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The distributional pattern of GC-rich regions and the physical mapping of ribosomal DNA (location of 35S and 5S rDNA) in the chromosomes of fourteen Amaranthus species were established using fluorochrome banding and fluorescence in situ hybridization (FISH). The karyotypes are composed of 2 n = 32 or 2 n = 34 chromosomes. Some species were also shown to exhibit infraspecific polymorphism in the chromosome number and consisted of accessions with both of these chromosome numbers. Two families of ribosomal genes, 35S and 5S rDNA, were separated onto different pairs in chromosome complements of most of the species examined. Both 35S and 5S rDNA sites were always located in the terminal part of the chromosomes and usually 35S rDNA sites were present in a lower number than the 5S rDNA sites in most of the species that were analyzed. Polymorphism of the rDNA site number was observed in three species. Fluorochrome banding revealed that CMA + /DAPI − bands were associated with the 35S rDNA sites in all of the species that were analyzed. In some amaranth accessions, 5S rDNA regions were also GC-rich. The possible mechanisms of the evolution of rDNA loci are discussed.

Background Polyploid hybrids represent a rich natural resource to study molecular evolution of plant genes and genomes. Here, we applied a combination of karyological and molecular methods to investigate chromosomal structure, molecular organization and evolution of ribosomal DNA (rDNA) in nightshade, Atropa belladonna (fam. Solanaceae), one of the oldest known allohexaploids among flowering plants. Because of their abundance and specific molecular organization (evolutionarily conserved coding regions linked to variable intergenic spacers, IGS), 45S and 5S rDNA are widely used in plant taxonomic and evolutionary studies. Results Molecular cloning and nucleotide sequencing of A. belladonna 45S rDNA repeats revealed a general structure characteristic of other Solanaceae species, and a very high sequence similarity of two length variants, with the only difference in number of short IGS subrepeats. These results combined with the detection of three pairs of 45S rDNA loci on separate chromosomes, presumably inherited from both tetraploid and diploid ancestor species, example intensive sequence homogenization that led to substitution/elimination of rDNA repeats of one parent. Chromosome silver-staining revealed that only four out of six 45S rDNA sites are frequently transcriptionally active, demonstrating nucleolar dominance. For 5S rDNA, three size variants of repeats were detected, with the major class represented by repeats containing all functional IGS elements required for transcription, the intermediate size repeats containing partially deleted IGS sequences, and the short 5S repeats containing severe defects both in the IGS and coding sequences. While shorter variants demonstrate increased rate of based substitution, probably in their transition into pseudogenes, the functional 5S rDNA variants are nearly identical at the sequence level, pointing to their origin from a single parental species. Localization of the 5S rDNA genes on two chromosome pairs further supports uniparental inheritance from the tetraploid progenitor. Conclusions The obtained molecular, cytogenetic and phylogenetic data demonstrate complex evolutionary dynamics of rDNA loci in allohexaploid species of Atropa belladonna. The high level of sequence unification revealed in 45S and 5S rDNA loci of this ancient hybrid species have been seemingly achieved by different molecular mechanisms. Electronic supplementary material The online version of this article (doi:10.1186/s12870-017-0978-6) contains supplementary material, which is available to authorized users.

Studies on Chenopodium chromosomes are scarce and restricted mainly to chromosome number estimation. To extend our knowledge on karyotype structure of the genus, the organization of 5S and 35S rRNA genes in Chenopodium chromosomes was studied. The rDNA sites were predominantly located at chromosomal termini, except in a few species where 5S rDNA sites were interstitial. The majority of the diploid species possessed one pair each of 35S and 5S rDNA sites located on separate chromosomes. Slightly higher diversity in rDNA site number was observed in polyploid accessions. One or two pairs of 35S rDNA sites were observed in tetraploids and hexaploids. Tetraploid species had two, four or six sites and hexaploid species had six or eight sites of 5S rDNA, respectively. These data indicate that, in the evolution of some polyploid species, there has been a tendency to reduce the number of rDNA sites. Additionally, polymorphism in rDNA site number was observed. Possible mechanisms of rDNA locus evolution are discussed. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, ••, ••–••.

