S. Chromosome studies in some Mediterranean angiosperms. Nord. J. Bot. 1973, 126, 266–268. [Google Scholar]
- Luque, T. Karyological studies on Spanish Boraginaceae. V.: Onosma L., Cerinthe L. and Alkanna Tausch (Lithospermeae). Bot. J. Linn. Soc. 1990, 102, 261–272. [Google Scholar] [CrossRef]
- Markova, M.; Goranova, V. Reports. In Mediterranean Chromosome Number Reports; Kamari, G., Felber, F., Garbari, F., Eds.; Flora Mediterranea: Palermo, Italy, 1995; pp. 289–317. [Google Scholar]
- Islam, M.M.; Deepo, D.M.; Nasif, S.O.; Siddique, A.B.; Hassan, O.; Siddique, A.B.; Paul, N.C. Cytogenetics and Consequences of Polyploidization on Different Biotic-Abiotic Stress Tolerance and the Potential Mechanisms Involved. Plants 2022, 11, 2684. [Google Scholar] [CrossRef] [PubMed]
- Berkov, S.; Pechlivanova, D.; Denev, R.; Nikolova, M.; Georgieva, L.; Sidjimova, B.; Bakalov, D.; Tafradjiiska, R.; Stoynev, A.; Momekov, G.; et al. GC-MS analysis of Amaryllidaceae and Sceletium-type alkaloids in bioactive fractions from Narcissus cv. Hawera. Rapid Commun. Mass Spectrom. 2021, 35, e9116. [Google Scholar] [CrossRef] [PubMed]
- Doyle, J.J.; Doyle, J.L. Isolation of plant DNA from fresh tissue. Focus 1990, 12, 13–15. [Google Scholar]
- Petrova, G.; Dzhambazova, T.; Moyankova, D.; Georgieva, D.; Michova, A.; Djilianov, D.; Möller, M. Morphological variation, genetic diversity and genome size of critically endangered Haberlea (Gesneriaceae) populations in Bulgaria do not support the recognition of two different species. Plant Syst. Evol. 2013, 300, 29–41. [Google Scholar] [CrossRef]
- Lynch, M.; Milligan, B.G. Analysis of population genetic structure with RAPD markers. Mol. Ecol. 1994, 3, 91–99. [Google Scholar] [CrossRef]
- Culley, T.M.; Wolfe, A.D. Population genetic structure of the cleistogamous plant species Viola pubescens Aiton (Violaceae), as indicated by allozyme and ISSR molecular markers. Heredity 2001, 86, 545–556. [Google Scholar] [CrossRef] [Green Version]
- Culley, T.M. Population Genetic Analysis of ISSR Data. Available online: https://homepages.uc.edu/~culleyt/Population%20Genetic%20Analysis%20of%20ISSR%20Data.pdf (accessed on 20 September 2022).
- Nei, M. Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics 1978, 89, 583–590. [Google Scholar] [CrossRef]
- Garcia-Vallve, S.; Palau, J.; Romeu, A. Horizontal gene transfer in glycosyl hydrolases inferred from codon usage in Escherichia coli and Bacillus subtilis. Mol. Biol. Evol. 1999, 16, 1125–1134. [Google Scholar] [CrossRef]
- Greilhuber, J.; Temsch, E.; Loureiro, J. Nuclear DNA content measurement. In Flow Cytometry with Plant Cells. Analysis of Genes, Chromosomes and Genomes; Doležel, J., Greilhuber, J., Suda, J., Eds.; Wiley-VCH: Weinheim, Germany, 2007; pp. 67–101. [Google Scholar]
Figure 1.
Chromatogram representative of methanolic extracted from A. tinctoria aerial parts. Peak 1: ononitol.
Figure 1.
Chromatogram representative of methanolic extracted from A. tinctoria aerial parts. Peak 1: ononitol.
Figure 2.
GC/MS spectra of (a) derivatized ononitol of methanolic extract, and (b) ononitol from the extract and MS spectra of standard of Golm Metabolome Database (GMD).
