Centaurea rupestris L.: Cytogenetics, Essential Oil Chemistry and Biological Activity

Centaurea species are used in eastern Mediterranean ethnopharmacology due to variety of bioactive compounds they comprise. Aim of this work was to characterize the Centaurea rupestris L. hydrodistilled essential oil chemical composition and test its biological activity: antimicrobial effect, antioxidant potential and inhibition of cholinesterases. Plant material authentication was by chromosome number counting and genome size assessment with the flow cytometry. Hydrodistilled essential oils were characterized using gas chromatography/mass spectrometry technique GC-MS and GC-FID. The antimicrobial effect was tested using disk diffusion and microdilution methods, antioxidant potential was tested with DPPH and FRAP methods and cholinesterases inhibition was tested with Ellman method. Genome size for C. rupestris species: sample A presented 2C=3.60 (0.10) pg and sample B 2C=3.62 (0.08) pg. The chromosome number was 2n=20 for both samples. The main essential oil constituents in isolated sample A oil, detected with GC-MS and GC-FID were: germacrene D (24.3 %), heptacosane (14.4 %), phytol (6.7 %), β-caryophyllene (5.0 %) and pentacosane (4.5 %). Sample B essential oil had the main constituents: hexadecanoic acid (18.7 %), heptacosane (13.8 %), α-linolenic acid (11.8 %), nonacosane (7.8 %) and germacrene D (5.4 %). Both samples of oil showed broad spectrum antimicrobial effect with good activity against emerging Gram-positive and Gram-negative opportunistic pathogens and pathogenic fungi which indicates the pharmaceutical potential of the C. rupestris essential oil. This work is licensed under a Creative Commons Attribution 4.0 International License

Cytogenetic characterization of Hydrangea involucrata Sieb. and H. aspera D. Don complex (Hydrangeaceae): genetic, evolutional, and taxonomic implications

The subsection Asperae of genus Hydrangea L. (Hydrangeaceae) has been investigated for three reasons: several ambiguous classifications concerning Hydrangea aspera have been published, unexpected differences in genome size among seven accessions have been reported Cerbah et al. (Theor Appl Genet 103:45–51, 2001), and two atypical chromosome numbers (2n = 30 for Hydrangea involucrata and 2n = 34 for H. aspera) have been found when all other species of the genus present 2n = 36. Therefore, these two species and four subspecies of Hydrangea in all 29 accessions were analyzed for their genome size, chromosome number, and karyotype features. This investigation includes flow cytometric measurements of nuclear DNA content and bases composition (GC%), fluorochrome banding for detection of GC- and AT-rich DNA regions, and fluorescent in situ hybridisation (FISH) for chromosome mapping of 5 S and 18 S-5.8 S-26 S rDNA genes. In the H. aspera complex, the genome size ranged from 2.98 (subsp. sargentiana) to 4.67 pg/2C (subsp. aspera), an exceptional intraspecific variation of 1.57-fold. The mean base composition was 40.5% GC. Our report establishes the first karyotype for the species H. involucrata, and for the subspecies of H. aspera which indeed present different formulae, offering an element of discrimination. FISH and fluorochrome banding revealed the important differentiation between these two species (H. involucrata and H. aspera) and among four subspecies of the H. aspera complex. Our results are in agreement with the Chinese classification that places the groups Kawakami and Villosa as two different species: Hydrangea villosa Rehder and Hydrangea kawakami Hayata. This knowledge can contribute to effective germplasm management and horticultural use

Genome size and chromosome number of Micromeria acropolitana (Lamiaceae), a steno-endemic from Greece

Abstract. The chromosome number 2n = 30, and nuclear DNA amount 2C = 0.79 pg, are determined for the first time for Micromeria acropolitana, a rare and endangered species from the Acropolis in Athens, Greece. The plant was considered extinct but rediscovered in 2006, a hundred years later. Its current status in the original habitat is assessed, and proposals for ensuring its survival presented

Evolutionary implications of heterochromatin and rDNA in chromosome number and genome size changes during dysploidy: a case study in Reichardia genus