Core histones are subjected to various post-translational modifications, and one of them, most intensively studied in plants, is the methylation of histone H3. In the majority of analyzed plant species, dimethylation of H3 at lysine 9 (H3K9me2) is detected in heterochromatin domains, whereas methylation of H3 at lysine 4 (H3K4me2) is detected in euchromatin domains. The distribution of H3K9me2 in the interphase nucleus seems to be correlated with genome size, chromatin organization, but also with tissue specificity. In this paper, we present the analysis of the pattern and level of histone H3 methylation for two allotetraploid and one diploid Brassica species. We have found that the pattern of H3K9me2 in interphase nuclei from root meristematic tissue is comparable within the analyzed species and includes both heterochromatin and euchromatin, but the level of modification differs not only among species but even among nuclei in the same phase of the cell cycle within one species. Moreover, the differences in the level of H3K9me2 are not directly coupled with DNA content in the nuclei and are probably tissue specific.

Background: Supernumerary chromosomes (B) comprise optional complement to basic (A) chromosome set. The presence of B-chromosomes may significantly reduce plant vigor and fertility. Potentially active genes constitute only small fraction of DNA of these chromosomes indicating that these effects are mediated by epigenetic mechanisms. One example is down-regulation of rDNA genes and condensation of their respective chromatin regions (demonstrated in squashed preparations using 2D microscopy). It may be postulated that the presence of B chromosomes leads to more extensive changes of local chromatin structure. Verification of hypothesis requires studying 3D spatial architecture of intact nuclei in tissue. Results: An image processing algorithm was developed and applied for isolation (from the confocal datasets) of regions corresponding to single nuclei. The nuclei were segmented using iterative global thresholding followed by growing and merging of regions belonging to different nuclei. The result of segmentation was verified by a human observer. Chromatin architecture was characterized quantitatively using global fluorescence intensity distribution measures (mean, variance) and local intensity distribution parameters (haraclick features, wavelet energy, run- length features). The sets of parameters corresponding to populations of nuclei with different number of B-chromo- somes were subjected to discriminate analysis. The distinct parameters were then correlated with depth in tissue at which a given nucleus was positioned. Conclusions: Combination of light microscopy with dedicated image processing and analysis framework made it possible to study chromatin architecture in nuclei containing various number of B chromosomes. These data indicate that alterations of 3D chromatin distribution occur globally in the interphase nuclei in the presence of Bs. The changes occur at the spatial scale comparable with the resolution limit of light microscopy and at larger distances.

The extent and significance of intraspecific genome size variation were analysed in quinoa (Chenopodium quinoa Willd.), a pseudocereal important for human consumption in the Andean region of South America. Flow cytometry, with propidium iodide as the DNA stain, was used to estimate the genome size of 20 quinoa accessions from Ecuador, Peru, Bolivia, Argentina, Chile and the USA. Limited genome size variation was found among the analysed accessions. The differences between the accessions were statistically significant but the maximum inter-accession difference between the populations with the largest and the smallest genome reached only 5.9%. The largest genome was found in population C4 from Chile (mean 3.077 pg/2C) and the smallest in the Peruvian population P2 (mean 2.905 pg/2C). The variation was not correlated with collection site; however, the quinoa accessions analysed in this study belonged to three distinct geographical groups: northern highland, southern highland and lowland.

A micronucleus test in combination with fluorescent in situ hybridization (FISH) using telomere-, centromere-specific probes and 5S and 25S rDNA was used for a detailed analysis of the effects of gamma ray irradiation on the root tip meristem cells of barley, Hordeum vulgare (2n = 14). FISH with four DNA probes was used to examine the involvement of specific chromosomes or chromosome fragments in gamma ray-induced micronuclei formation and then to explain their origin. Additionally, a comparison of the possible origin of the micronuclei induced by physical and chemical treatment: maleic hydrazide (MH) and N-nitroso-N-methylurea (MNU) was done. The micronuclei induced by gamma ray could originate from acentric fragments after chromosome breakage or from whole lagging chromosomes as a result of a dysfunction of the mitotic apparatus. No micronuclei containing only centromeric signals were found. An application of rDNA as probes allowed it to be stated that 5S rDNA–bearing chromosomes are involved in micronuclei formation more often than NOR chromosomes. This work allowed the origin of physically- and chemically-induced micronuclei in barley cells to be compared: the origin of micronuclei was most often from terminal fragments. FISH confirmed its usefulness in the characterization of micronuclei content, as well as in understanding and comparing the mechanisms of the actions of mutagens applied in plant genotoxicity.

