Phytochemical and molecular characterization of centaury (Centaurium erythraea Rafn) populations from the Balkan Peninsula

Kičica (Centaurium erythraea Rafn) je farmakološki atraktivna biljna vrsta koja pokazuje čitav spektar bioloških aktivnosti, a čiji je biodiverzitet nedovoljno istražen. Osnovni cilj predstavljenog istraživanja jeste analiza genetičkog i fitohemijskog diverziteta kičice na teritoriji Balkanskog poluostrva. U tom smislu, formirana je kolekcija semena kičice poreklom iz prirodnih populacija, koja oslikava diverzitet vrste na ovom području. Istraživanja su uključila 43 populacije ove vrste, od kojih je 41 poreklom sa Balkanskog poluostrva. Za procenu genetičkog diverziteta optimizovane su tehnike RAPD (Random Amplified Polymorphic DNA) i TRAP (Target Region Amplification Polymorphism) molekularnih marker sistema. DNK upotrebljena u ovim analizama je izolovana iz biljaka gajenih u uslovima in vitro, kako bi se obezbedila autentičnost DNK kičice i eliminisala mogućnost kontaminanacije stranom DNK. Za potrebe dobijanja što većeg broja informativnih jedinica, upotrebljeno je 16 desetomernih prajmera u slučaju RAPD markera, pri čemu je dobijeno ukupno 408 različitih PCR produkata, dok su za TRAP markere upotrebljene kombinacije tri specifična i tri arbitrarna prajmera i dobijeno je 767 fragmenata DNK. Specifični prajmeri korišćeni u TRAP analizama, konstruisani su na osnovu sekvenci RNK kičice sa visokim stepenom homologije sa DNK sekvencama za sledeće enzime: geraniol 10-hidroksilaza, citohrom P450 reduktaza i sekologanin sintaza, objavljenih u elektronskim bazama podataka. Primenom molekularnih markera dobijen je visok procenat polimorfizma, a informativnost odabranih prajmera ili kombinacija prajmera je potvrđena pomoću više parametara. Ustanovljena je vrlo niska unutarpopulaciona genetička varijabilnost kičice, što može da se objasni velikim udelom samooplodnje i geitonogamije u nastanku Rezime/Summary semena kod prirodnih populacija, kao i snažnim genetičkim driftom. S obzirom da se kičica javlja u malim i fragmentisanim populacijama, između kojih verovatno ne postoji protok gena, očekivana je i visoka genetička diferenciranost analiziranih populacija, što je potvrđeno i metodama molekularnih markera. Osim toga, molekularnim markerima vrlo uspešno su razdvojene diploidne od tetraploidnih populacija kičice. Tetraploidne populacije se prilično jasno diferenciraju u posebne grupe koje su povezane sa njihovim geografskim poreklom, naročito kada su primenjeni RAPD markeri. Značajna korelacija pokazana je između genetičkih sličnosti populacija kičice sa Balkanskog poluostrva, dobijenih pomoću dva tipa molekularnih markera i geografskih distanci lokaliteta sa kojih ove populacije potiču. Određivanje i kvantifikacija osnovnih jedinjenja sekundarnog metabolizma kičice, sekoiridoidnih glikozida, dalo je značajan doprinos proceni varijabilnosti analiziranih populacija kičice. Sadržaj svercijamarina, dominantnog sekoiridoidnog glikozida u nadzemnim delovima biljaka gajenih u uslovima staklare, pokazao se kao najznačajniji faktor međupopulacione varijabilnosti kičice. Uz to, pokazana je značajna korelacija između sadržaja sekoiridoidnih glikozida i genetičke sličnosti populacija, korišćenjem oba tipa molekularnih markera, s tim što je snažnija korelacija primećena u slučaju TRAP markera. Predstavljena procena genetičkog i fitohemijskog diverziteta kičice daju osnovu za eventualnu zaštitu ukupnog biodiverziteta ove vrste na Balkanskom poluostrvu, kao i za odabir genotipova za zasnivanje i unapređenje njene plantažne proizvodnje.Centaury (Centaurium erythraea Rafn) is a medicinal and pharmacologically attractive plant species which has a wide range of biological activity. The main objective of presented study was to estimate genetic and phytochemical diversity of centaury within the Balkan Peninsula. With that purpose, seed collection of natural centaury populations was established. The study included 43 populations of C. erythraea, among which 41 originated from the Balkan Peninsula. Molecular marker techniques, such as RAPD (Random Amplification Polymorphic DNA) and TRAP (Target Region Amplification Polymorphism) were optimized to investigate the genetic diversity among these populations. DNA used in these analyses originated from in vitro grown plants to ensure the authenticity of centaury DNA by eliminating the possibility of its contamination by extraneous DNA. To acquire sufficient informational characters, 16 decameric primers were applied in RAPD assays, which produced 408 different PCR products, while TRAP assays included the combinations of 3 specific and 3 arbitrary primers, resulting in 767 fragments of DNA. Specific primers used for TRAP analyses were constructed according to the RNA sequences, showing high homology with DNA sequences published in electronic data bases for following enzymes: geraniol 10-hydroxylase, cytochrome P450 reductase and secologanin synthase. Molecular markers provided a high percentage of polymorphism. The informativeness of single primers (RAPD) of primer combinations (TRAP) was confirmed by various parameters. Low within-population genetic variability of natural populations of centaury could be explained by the high levels of selfing or geitonogamy in the process of fertilization and by strong genetic drift. Due to the fact that centaury grows in small, fragmented populations and that gene flow between them probably does not exist, a high genetic differentiation of analyzed populations was expected and this was confirmed by molecular markers. Moreover, molecular markers Резиме/Summary efficiently differentiated diploid populations from tetraploids. Tetraploid populations were clearly clustered into groups related to their geographic origin, especially when RAPD markers were analyzed. Genetic similarity data between the Balkan Peninsula populations, obtained with either RAPD or TRAP markers, significantly correlated with the geographic distances between localities of their origin. Identification and quantification of secondary metabolite compounds, secoiridoid glycosides, highly contributed to the estimation of diversity of centaury populations in the analyzed region. A dominant secoiridoid glycoside in aerial parts of plants grown under greenhouse conditions was swertiamarin, which pointed to the interpopulation variability of centaury. Furthermore, a significant correlation between the secoiridoid glycosides content and genetic similarity of populations has been obtained, though a higher correlation was recorded for TRAP markers. The genetic and phytochemical diversity estimation of centaury from the Balkan Peninsula provides a basis for future biodiversity conservation efforts and also for high-productive genotype selection and field production improvement