Figure 2.
GC/MS spectra of (a) derivatized ononitol of methanolic extract, and (b) ononitol from the extract and MS spectra of standard of Golm Metabolome Database (GMD).
Figure 3.
Cluster dendrogram based on the Nei’s genetic distances among populations. Codes of the samples (AT1, AT3, AT4, AT5, AT6, AT9) are given according to Table 6.
Figure 3.
Cluster dendrogram based on the Nei’s genetic distances among populations. Codes of the samples (AT1, AT3, AT4, AT5, AT6, AT9) are given according to Table 6.
Table 1.
Comparative summary data on metabolite profile of the aerial parts and roots of the studied A. tinctoria populations.
Table 1.
Comparative summary data on metabolite profile of the aerial parts and roots of the studied A. tinctoria populations.
| Identified Compounds | Roots * | Aerial Parts * |
|---|---|---|
| Phenolic acids | ||
| 4-Hydroxybenzoic acid | 1.4 | 4.7 |
| Vanilic acid | 0.4 | 0.3 |
| Protocatechuic acid | 1.0 | 1.3 |
| Ferulic acid | 0.5 | 1.1 |
| Caffeic acid | 11.5 | 38.1 |
| Organic acids | ||
| Succinic acid | 10.8 | 65.5 |
| Glyceric acid | 5.9 | |
| Malic acid | 8.0 | 12.9 |
| Sugars and polyols | ||
| Fructose 1 | 163.9 | 106.7 |
| Fructose 2 | 226.8 | 56.3 |
| Glucose | 281.2 | 137.6 |
| Ononitol | 2313.1 | |
| Myo-Inositol | 60.7 | 27.8 |
| Sucrose | 2272.1 | 1232.1 |
| Lipids | ||
| Hexadecanoic acid | 45.1 | 70.4 |
| Octadecanoic acid | 12.4 | 18.2 |
| Teracosanol | 5.0 | 1.9 |
| Hexacosanol | 3.7 | |
| Campestrol | 8.1 | 4.6 |
| Stigmasterol | 3.1 | |
| β-Sitosterol | 43.3 | 24.9 |
* The presented values are mean of all studied populations. The amounts of metabolites are represented as response ratio that represents peak area ratios using 3,5 dichloro-4-hidroxybenzoic acid (50 μg) as quantitative internal standard.
Table 2.
Spectrophotometric determination on the alkannin content in the studied samples of Alcanna tinctoria roots.
Table 2.
Spectrophotometric determination on the alkannin content in the studied samples of Alcanna tinctoria roots.
| Sample 1 | Alkannin Content to Air Dry Weight, g [%] |
|---|---|
| AT0 | 4.4 ± 0.1 |
| AT1 | 2.4 ± 0.1 |
| AT2 | 3.8 ± 0.1 |
| AT3 | 5.9 ± 0.1 |
| AT4 | 3.6 ± 0.1 |
| AT5 | 2.9 ± 0.1 |
| AT6 | 1.9 ± 0.1 |
| AT7 | 1.8 ± 0.5 |
| AT8 | 4.2 ± 0.9 |
| AT9 | 1.4 ± 0.4 |
| AT10 | 3.2 ± 0.2 |
| AT11 | 1.2 ± 0.1 |
1 Codes of the samples (AT1–AT11) are given according to Table 6 (Section 4). AT0—a control (commercial product).
Table 3.
Natural populations included in the genetic studies and polymorphism and diversity.
| Population (Abbreviation) | Sample Size | Percent of Polymorphic Bands | Gene Diversity He ± SD |
|---|---|---|---|
| AT5 | 8 | 69.1 | 0.236 ± 0.110 |
| AT1 | 12 | 72.1 | 0.228 ± 0.056 |
| AT3 | 7 | 59.3 | 0.298 ± 0.060 |
| AT6 | 2 | 55.3 | 0.220 ± 0.110 |
| AT4 | 8 | 58.2 | 0.248 ± 0.096 |
| AT9 | 2 | 55.3 | 0.224 ± 0.132 |
| Mean | 6.5 | 61.5 | 0.242 |
Table 4.