The allopolyploid origin of several bryophyte species was reported approximately 20 years ago. Among them the best characterized is Pellia borealis, a hybrid of two cryptic sibling species: Pellia epiphylla-species N and P. epiphylla-species S. Genomes of the allopolyploid liverwort P. borealis and its progenitors were investigated using conventional as well as molecular cytogenetic techniques. The nuclear DNA content and cell cycle phase in the thallus nuclei was established using flow cytometry. Fluorescent differential staining, C-banding and fluorescent in situ hybridization (FISH) with 26S and 5S ribosomal DNA (rDNA) probes revealed new features of the chromosomes in the P. epiphylla–P. borealis complex. Some characteristics found in both the polyploid and haploid karyotypes support earlier suggestions about the allopolyploid origin of P. borealis. The banding pattern observed on P. borealis chromosomes suggests the occurrence of structural changes in the allopolyploid genome. The number and ...

In this study, a novel repetitive sequence pTaq10 was isolated from the Taq I digest of the genomic DNA of the pseudocereal Chenopodium quinoa. Sequence analysis indicated that this 286-bp monomer is not homologous to any known retroelement sequence. FISH and Southern blot analysis showed that this sequence is characterized by an interspersed genomic organization. After FISH, hybridization signals were observed as small dots spread throughout all of the chromosomes. pTaq hybridization signals were excluded from 45S rRNA gene loci, but they partly overlapped with 5S rDNA loci. pTaq10 is not a species-specific sequence, as it was also detected in C. berlandieri.

The present study is a rare example of a detailed characterization of chromosomal aberrations by identification of individual chromosomes (or chromosome arms) involved in their formation in plant cells by using fluorescent in situ hybridization (FISH). In addition, the first application of more than 2 DNA probes in FISH experiments in order to analyse chromosomal aberrations in plant cells is presented. Simultaneous FISH with 5S and 25S rDNA and, after reprobing of preparations, telomeric and centromeric DNA sequences as probes, were used to compare the cytogenetic effects of 2 chemical mutagens: N-nitroso-N-methylurea (MNU) and maleic hydrazide (MH) on root tip meristem cells of Hordeum vulgare (2n=14). The micronucleus (MN) test combined with FISH allowed the quantitative analysis of the involvement of specific chromosome fragments in micronuclei formation and thus enabled the possible origin of mutagen-induced micronuclei to be explained. Terminal deletions were most frequently caused by MH and MNU. The analysis of the frequency of micronuclei with signals of the investigated DNA probes showed differences between the frequency of MH- and MNU-induced micronuclei with specific signals. The micronuclei with 2 signals, telomeric DNA and rDNA (5S and/or 25S rDNA), were the most frequently observed in the case of both mutagens, but with a higher frequency after treatment with MH (46%) than MNU (37%). Also, 10% of MH-induced micronuclei were characterized by the presence of only telomere DNA sequences, whereas there were almost 3-fold more in the case of MNU-induced micronuclei (28%). Additionally, by using FISH with the same probes, an attempt was made to identify the origin of chromosome fragments in mitotic anaphase.

• Background and Aims The Brassicaceae family encompasses numerous species of great agronomic importance, belonging to such genera, as Brassica, Raphanus, Sinapis and Armoracia. Many of them are characterized by extensive intraspecific diversity of phenotypes. The present study focuses on the polymorphism of number, appearance and chromosomal localization of ribosomal DNA (rDNA) sites and, when possible, in relation to polyploidy, in 42 accessions of Brassica species and ten accessions of Diplotaxis, Eruca, Raphanus and Sinapis species. • Methods Chromosomal localization of ribosomal DNA was carried out using dual colour fluorescence in situ hybridization (FISH) with 5S rDNA and 25S rDNA sequences as probes on enzymatically digested root-tip meristematic cells. • Key Results Loci for 5S and 18S–5.8S–25S rDNA were determined for the first time in six taxa, and previously unreported rDNA constellations were described in an additional 12 accessions. FISH revealed frequent polymorphism in number, appearance and chromosomal localization of both 5S and 25S rDNA sites. This phenomenon was most commonly observed in the A genome of Brassica, where it involves exclusively pericentromeric sites of 5S and 25S rRNA genes. The intraspecific polymorphism was between subspecies/varieties or within a variety or cultivar (i.e. interindividual). • Conclusions The number of rDNA sites can differ up to 5-fold in species with the same chromosome number. In addition to the eight previously reported chromosomal types with ribosomal genes, three new variant types are described. The extent of polymorphism is genome dependent. Comparing the A, B and C genomes revealed the highest rDNA polymorphism in the A genome. The loci carrying presumably inactive ribosomal RNA genes are particularly prone to polymorphism. It can also be concluded that there is no obvious polyploidization-related tendency to reduce the number of ribosomal DNA loci in the allotetraploid species, when compared with their putative diploid progenitors. The observed differences are rather caused by the prevailing polymorphism within the diploids and allotetraploids. This would make it difficult to predict expected numbers of rDNA loci in natural polyploids.