Genetic patterns in range-edge populations of Vaccinium species from the central Balkans: implications on conservation prospects and sustainable usage

L., L. and L. are perennial, cold-adapted clonal shrubs distributed throughout Europe, northern Asia and North America. Due to their usage in food (berries) and pharmaceutical industry (berries and leaves), their natural populations are exposed to anthropogenic and other impacts that affect their genetic make-up. We analyzed 14 fragmentary distributed and small-sized peripheral populations of these species from the Balkans, which represents the southeastern-European marginal area of their wide European distributions, using RAPD molecular markers. The contemporary genetic patterns in all three species within the Balkans were generally similar, and in comparison to previous reports on populations of these species found in northward Europe, where they have a more continuous distribution, the levels of genetic diversity were more or less halved, genetic differentiation was several times higher, gene flow exceptionally low, and the expected prevalence of clonal individuals was lacking. The population dynamics of all three species within the Balkans was complex and distinct, and was characterized by a past admixture of individuals from discrete populations of the same species and interspecific hybridisation not only between and but also between and , the latter not being reported to date. Conservation measures suitable for preservation of presumably genetically distinct portions of the Balkansâ gene pools of studied species have been suggested, while the utility of interspecific hybrids in breeding programs and/ or in food/pharmaceutical industry is yet to be assessed. Vaccinium myrtillus Vaccinium uliginosum Vaccinium vitis-idaea V. myrtillus V. vitis-idaea V. uliginosum V. vitis-idae

Effects of salinity on in vitro growth and photosynthesis of common centaury (Centaurium erythraea Rafn.)

