Karyotype Variability in Wild Narcissus poeticus L. Populations from Different Environmental Conditions in the Dinaric Alps.

Narcissus poeticus L. (Amaryllidaceae), a facultative serpentinophyte, is a highly variable species and particularly important ancestor of cultivated daffodils, but is rarely studied in field populations. This study, based on natural populations in the Balkans, focused on karyotype variability, genome size, ploidy and the presence of B chromosomes. Thirteen native populations from different environmental and soil conditions were collected and analyzed using flow cytometry to estimate nuclear genome size, fluorescence in situ hybridization (FISH) for physical mapping of rDNA, fluorochrome labeling (chromomycin and Hoechst) for heterochromatin organization and silver nitrate staining of nucleoli for determining rRNA gene activity. The organization of rDNA and natural triploids is reported here for the first time. The presence of individuals with B chromosomes (in 9/13 populations) and chromosomal rearrangements was also detected. The observed B chromosome showed three different morphotypes. The most frequent submetacentric type showed four different patterns, mainly with active ribosomal genes. The results obtained show that N. poeticus has a dynamic genome with variable genome size due to the presence of polyploidy, B chromosomes and chromosomal rearrangements. It is hypothesized that the observed changes reflect the response of the genome to different environmental conditions, where individuals with B chromosomes appear to have certain adaptive advantages.

Molecular Cytogenetics (Fluorescence In Situ Hybridization - FISH and Fluorochrome Banding): Resolving Species Relationships and Genome Organization.

Fluorochrome banding (chromomycin, Hoechst, and DAPI) and fluorescence in situ hybridization (FISH) are excellent molecular cytogenetic tools providing various possibilities in the study of chromosomal evolution and genome organization. The constitutive heterochromatin and rRNA genes are the most widely used FISH markers. The rDNA is organized into two distinct gene families (18S-5.8S-26S and 5S) whose number and location vary within the complex of closely related species. Therefore, they are widely used as chromosomal landmarks to provide valuable evidence concerning genome evolution at chromosomal levels.

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

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.

When sexual meets apomict: genome size, ploidy level and reproductive mode variation of Sorbus aria s.l. and S. austriaca (Rosaceae) in Bosnia and Herzegovina.

BACKGROUND AND AIMS: Allopolyploidy and intraspecific heteroploid crosses are associated, in certain groups, with changes in the mating system. The genus Sorbus represents an appropriate model to study the relationships between ploidy and reproductive mode variations. Diploid S. aria and tetraploid apomictic S. austriaca were screened for ploidy and mating system variations within pure and sympatric populations in order to gain insights into their putative causalities. METHODS: Flow cytometry was used to assess genome size and ploidy level among 380 S. aria s.l. and S. austriaca individuals from Bosnia and Herzegovina, with 303 single-seed flow cytometric seed screenings being performed to identify their mating system. Pollen viability and seed set were also determined. KEY RESULTS: Flow cytometry confirmed the presence of di-, tri- and tetraploid cytotype mixtures in mixed-ploidy populations of S. aria and S. austriaca. No ploidy variation was detected in single-species populations. Diploid S. aria mother plants always produced sexually originated seeds, whereas tetraploid S. austriaca as well as triploid S. aria were obligate apomicts. Tetraploid S. aria preserved sexuality in a low portion of plants. A tendency towards a balanced 2m : 1p parental genome contribution to the endosperm was shared by diploids and tetraploids, regardless of their sexual or asexual origin. In contrast, most triploids apparently tolerated endosperm imbalance. CONCLUSIONS: Coexistence of apomictic tetraploids and sexual diploids drives the production of novel polyploid cytotypes with predominantly apomictic reproductive modes. The data suggest that processes governing cytotype diversity and mating system variation in Sorbus from Bosnia and Herzegovina are probably parallel to those in other diversity hotspots of this genus. The results represent a solid contribution to knowledge of the reproduction of Sorbus and will inform future investigations of the molecular and genetic mechanisms involved in triggering and regulating cytotype diversity and alteration of reproductive modes.

Molecular cytogenetics (FISH and fluorochrome banding): resolving species relationships and genome organization.

Fluorochrome banding (chromomycin, Hoechst, and DAPI) and fluorescence in situ hybridization (FISH) are excellent molecular cytogenetic tools providing various possibilities in the study of chromosomal evolution and genome organization. The constitutive heterochromatin and rRNA genes are the most widely used FISH markers. The rDNA is organized into two distinct gene families (18S-5.8S-26S and 5S) whose number and location vary within the complex of closely related species. Therefore, they are widely used as chromosomal landmarks to provide valuable evidence concerning genome evolution at chromosomal levels.

Physical mapping of 5S and 18S-5.8S-26S RNA gene families in polyploid series of Cenchrus ciliaris Linnaeus, 1771 (Poaceae).

The Buffelgrass (Cenchrus ciliaris L., Poaceae) is one of the most important pasturage grasses due to its high productivity and good forage qualities. This species possess a high adaptability to bioclimatic constraints of arid zones and may be used for the restoration of degraded arid ecosystems. Tunisian populations present three ploidy levels (4x, 5x and 6x) with a basic chromosome number x=9. This study reported for the first time the distribution of the ribosomal genes (rRNA) for pentaploid and hexaploid cytotypes of Cenchrus ciliaris. Molecular cytogenetic study using double fluorescence in situ hybridization has shown that the two rDNA families, 5S and 18S-5.8S-26S (18S), displayed intraspecific variation in number of loci among different ploidy levels. Each ploidy level was characterized by specific number of both 5S and 18S rDNA loci (two loci in tetraploid, five in pentaploid and six in hexaploid level). For three studied cytotypes (4x, 5x and 6x) all 5S rDNA loci were localized on the subcentromeric region of chromosomes, while 18S loci were situated on the telomeric region of short chromosome arms. Data of the FISH experiments show proportional increase of ribosomal loci number during polyploidization processes.