Like those of most angiosperms, vegetative tissues of Arabidopsis thaliana undergo high levels of endopolyploidization. One such tissue is the anther tapetum which plays a role in male sporo- and gametogenesis. The degree of polyploidization of the tapetum varies from species to species. Although the role of this process is not yet fully understood, it may be linked to functioning of the tapetum, increasing the copy number of genes needed for the synthesis of specific factors required by developing pollen mother cells (PMCs) and pollen grains. The present study focused on polyploidization during the development of the tapetum of Arabidopsis thaliana . The aim was to outline the mode of tapetum polyploidization in this model plant species and to establish an efficient method for analysing ploidy levels in differentiated cells. The course and degree of tapetum polyploidization in Arabidopsis was analysed in interphase nuclei using fluorescence in situ hybridization (FISH) with repetitive DNA (45S rDNA). The stages of development of the tapetum were analysed alongside meiosis in PMCs. The majority of tapetal cells undergo two, maximally three, rounds of divisions. Tapetal nuclei have usually divided by metaphase I of meiosis of PMCs. The pattern of tapetum polyploidization was similar in diploid and autotetraploid plants and is thus not affected by increasing amounts of maternal plant DNA. The tapetum of autotetraploid plants exhibits a higher frequency of additional division than seen in diploid plants.

Molecular cytogenetics, particularly the localization of DNA sequences by in situ hybridization, has increased our understanding about the genomic structure of plants and animals. We demonstrate here the application of an improved nonfluorescent in situ hybridization system detection (DAKO GenPoint system) to plant chromosomes. Using this system, highly repetitive 18S-25S rRNA genes were mapped on Vicia faba chromosomes (2n = 12). The modified method of this horseradish peroxidase based enzymatic detection system gave satisfactory results that are comparable to fluorescent signal detection.

The chromosome behaviour has been compared in three Crepis capillaris callus culture lines and the roots regenerated from these calli. The calli were obtained from explants derived from plants without and with two B chromosomes and the hairy roots were obtained from plants transformed with Agrobacterium rhizogenes. Cytological studies demonstrated that the presence of additional DNA as B chromosomes or as T-DNA had an influence on the numerical and structural variability of the standard chromosome in long-term callus cultures and in regenerated organs. The callus with two B chromosomes displayed higher levels of polyploidyzation than callus without B chromosomes. The roots regenerated from both these calli were only diploid, while roots regenerated from transformed callus were also polyploid.

Plant growth and development is precisely programmed and achieved through three processes: cell division (proliferation), growth and differentiation. These three processes may overlap during plant organ development, when some cells start to differentiate while others continue to divide e.g. leaf epidermal cells (Harashima and Schnittger 2010). Dividing cells, called meristematic cells, increase their number and supply new cells for post-embryonic plant development. Outside the meristems non-dividing cells expand and differentiate. Cell proliferation and expansion result in varied but determined cell sizes specific for the plant, organ and tissue. The next phase in plant development is cell-type specification along with the differentiation processes. The control of all processes and the determination of final cell mass and size are poorly understood but there is increasing knowledge about the molecular mechanisms underpinning the regulatory systems. Cell sizes in plants are usually closely related to their function. There are two strategies to enlarge cell size: one is based on water uptake and vacuolar growth and the other is to increase the nuclear DNA content or the level of polyploidy, this gives rise to endopolyploidy.

We analyzed DNA damage, mitotic activity and polyploidization in Crepis capillaris callus cells during short- and long-term in vitro culture, and the influence of plant growth regulators on these processes. Changes in the concentration of growth regulators altered the stability of callus. The level of DNA damage was highly dependent on the growth regulator composition of the medium. Cytokinin at high concentrations damaged DNA in the absence of auxin. Short- and long-term callus differed in sensitivity to growth regulators. Mitotic activity changed when callus was transferred to medium with modified growth regulators. Callus cell nuclear DNA content increased with age and in response to plant growth regulators. Hormones played a role in the genetic changes in C. capillaries callus culture. We demonstrated the usefulness of C. capillaris callus culture as a model for analyzing the effect of culture conditions, including plant growth regulators, on genetic stability.