The effects of salinity on in vitro growth, morphogenesis, photosynthetic rate (Pn), PSII efficiency (Fv/Fm), and chlorophyll content were investigated in Centaurium erythraea Rafn. Root growth was more adversely affected by increasing NaCl concentration than was shoot growth. High salt concentrations were effective in induction of axillary and adventitious buds on shoots, with 400 mM NaCl being the most efficient one. Values of Pn and Fv/Fm increased at moderate salt levels, but decreased when 400 mM NaCl was applied. Chlorophyll a and b contents and total chlorophyll had a decreasing trend with increasing supply of NaCl in the growth medium. .Praćen je efekat NaCl (u rasponu koncentracija od 0 do 400 mM) na rast, morfogenezu, intenzitet fotosinteze (Pn), efikasnost fotosistema II (Fv/Fm) i sadržaj hlorofila a i b kod vrste Centaurium erythraea Rafn. u in vitro uslovima. Utvrđeno je da su koreni kičice osetljiviji na visoke koncentracije soli u hranljivoj podlozi u poređenju sa izdancima. Visoke koncentracije soli su se pokazale efikasnim u pogledu indukcije formiranja aksilarnih i adventivnih pupoljaka na izdancima. U uslovima umerenog stresa (100 i 200 mM NaCl), vrednosti Pn i Fv/Fm rastu u poređenju sa vrednostima dobijenim kod kontrolne grupe biljaka. Primena 400 mM NaCl dovela je do opadanja vrednosti Pn i Fv/Fm ispod kontrolnih vrednosti. Sadržaj hlorofila a i b, kao i ukupnog hlorofila opada sa porastom sadržaja soli u hranljivoj podlozi. Odnos Chl a/b takođe opada. Može se zaključiti da je Centaurium erythraea Rafn vrsta koja je otporna na povećani salinitet podloge u kojoj raste. Prema tome, može se pretpostaviti da je komercijalno gajenje ove vrste kičice ostvarivo na zaslanjenim terenima. .nul

Supplementary data for article: Mišić, D.; Šiler, B.; Gašić, U.; Avramov, S.; Živković, S.; Živković, J. N.; Milutinović, M.; Tešić, Ž. Simultaneous UHPLC/DAD/(+/-)HESI-MS/MS Analysis of Phenolic Acids and Nepetalactones in Methanol Extracts of Nepeta Species: A Possible Application in Chemotaxonomic Studies. Phytochemical Analysis 2015, 26 (1), 72–85. https://doi.org/10.1002/pca.2538

Supporting information for: [https://doi.org/10.1002/pca.2538]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/1663

Genetic diversity of Nepeta rtanjensis based on RAPD data

Nepeta rtanjensis Diklić et Milojević (Lamiaceae) is an endemic, herbaceous plant, found only in a very small area of Mount Rtanj in Southeast Serbia. The whole species is represented by several hundred specimens distributed within three known populations: Greda, Mirčin del and Javor. Like many species of the genus Nepeta, N. rtanjensis is also famous for its secondary metabolite production (mainly nepetalactones and phenolics) and it is well known that these compounds show antibacterial, antifungal, cytotoxic, phytotoxic and antioxidant activities. Because of its limited distribution and the status of critically endangered species, it is important to find as much as possible information about the population genetic structure of this species. Therefore, we performed randomly amplified polymorphic DNA (RAPD) analysis to get an insight into the genetic diversity and variation of this species within and between the populations. DNA material used in this process was isolated from leaves collected from plants in their natural habitats. To our knowledge, this is the first employment of RAPD markers in the assessment of the N. rtanjenis genetic structure. Genetic variability between populations was not detected, but only small variations between individual plants from the same populations were recorded. These preliminary data give us a good base for further studies towards elucidating the genetic structure of this rare species using more reliable EST-SSR markers, designed based on N. rtanjensis transcriptome

Supplementary data for article: Banjanac, T.; Dragićević, M.; Šiler, B.; Gašić, U.; Bohanec, B.; Nestorović Živković, J.; Trifunović, S.; Mišić, D. Chemodiversity of Two Closely Related Tetraploid Centaurium Species and Their Hexaploid Hybrid: Metabolomic Search for High-Resolution Taxonomic Classifiers. Phytochemistry 2017, 140, 27–44. https://doi.org/10.1016/j.phytochem.2017.04.005

Supplementary material for: [https://doi.org/10.1016/j.phytochem.2017.04.005]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2477

DNA barcoding as new diagnostic tool to lethal plant poisoning in herbivorous mammals

Reliable identification of plant species in the digestive tract of a deceased animal often represents the major key to diagnose a lethal intoxication with poisonous plants in veterinary pathology. In many cases, identification of the species is challenging or even impossible because the diagnostic morphological features have been degraded, and because the interpretation of such features requires a considerable expertise in plant anatomy and biodiversity. The use of DNA barcoding markers can support or even replace classical morphological assessment. While these markers have been widely used for plant taxonomy, their forensic application to clarify causes of animal poisoning is novel. In addition, we use specific single-nucleotide polymorphisms as fingerprints. This allows for a clear decision even in cases, where the conventionally used statistical e-values remain ambiguous. In the current work, we explore the feasibility of this strategy in a couple of exemplary cases, either in concert with anatomical diagnostics, or in cases where visual species identification is not possible, or where chemical toxin detection methods are not well established, complex, time consuming and expensive

Rapid in vitro selection of salt-tolerant genotypes of the potentially medicinal plant Centaurium maritimum (L.) fritsch

