Edina Muratović

Chromosomal differentiation and genome size in three European mountain Lilium species

Abstract. Three related and taxonomically close species of the genus Lilium (L. pyrenaicum Gouan, L. pomponium L. and L. carniolicum Bernh.), all of them with 2n=24 chromosomes, have been studied for chromosomal differentiation, using fluorochrome banding and fluorescence in situhybridization (FISH), and for genome size and GC percentage using flow cytometry. The total DNA content of L. pomponium (2C=70.26 pg) was about 5% higher than that of L. pyrenaicum (2C=67.74) and L. carniolicum (2C=67.37 pg), while GC percentage was higher in this last species (36.60%) than in L. pomponium (35.56%) and lower than in L. pyrenaicum (37.92%). Silver staining, fluorochrome banding with chromomycin A3 (CMA) and fluorescence in situ hybridization (FISH) clearly pointed out the number of nucleoli, the number and position of GC-rich bands and the number and location of rDNA sites thus permitting distinction of the three species at chromosomal level. Two families of ribosomal genes, 18S-5.8S-26S (18S) and 5S rRNA genes, were separated onto different pairs in chromosome complements of examined species. Chromosome regions containing both kinds of rRNA genes were also GC-rich regions. The results revealed a clear interspecific differentiation at the chromosomal level and permitted the discussion about relationships among the species.

Different karyotype patterns among allopatric Pinus nigra (Pinaceae) populations revealed by molecular cytogenetics

To examine variation and taxonomic recognition of Pinus nigra (European black pine) at the intraspecific level, chromosomal distribution of 5S and 18S-5.8S-26S rDNA loci revealed by fluorescent in situ hybridisation (FISH) and fluorochrome banding with chromomycin A(3) and DAPI were analysed among allopatric populations belonging to different subspecies. Despite prevalent opinion on predominantly conserved and homogenous conifer karyotypes, several patterns were observed. Surprisingly, interstitial 18S rDNA loci and DAPI heterochromatin staining after FISH showed variations in distribution and localisation. Three subspecies shared a pattern with nine 18S rDNA loci (ssp. nigra, pallasiana and laricio) while ssp. dalmatica and salzmannii had eight rDNA loci. DAPI banding displayed two patterns, one with a high number of signals (ssp. nigra, pallasiana and dalmatica) and the other with a lower number of signals (ssp. salzmannii and laricio). We conclude that our results cannot provide proof for either classification scheme for the P. nigra complex, but rather demonstrate the variability of different heterochromatin fractions at the intraspecific level.

Does Lilium bosniacum merit species rank? A classical and molecular-cytogenetic analysis

Abstract.Four populations of the Balkan endemic taxon Lilium bosniacum from Bosnia and Herzegovina were investigated. Conventional karyological study did not reveal any important differences. Molecular-cytogenetic studies pointed out certain interpopulation and intrapopulation variability, and important differences in organization of ribosomal genes in regard to its closest relative L. carniolicum. The results of fluorochrome banding and FISH experiment for L. bosniacum were reported here for the first time. Differences occurred in the number and position of 18S-5.8S-26S ribosomal gene loci for some individuals from population Kladanj. Heterochromatic bands revealed with DAPI after FISH experiment were constant. All investigated populations possess the same number of active NORs except some individuals from Kladanj population. Genome size and GC-bases percentage, estimated by flow cytometry, did not show any significant difference among the populations. However, the present results reveal clear interspecific differentiation between two endemics, L. carniolicum and L. bosniacum.

Molecular cytogenetics and flow cytometry reveal conserved genome organization in Pinus mugo and P. uncinata

Abstract• IntroductionThe most common representatives of the European mountain pine complex (Pinus mugo s.l.) are P. mugo s.s. and Pinus uncinata.• Materials and methodsGenome characterization of P. mugo and P. uncinata was studied using fluorescence in situ hybridization of 5S and 18–5.8–26S rDNA, fluorochrome banding for heterochromatin characterization, and flow cytometry for DNA content measurement.• Results and discussionDistribution of 5S and 18S rDNA showed identical patterns for both pine species. In contrast, heterochromatin patterns revealed slight differences in the number and position of bands between these two pines. Genome size analysis of 21 P. mugo populations and one P. uncinata population revealed no significant variations across seven European countries. The mean genome size (2C DNA) for the 21 P. mugo populations was 42.56 ± 0.79 pg, equivalent to 41.62 × 103 Mbp, and ranged from 41.08 to 43.95 pg. No relationships were observed between nuclear DNA content and geographic origin of the studied populations.• ConclusionsOur results reveal that the mechanisms shaping molecular cytogenetic organization and genome size did not profoundly differentiate the genomes of P. mugo and P. uncinata. Observed variations in heterochromatin patterns indicate ongoing divergence processes in the genomes of the two pines.