The chromosomal organization of two novel repetitive DNA sequences isolated from the Chenopodium quinoa Willd. genome was analyzed across the genomes of selected Chenopodium species. Fluorescence in situ hybridization (FISH) analysis with the repetitive DNA clone 18–24J in the closely related allotetraploids C. quinoa and Chenopodium berlandieri Moq. (2n = 4x = 36) evidenced hybridization signals that were mainly present on 18 chromosomes; however, in the allohexaploid Chenopodium album L. (2n = 6x = 54), cross-hybridization was observed on all of the chromosomes. In situ hybridization with rRNA gene probes indicated that during the evolution of polyploidy, the chenopods lost some of their rDNA loci. Reprobing with rDNA indicated that in the subgenome labeled with 18–24J, one 35S rRNA locus and at least half of the 5S rDNA loci were present. A second analyzed sequence, 12–13P, localized exclusively in pericentromeric regions of each chromosome of C. quinoa and related species. The intensity of the FISH signals differed considerably among chromosomes. The pattern observed on C. quinoa chromosomes after FISH with 12–13P was very similar to GISH results, suggesting that the 12–13P sequence constitutes a major part of the repetitive DNA of C. quinoa.

Cytogenetic analysis of several populations of Centaurea jacea (2n = 4x = 44), C. oxylepis (2n = 4x = 44) and C. phrygia (2n = 2x = 22) was performed using flow cytometry, differential chromosome staining and FISH. In all species Arabidopsis-type telomeric repeats hybridized only to the terminal part of chromosomes. In C. phrygia three pairs and in C. oxylepis six pairs of chromosomes revealed the hybridization signals of 45S rDNA. Centaurea jacea showed polymorphism in the 45S rDNA loci number, five or six pairs of sites were observed. 5S rDNA loci were located in two pairs of chromosomes in C. phrygia. In C. jacea and C. oxylepis the number and position of 5S rDNA loci were the same: three pairs located interstitially and one terminally. The genome size of the diploid C. phrygia was established as 2.14 pg/2C. The genomes of tetraploid species were nearly two times larger and genome size polymorphism was observed among C. jacea populations.

It is important for the prevention of DNA changes caused by environment to understand the biological consequences of DNA damages and their molecular modes of action that lead to repair or alterations of the genetic material. Numerous genotoxicity assay systems have been developed to identify DNA reactive compounds. The available data show that plant bioassays are important tests in the detection of genotoxic contamination in the environment and the establishment of controlling systems. Plant system can detect a wide range of genetic damage, including gene mutations and chromosome aberrations. Recently introduced molecular cytogenetic methods allow analysis of genotoxicity, both at the chromosomal and DNA level. FISH gives a new possibility of the detection and analysis of chromosomal rearrangements in a great detail. DNA fragmentation can be estimated using the TUNEL test and the single cell gel electrophoresis (Comet assay).

It is important for the prevention of DNA changes caused by environment to understand the biological consequences of DNA damages and their molecular modes of action that lead to repair or alterations of the genetic material. Numerous genotoxicity assay systems have been developed to identify DNA reactive compounds. The available data show that plant bioassays are important tests in the detection of genotoxic contamination in the environment and the establishment of controlling systems. Plant system can detect a wide range of genetic damage, including gene mutations and chromosome aberrations. Recently introduced molecular cytogenetic methods allow analysis of genotoxicity, both at the chromosomal and DNA level. FISH gives a new possibility of the detection and analysis of chromosomal rearrangements in a great detail. DNA fragmentation can be estimated using the TUNEL test and the single cell gel electrophoresis (Comet assay)., Za prevenciju oštećivanja molekule DNA zbog onečišćenja okoliša nužno je razumijevanje bioloških posljedica nastalih oštećenjem DNA i molekularnih mehanizama djelovanja genotoksikanata koji vode ili popravku ili promjenama genskog materijala. Do danas je usavršen niz testova za detekciju genotoksičnog djelovanja koji omogućuju identifikaciju supstancija koje reagiraju s molekulom DNA. Raspoloživi podaci pokazuju da su biljni biotestovi vrlo važni u detekciji genotoksičnog onečišćenja okoliša, kao i za uspostavljanje nadzornih sustava u okolišu. Biljni test-sustav može otkriti široki raspon genskog oštećenja uključujući mutacije gena i kromosomske aberacije. Nove molekularno-citogenetičke metode omogućuju analizu genotoksičnog djelovanja na razini kromosoma i molekule DNA. Metoda FISH-a ("fluorescent in situ hybridization") pruža nove mogućnosti za detekciju i analizu kromosomskih preraspodjela. Lomovi u molekuli DNA mogu se uspješno detektirati s pomoću metode TUNEL i gel-elektroforeze pojedinačnih stanica (komet test).