In this study we showed that constitutive heterochromatin, GC-rich DNA and rDNA are implicated in chromosomal rearrangements during the basic chromosome number changing (dysploidy) in Reichardia genus. This small Mediterranean genus comprises 8-10 species and presents three basic chromosome numbers (x = 9, 8 and 7). To assess genome evolution and differentiation processes, studies were conducted in a dysploid series of six species: R. dichotoma, R. macrophylla and R. albanica (2n = 18), R. tingitana and R. gaditana (2n = 16), and R. picroides (2n = 14). The molecular phylogeny reconstruction comprised three additional species (R. crystallina and R. ligulata, 2n = 16 and R. intermedia, 2n = 14). Our results indicate that the way of dysploidy is descending. During this process, a positive correlation was observed between chromosome number and genome size, rDNA loci number and pollen size, although only the correlation between chromosome number and genome size is still recovered significant once considering the phylogenetic effect. Fluorescent in situ hybridisation also evidenced changes in number, position and organisation of two rDNA families (35S and 5S), including the reduction of loci number and, consequently, reduction in the number of secondary constrictions and nuclear organising regions from three to one per diploid genome. The potential mechanisms of chromosomal and genome evolution, strongly implicating heterochromatin, are proposed and discussed, with particular consideration for Reichardia genus

Flow cytometry as tool in plant sciences, with emphasis on genome size and ploidy level assessment

Flow cytometry has become the method of choice to measure the DNA content (genome size) in plants. Ease of sample preparation, fast acquisition, and accurate measurements have made the method popular in the domains of plant cell biology, systematics, evolution, genetics and biotechnology. Although the cell wall is a problem when isolating plant cells, cytometry remains a powerful tool in plant sciences. Based on our 30-years’ experience in this field, this review will focus at first on genome size measurement using simply isolated nuclei: the good practice for acquisition, nuclei isolation, appropriate buffers, kind of tissues to use. The second part will briefly review what kind of measurements it is possible to make in plant cytometry, and for what purpose: base composition, ploidy level, cell cycle, endoreplication, seed screening, and nuclei/chromosomes sorting. We will address troubleshooting. The commonly-used mathematic tools will be discussed

Nuclear and chloroplast DNAs reveal diverse origins and mis-identifications of Juniperus cultivars from Windsor Gardens, UK, Part 3 of 3

Ploidy was determined for 15 plants labeled as Juniperus squamata at the Windsor Gardens, UK and revealed 12 were tetraploids (2n=4x=44) and 3 were diploids (2n=2x=22). nrDNA (ITS) and cp DNA sequencing the tetraploids found: 4 J. squamata (4x); 4 J. tibetica (4x) x J. squamata (4x); 2 J. sabina var. balkanensis (4x) x J. squamata (4x); and one J. chinensis var. sargentii (4x) x J. squamata (4x). Sequencing the 3 diploids revealed: 2 J. pingii (2x) x J. pingii (2x); and 1 J. pingii (2x)? x J. komarovii(2x)? Ploidy analyses of 18 additional cultivars, putatively from Juniperus davurica, J. recurva, J. rushforthiana, J. sabina, and J. virginiana revealed 6 diploids, 5 triploids and 7 tetraploids. Cultivar \u27Musgrave\u27 (4x), by DNA, was identical to J. xpfitzeriana \u27Wilhelm Pfitzer\u27 (4x). The DNA of the 5 triploids were all nearly identical to J. xpfitzeriana \u27Wilhelm Pfitzer\u27 (4x). \u27Tamariscifolia\u27 and \u27Variegata\u27 both had J. sabina var. sabina as their maternal parent, but the first had J. sabina var. balkanensis as the male parent and the second had J. sabina var. sabina as the male parent. Thus, \u27Tamariscifolia\u27 is the first discovery of a J. sabina var. balkanensis x J. s. var. sabina hybrid in cultivation. None of the 3 \u27davurica\u27 cultivars proved to be J. davurica, but rather J. chinensis var. procumbens x J. chinensis var. sargentii. Cultivars J. indica and recurva \u27densa\u27 were shown to be J. indica var. caespitosa. recurva \u27 Embley Park\u27 appears to be J. coxii x J. squamata var. wilsonii. J. wallichiana (=J. indica) 15460 was found to be J. rushforthiana, whereas J. wallichiana (15487) was discovered to be J. indica x J. rushforthiana. Cultivar virginiana \u27cannaertii\u27 was shown to be J. virginiana. Botanic gardens provide a great opportunity for species to hybridize with other species that are not in contact in nature. The species care and suitable habitat provided in a garden setting, as well as vegetative propagation methods have allowed the preservation of those rare hybrids). Identification of juniper hybrids and variants is quite imprecise. DNA barcoding of cultivated plants in botanic gardens would greatly facilitate the recognition, study and utilization of rare hybrids and somatic mutations