We investigated differences of salinity tolerance between 'salt-tolerant' (ST) and 'salt-sensitive' (SS) genotypes of yellow centaury [Centaurium maritimum (L.) Fritsch] selected during the germination phase. The ability of in vitro cultured C. maritimum to complete the whole ontogenetic cycle in less than 6 months enabled us to deterine salinity tolerance during different growth phases. Based on the physiological attributes measured in this study (growth, morphogenesis, photosynthesis, flowering, seed germination), it can be concluded that C. maritimum genotypes differing in salinity tolerance showed a variable response to elevated salt concentrations during both the vegetative and the generative growth phase.Vršena su istraživanja u cilju utvrđivanja razlika u tolerantnosti na povećani salinitet između in vitro selekcionisanih 'salt tolerant' (ST) i 'salt sensitive' (SS) genotipova žute kičice [Centaurium maritimum (L.) Fritsch]. Sposobnost vrste C. maritimum da kompletira ontogenetski ciklus u in vitro uslovima za manje od 6 meseci, omogućila je praćenje tolerancije na povećani salinitet tokom različitih faza razvića. Ako se uzmu u obzir svi fiziološki parametri koji su praćeni u ovom istraživanju (rastenje, morfogeneza, fotosinteza, cvetanje, klijanje semena), može se zaključiti da selekcionisani STi SS genotipovi zaista pokazuju različit odgovor u uslovima u uslovima povećanog saliniteta, kako tokom vegetativne faze, tako i tokom generativne faze razvića.Projekat ministarstva br. 14303

Pursuit for est microsatellites in a tetraploid model from de novo transcriptome sequencing

Available scientific literature reports very few microsatellite markers derived from tetraploid genomes using de novo transcriptome sequencing, mostly because their gain usually represents a major computational challenge due to complicated combinatorics during assembly of sequence reads. Here we present a novel approach for mining polymorphic microsatellite loci from transcriptome data in a tetraploid species with no reference genome available. Pairs of 114 bp long de novo sequenced transcriptome reads of Centaurium erythraea were merged into short contigs of 170-200 bp each. High accuracy assembly of the pairs of reads was accomplished by a minimum of 14 bp overlap. Sequential bioinformatics operations involved fully free and open-source software and were performed using an average personal computer. Out of the 13 150 candidate contigs harboring SSR motifs obtained in a final output, we randomly chose 16 putative markers for which we designed primers. We tested the effectiveness of the established bioinformatics approach by amplifying them in eight different taxa within the genus Centaurium having various ploidy levels (diploids, tetraploids and hexaploids). Nine markers displayed polymorphism and/or transferability among studied taxa. They provided 54 alleles in total, ranging from 2 to 14 alleles per locus. The highest number of alleles was observed in C. erythraea, C. littorale and a hybridogenic taxon C. pannonicum. The developed markers are qualified to be used in genetic population studies on declining natural populations of Centaurium species, thus providing valuable information to evolutionary and conservation biologists. The developed cost-effective methodology provides abundant de novo assembled short contigs and holds great promise to mine numerous additional EST-SSR-containing markers for possible use in genetics population studies of tetraploid taxa within the genus Centauriu

Phenotypic and Genetic Variation of an Interspecific Centaurium Hybrid (Gentianaceae) and Its Parental Species.

Interspecific hybridization is one of the major actuators of evolutionary changes in plants. As the result of allopolyploid hybridization, offspring may gain different ploidy levels in comparison to parental species, which can provide them instant reproductive isolation. Two tetraploid sister species, Centaurium erythraea and C. littorale, readily cross-fertilize, resulting in hybrids of various ploidy. In northern Serbia, two stable populations of a hexaploid taxon C. pannonicum have been documented. It has been proposed previously that this taxon emerged after an interspecific hybridization event between two tetraploid sister-species: C. erythraea and C. littorale subsp. compressum. The existing populations of the hybridogenic taxon, as well as neighboring populations of the two parental taxa were here characterized by both morphometrics and molecular markers (EST-SSR and trnL-F). Three leaf and two flower characteristics were found to be informative in delimitation of the parental taxa and in their discernment from hybrid individuals, the latter having intermediate values. Eight microsatellite markers were found to have good ability to distinguish studied taxa, placing C. pannonicum in closer relationship with C. erythraea. Conversely, trnL-F plastid marker nominated C. littorale subsp. compressum to be the donor of the C. pannonicum plastid DNA. Reproductive isolation of the hexaploid hybrid individuals from the parental species should be examined as the next logical step in describing the new species