Interspecific alien chromosome addition lines can be very useful for gene mapping and studying chromosome homoeology between closely related species. In this study we demonstrate a simple but robust manner of identifying individual C-genome chromosomes (C5, C8 and C9) in the A-genome background through the simultaneous use of 5S and 25S ribosomal probes on mitotic and meiotic chromosomes of three different Brassica rapa-B. oleracea var. alboglabra monosomic addition lines. Sequential silver staining and fluorescence in situ hybridisation indicated that 18S-5.8S-25S rRNA genes on the additional chromosome C9 are expressed in the A-genome background. Meiotic behaviour of the additional chromosomes was studied in pollen mother cells at diakinesis and metaphase I. In all of the addition lines the alien chromosome was most frequently observed as a univalent. The alien chromosome C5, which carries an intercalary 5S rDNA locus, occasionally formed trivalents that involved either rDNA- or non rDNA-carrying chromosomes from the A genome. In the case of chromosomes C8 and C9, the most frequently observed intergenomic associations involved the regions occupied by 18S-5.8S-25S ribosomal RNA genes. It is possible that not all such associations represent true pairing but are remnants of nucleolar associations from the preceding interphase. Variations in the numbers and distribution of 5S and 25S rDNA sites between cultivars of B. oleracea, B. oleracea var. alboglabra and B. rapa are discussed.

Twelve callus lines of Arabidopsis thaliana were derived from four types of explants excised from diploid plants of two ecotypes (Columbia and Wilna) and autotetraploid plants of the Wilna ecotype. Cytogenetic analysis of the chromosome variation in particular callus lines was carried out for primary culture and callus during 5 months of culture. Ploidy levels of interphase nuclei were estimated by counting the number and size of chromocentres and nuclei of interphase cells. The first polyploid cells in all callus lines were observed during callogenesis. In primary culture the ploidy level ranged between 2 and 15x (10-75 chromosomes). The frequency of polyploid cells was higher in the 5-month old callus culture, but the ploidy level was the same. In the callus lines derived from autotetraploid plants, cells with reduced chromosome number appeared quite frequently along with diploid and polyploid cells.

The presence of a large number of pollutants, including mutagenic agents in the environment is a problem of a major concern. Rapid progress in plant biotechnology, especially in the development of cell transformation methods, including the production of transformed roots -- 'hairy roots' -- has opened new possibilities to use transformed root cultures in plant bioassays for the evaluation mutagenic effects of different agents. We have used Crepis capillaris hairy roots for evaluation of cytogenetic effects of mutagenic treatment. Effects of maleic acid hydrazide (MH) and X-ray treatment were analysed in chromosomal aberration, sister chromatid exchange (SCE) and TUNEL tests. Comparison of cytogenetic effects in hairy roots and roots of seedlings showed a much higher sensitivity of hairy roots, which makes them convenient material for monitoring DNA damage after mutagenic treatment.

Crepis capillaris (2n=6) is an excellent plant for the assay of chromosome aberrations after mutagenic treatment. It has simple karyotype: three pairs of morphologically distinct and relatively large chromosomes. The frequency of structural chromosome aberrations and micronuclei in root meristem cells has been used for evaluation of the genotoxicity of chemicals and environmental pollutants. The introduction of fluorescence in situ hybridization method allows more detailed detection and localization of chromosomal rearrangements not only in mitotic but also in interphase nuclei. We demonstrate a few examples of the detection of chromosomal aberrations using rDNA and telomeric sequences as probes for in situ hybridization to C. capillaris chromosomes.