Discovery of Juniperus sabina var. balkanensis R. P. Adams and A. N. Tashev in Macedonia, Bosnia-Herzegovina, Croatia and Central and Southern Italy and relictual polymorphisms found in nrDNA

Additional analyses of trnS-trnG and nrDNA from specimens from Bosnia-Herzegovina, southern and central, Italy, Croatia and Macedonia revealed the presence of J. sabina var. balkanensis in these areas west of the previously known populations in Greece, Bulgaria and western Turkey. Careful chromatogram analysis of eight (8) polymorphic sites in nrDNA revealed that nearly all of the populations of both var. balkanensis and var. sabina contained from 2 to 8 polymorphic sites. For these 8 heterozygous sites, two exclusive patterns were found in J. sabina. One type (GGACCCAG) was found in 16/62 plants and type 2 (ACGACAGT) was found in 4/62 plants. The majority of the plants examined (42/62) were heterozygous for 1 to 8 sites. These two nrDNA types appear to have arisen via hybridization with a J. thurifera ancestor. The two types appear in both v. sabina and v. balkanensis populations. Extant putative hybrids appear to have formed by crosses between present day type 1 and type 2 nrDNA. Publishe

Discovery of Juniperus sabina var. balkanensis R. P. Adams and A. N. Tashev in Macedonia, Bosnia-Herzegovina, Croatia and Central and Southern Italy and relictual polymorphisms found in nrDNA

Additional analyses of trnS-trnG and nrDNA from specimens from Bosnia-Herzegovina, southern and central, Italy, Croatia and Macedonia revealed the presence of J. sabina var. balkanensis in these areas west of the previously known populations in Greece, Bulgaria and western Turkey. Careful chromatogram analysis of eight (8) polymorphic sites in nrDNA revealed that nearly all of the populations of both var. balkanensis and var. sabina contained from 2 to 8 polymorphic sites. For these 8 heterozygous sites, two exclusive patterns were found in J. sabina. One type (GGACCCAG) was found in 16/62 plants and type 2 (ACGACAGT) was found in 4/62 plants. The majority of the plants examined (42/62) were heterozygous for 1 to 8 sites. These two nrDNA types appear to have arisen via hybridization with a J. thurifera ancestor. The two types appear in both v. sabina and v. balkanensis populations. Extant putative hybrids appear to have formed by crosses between present day type 1 and type 2 nrDNA

Mediterranean plant karyological data

For the first time, the chromosome number was determined for 5 species of the genus Pyrus L. (Rosaceae) from Armenia, viz. P. daralaghezii, P. hyrcana var. yeghegisi, P. medvedevii, P. oxiprion, P. takhtadzhianii, and the previous count of chromosome number for P. caucasica was confirmed. All the explored species have a diploid chromosome number 2n = 34 with the basic chromosome number x = 17.This study has been funded by the projects Proyectos Intramurales Especiales (202330E114) and Functional and evolutionary insights into reproductive biology: the capitulum of Asteraceae as a model system (COMPOSITAE) (PID2020-116480GB-100), and by the Catalan government (2021SGR00315)Introduction Materials and methods Results and discussio

Polyploidy in the Conifer Genus Juniperus: An Unexpectedly High Rate

Recent research suggests that the frequency of polyploidy may have been underestimated in gymnosperms. One notable example is in the conifer genus Juniperus, where there are already a few reports of polyploids although data are still missing for most species. In this study, we evaluated the extent of polyploidy in Juniperus by conducting the first comprehensive screen across nearly all of the genus. Genome size data from fresh material, together with chromosome counts, were used to demonstrate that genome sizes estimated from dried material could be used as reliable proxies to uncover the extent of ploidy diversity across the genus. Our analysis revealed that 16 Juniperus taxa were polyploid, with tetraploids and one hexaploid being reported. Furthermore, by analyzing the genome size and chromosome data within a phylogenetic framework we provide the first evidence of possible lineage-specific polyploidizations within the genus. Genome downsizing following polyploidization is moderate, suggesting limited genome restructuring. This study highlights the importance of polyploidy in Juniperus, making it the first conifer genus and only the second genus in gymnosperms where polyploidy is frequent. In this sense, Juniperus represents an interesting model for investigating the genomic and ecological consequences of polyploidy in conifers