Biochemical status of in vitro regenerated Lilium bosniacum and Lilium cattaniae plantlets

Lilium cattaniae (Vis.) Vis. and Lilium bosniacum (G. Beck) Beck ex Fritsch, endemic species of Balkan Dinaric Alps, were micropropagated from seeds collected from their natural habitats. The relationship between peroxidase activity, photosynthetic status and differentiation of Lilium cattaniae and L. bosniacum in vitro was investigated. Peroxidase activity recorded for somatic embryos of Lilium cattaniae obtained on Murashige and Skoog (MS) medium containing 9.05 mM 2.4-dichlorophenoxyacetic acid (2.4-D) and 4.44 mM N6-bezyladenin (BA), was about two times higher than for any other treatment. Photosynthetic status of plantlets obtained through regeneration was explant-specific and generally higher for plantlets regenerated from basal leaf explants than from bulb explants. The accumulation of anthocyanin was detected in some regenerated shoots and more often in plantlets obtained through regeneration from bulb explants. High frequency of somatic embryo formation was recorded for L. cattaniae on MS medium containing 9.05 mM 2.4-D and 4.44 mM BA. The peroxidase activity for L. bosniacum plantlets obtained through regeneration showed treatment-specific elevations. We consider that basal leaf parts are suitable for successful regeneration of these two lilies and that elevation in peroxidase activity is a good indicator of somatic embryogenesis in both lilies.

Epigenetic Differentiation of Natural Populations of Lilium bosniacum Associated with Contrasting Habitat Conditions

Abstract Epigenetic variation in natural populations with contrasting habitats might be an important element, in addition to the genetic variation, in plant adaptation to environmental stress. Here, we assessed genetic, epigenetic, and cytogenetic structure of the three Lilium bosniacum populations growing on distinct habitats. One population was growing under habitual ecological conditions for this species and the other two were growing under stress associated with high altitude and serpentine soil. Amplified fragment length polymorphism and methylation-sensitive amplification polymorphism analyses revealed that the three populations did not differentiate genetically, but were clearly separated in three distinct clusters according to DNA methylation profiles. Principal coordinate analysis showed that overall epigenetic variation was closely related to habitat conditions. A new methylation-sensitive amplification polymorphism scoring approach allowed identification of mainly unmethylated (φST = 0.190) and fully CpG methylated (φST = 0.118) subepiloci playing a role in overall population differentiation, in comparison with hemimethylated sites (φST = 0.073). In addition, unusual rDNA repatterning and the presence of B chromosomes bearing 5S rDNA loci were recorded in the population growing on serpentine soil, suggesting dynamic chromosome rearrangements probably linked to global genome demethylation, which might have reactivated some mobile elements. We discuss our results considering our earlier data on morphology and leaf anatomy of several L. bosniacum populations, and suggest a possible role of epigenetics as a key element in population differentiation associated with environmental stress in these particular lily populations.

Media composition affects seed dormancy, apical dominance and phenolic profile of Knautia sarajevensis (Dipsacaceae), Bosnian endemic

Abstract Knautia sarajevensis is an endemic plant of the Dinaric Alps and is mainly distributed on Bosnian Mountains. Due to the quite large flower heads and easy maintenance, this plant has a potential use as a substitute ornamental plant for K. arvensis in perennial beds. The current study evaluated the germination process in different treatments in an attempt to suppress dormancy and increase germination rate, and to develop a successful protocol for micropropagation. An over 60% germination rate was achieved through cultivation of seeds on MS basal medium with reduced mineral nutrient composition and the absence of sucrose. On the other hand, a below 10% germination rate was achieved with untreated seeds. Suppression of apical dominance was achieved through application of high concentrations of kinetin, apical shoot decapitation or cultivation of shoots in liquid media. Overall, liquid cultures were more successful as a micropropagation system for this plant. Shoots spontaneously developed roots on multiplication treatments and were successfully acclimatized. Moreover, phenolic compound profile was analysed in the light of the possible medicinal potential of this plant. Variable amounts of total phenolic compounds as well as individual phenolics were recorded, according to treatment and solidification of media. An increase in rosmarinic acid content was reported for kinetin treatments and acclimatized plants comparing to mother plants in natural habitat. The present study shows that choice of cytokinin concentration, explant type as well as culture type influences not only shoot proliferation and apical dominance suppression but also in vitro production of phenolics.