Molecular cytogenetic analysis of Lupinus angustifolius and Lupinus cosentinii was performed using flow cytometry, fluorescence in situ hybridisation (FISH) and differential chromosome staining. Genome size was determined as 2.07 pg for L. angustifolius and 1.54 pg for L. cosentinii. Analysis of nuclear DNA amount in cells during plant development has shown endopolyploidisation in different organs. The highest level of endopolyploidy was in cotyledons and reached 32C in L. angustifolius and 64C in L. cosentinii. Both of the investigated Lupinus species belong to the polysomatic type of plants. Double FISH with rDNA probes provided chromosomal landmarks for 10 out of 40 chromosomes for L. angustifolius and 8 out of 32 chromosomes for L. cosentinii. In L. angustifolius, the number and localisation of 25S rDNA hybridisation signals precisely corresponded to the chromomycin A3 (CMA(+)) bands, while in L. cosentinii both 25S and 5S rDNA loci overlapped with CMA(+) bands. Silver staining revealed that only 45S rRNA genes located in secondary constriction regions were transcriptionally active. FISH with Arabidopsis-type telomeric arrays revealed the presence of signals at termini of all chromosomes. Despite the application of different DNA probes for FISH and different chromosome staining, a relatively small proportion of chromosomes in the Lupinus karyotypes can be distinguished. Identification of all chromosomes requires the use of more chromosome-specific markers.

Species of Brassica have small, morphologically similar chromosomes, which makes karyotyping difficult using conventional cytogenetic methods. Molecular cytogenetic methods, like fluorescence in situ hybridisation (FISH) have the potential to improve karyotyping through the use of chromosome- or genome-specific markers. Simultaneous application of more than one DNA probe can greatly enrich the results obtained compared with separate single target FISH experiments. This paper demonstrates the use of multicolour fluorescence in situ hybridisation with 5S and 25S rDNA for karyotyping three amphidiploid species: B. napus, B. juncea and B. carinata. Using this method, it was possible to identify eight out of nineteen pairs of chromosomes in B. napus, ten out of eighteen pairs in B. juncea and six out of sixteen pairs in B. carinata. Additionally, rDNA sites allow the determination of the genomic origin of all marked chromosomes in B. napus and B. juncea.

Experiments were performed on Crepis capillaris callus lines with 0, 1 and 2 B chromosomes and on hairy root lines without or with 1 and 2 B chromosomes. Comparison of HPLC results for DNA from calli differing in number of B chromosomes did not reveal any significant differences in methylation level (30.4 +/- 1.1%, 30.9 +/- 1.2%, 31.7 +/- 1.7% in lines without or with one or two B chromosomes respectively) which could be attributed to the number of B chromosomes. Restriction patterns obtained after DNA digestion with HhaI, HpaII, MspI or HaeIII (i.e. restriction enzymes sensitive to cytosine methylation) were similar in calli and apical root segments and also did not depend on the presence or number of B chromosomes. Methylation of B chromosomes higher than that of A chromosomes was demonstrated by fluorescent in situ nick translation driven by HpaII, MspI or HaeIII in metaphase chromosomes. After short digestion (I and 3 h), B chromosomes, in contrast to A chromosomes, were weakly labelled or not labelled at all, which indicates longer distances between target sequences containing unmethylated cytosine in the former.

Cytogenetics is complementary to genetic and molecular analysis of plant genome structure and function. Since its beginning it has been mainly used for identification of chromosomes in plant genomes and karyotype construction (karyotyping). The cytological techniques applied to karyotyping should be easy, reproducible, and should be able to identify major structural rearrangements that can occur between chromosomes. Most significant for the progress in plant cytogenetics was the development of the squash method for chromosome preparation (Darlington, 1937), chromosome banding techniques (Caspersson et al., 1968) and in situ hybridization (ISH) techniques (Pardue and Gall, 1969), especially fluorescent in situ hybridization (FISH) (Pinkel et al., 1986). These methods have had a major impact on investigation and understanding of plant genome organisation.