DNA content in fresh leaves.
| Sample | 1C [pg] 1st Run | 1C [pg] 2nd Run | 1C [pg] 3rd Run | 1C [pg] 4th Run | 1C [pg] 5th Run | 1C [pg] Mean | CV [%] |
|---|---|---|---|---|---|---|---|
| AT 5 | 1.333 | 1.327 | 1.323 | 1.324 | 1.324 | 1.326 | 0.33 |
| AT 7 | 1.326 | 1.326 | 1.325 | - | - | 1.326 | 0.02 |
Table 5.
DNA content in the seeds of the studied population of A. tinctoria.
| Sample | No. of Seeds Per Run | 1C [pg] | CV Alkanna [%] |
|---|---|---|---|
| AT1 | 10 | 1.29 | 6.6 |
| AT2 | 10 | 1.31 | 6.1 |
| AT3 | 10 | 1.43 | 7.5 |
| AT4 | 10 | 1.42 | 8.4 |
| AT5 | 10 | 1.28 | 5.8 |
| AT6 | 10 | 1.39 | 5.3 |
| AT7 | 10 | 1.33 | 4.7 |
| AT8 | 10 | 1.43 | 8.5 |
| AT9 | 10 | 1.30 | 5.1 |
| AT10 | 10 | 1.32 | 8.0 |
| AT11 | 10 | 1.49 | 10.7 |
Table 7.
ISSR primers studied for the study of genetic diversity of A. tinctoria.
| ISSR Primer (Name and Sequence) | Optimal Annealing Temperature | Total Number of Bands | Number of Polymorphic Bands | |
|---|---|---|---|---|
| UBC-807 | 5′-AGAGAGAGAGAGAGAGT-3′ | 59 | 11 | 8 |
| UBC-811 | 5′-GAGAGAGAGAGAGAGAC-3′ | 53 | 12 | 9 |
| UBC-827 | 5′-ACACACACACACACACG-3′ | 57 | 8 | 5 |
| UBC-835 | 5′-AGAGAGAGAGAGAGAGYC-3′ | 54 | 9 | 6 |
| UBC-845 | 5′-CTCTCTCT CTCTCTCTRG-3′ | 54 | 13 | 9 |
| UBC-846 | 5′-CACACACACACACACAAGT-3′ | 54 | 10 | 8 |
| UBC-856 | 5′-ACACACACACACACACCTA-3′ | 54 | 11 | 8 |
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
MDPI and ACS Style
Nikolova, M.; Aneva, I.; Zhelev, P.; Semerdjieva, I.; Zheljazkov, V.D.; Vladimirov, V.; Stoyanov, S.; Berkov, S.; Yankova-Tsvetkova, E. Metabolic Profiles, Genetic Diversity, and Genome Size of Bulgarian Population of Alkanna tinctoria. Plants 2023, 12, 111. https://doi.org/10.3390/plants12010111
AMA Style
Nikolova M, Aneva I, Zhelev P, Semerdjieva I, Zheljazkov VD, Vladimirov V, Stoyanov S, Berkov S, Yankova-Tsvetkova E. Metabolic Profiles, Genetic Diversity, and Genome Size of Bulgarian Population of Alkanna tinctoria. Plants. 2023; 12(1):111. https://doi.org/10.3390/plants12010111
Chicago/Turabian StyleNikolova, Milena, Ina Aneva, Petar Zhelev, Ivanka Semerdjieva, Valtcho D. Zheljazkov, Vladimir Vladimirov, Stoyan Stoyanov, Strahil Berkov, and Elina Yankova-Tsvetkova. 2023. "Metabolic Profiles, Genetic Diversity, and Genome Size of Bulgarian Population of Alkanna tinctoria" Plants 12, no. 1: 111. https://doi.org/10.3390/plants12010111
Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.