Recent development of cytogenetic techniques has facilitated significant progress in Arabidopsis thaliana karyotype studies. Double-target FISH with rRNA genes provides makers that allow individual chromosome in the genome to be distinguished. Those studies have revealed that the number and position of rDNA loci is ecotype-specific. Arabidopsis is believed to be a true diploid (x = 5) with numerous ecotypes (accessions) and only a very few natural polyploid populations reported. Few studies were undertaken to induce polyploidy in Arabidopsis, however none of those gave the cytogenetic characteristics of polyploid plants. Our analysis of chromosome pairing of colchicine-induced autotetraploid Arabidopsis (Wilna ecotype) revealed preferential bivalent pairing in PMCs (pollen mother cells). In order to attempt to explain this phenomenon, first of all more detailed cytogenetic studies of autopolyploid plants have been undertaken. The localization of 45S and 5S rDNA loci in the diploid and autotetraploid plants revealed that Wilna ecotypes belongs to the group of Arabidopsis accessions with only two 5S rDNA loci present in a genome. Furthermore, the rearrangement of 45S rDNA locus in autopolyploid, when compared to the diploid plants of the same ecotype, was revealed. These results are interesting also in the context of the recently emphasised role of polyploidy in plant evolution and speciation. Arabidopsis, despite having small chromosomes, is a good system to study chromosome behaviour in relation to diploidization of autopolyploids and to evaluate the degree of chromosomal rearrangements during this process.

Chenopodium quinoa Wild, and Amaranthus caudatus L„ two plant species from South America, have small and numerous chromosomes. Looking for chromosome markers to distinguish pairs of homologous chromosomes double fluorescence staining, in situ hybridization with 45S rDNA and silver staining were applied. Fluorescent in situ hybridization with 45S rDNA has shown two sites of hybridization occurring on one pair of chromosomes in quinua genome (lines PQ-I. PQ-X). The number of rDNA loci in Amaranthus caudatus L. genome depends on the accession. Kiwicha 3 line has one pair of chromosomes with signals and Kiwicha Molincra cultivar two pairs. All observed rDNA loci were active. After chromomycin/ DAPl staining in all cases, except Kiwicha Molinera cultivar, the CMA, positive bands co-localized with signals of in situ hybridization with rDNA. In Kiwicha Molinera the number of CMA+ bands was higher than the number of 45S rDNA signals after FISH. Genomy dwóch południowoamerykańskich gatunków. Chenopodiuma quinoa Wild, i Amaranthus caudatus L.. charakteryzują się dużą liczbą niewielkich i morfologicznie mało zróżnicowanych chromosomów. Dla opracowania kariotypu tych gatunków niezbędne jest znalezienie cytogenetycznych markerów pozwalających odróżnić pary chromosomów homologicznych. W tym celu zastosowano barwienie fluorescencyjne (CMA,/DAPI) i hybrydyzację in situ z 45S rDNA oraz srebrzenie dla określenia jąderkowej aktywności transkrypcyjnej. Po fluorescencyjnej hybrydyzacji in situ z. 45S rDNA w genomie C. quinoa (linie PQ-I. PQ-8) obserwowano dwa sygnały występujące w jednej parze chromosomów. Liczba loci rDNA w genomie A. caudatus była różna. W linii Kiwicha .3 obserwowano dwa sygnały a u odmiany Kiwicha Molinera cztery. Wszystkie obserwowane loci rDNA są aktywne transkrypcyjnic. We wszystkich przypadkach, za wyjątkiem odmiany Kiwicha Molinera, pozytywne prążki CM A, pokrywały się z sygnałami hybrydyzacji in situ. U Kiwicha Molinera liczba pozytywnych prążków CMA( była większa niż liczba sygnałów po hybrydyzacji z 45S rDNA.

Two cytotypes of Chenopodium album, diploid (2n=2x=18) and hexaploid (2n=6x=54), were analysed using flow cytometry and a FISH experiment. The genome size was indicated as 1.795 pg for the diploid and 3.845 pg for the hexaploid plants which suggested genome downsizing in the evolution of hexaploid cytotype. Double FISH with 25S rDNA and 5S rDNA allowed three to five homologue chromosome pairs to be distinguished depending on the cytotype. The Variation in size and number of rDNA sites between the polyploid C. album and its putative diploid ancestor indicated that rDNA loci underwent rearrangements after polyploidization. Flow cytometry measurements of the relative nuclear DNA content in the somatic tissue of C. album revealed extensive endopolyploidization resulting in tissues comprising a mixture of cells with a different DNA content (from 2C to 32C) in varying proportions. The pattern of endopolyploidy was characteristic for the developmental stage of the plant and for the individual organ. Polysomaty was not observed in the embryo tissues however endopolyploidization had taken place in most tested organs of seedlings. The endopolyploidy in diploid and hexaploid C. album was compared to find any relationship between the pattern of polysomaty and polyploidy level in this species. This revealed that polyploid plants showed a decline in the number of endocycles as well as in the frequency of endopolyploidy cells compared to diploid plants.