5S rDNA Loci Research Articles

Highly divergent karyotypes and barcoding of the East African genus Gonatoxia Karsch (Orthoptera: Phaneropterinae)

East Africa is a hotspot of biodiversity of many orthopteran taxa, including bushcrickets. Gonatoxia Karsch, 1889 species are fully alate Phaneropterinae, which are perfectly adapted to the foliage of forests. We examined five species using combined cytogenetic and molecular data to determine the inter- and intraspecific genetic diversity. The variation in the diploid number of chromosomes in males ranged from 2n = 28 + X0 and 26 + X0 to 2n = 6 + X0. Fluorescence in situ hybridization showed from one to many 18S rDNA loci as well as interstitial sequences, especially in G. helleri. 18S rDNA loci coincided with active NOR and C-banding patterns. The isolation of populations of the species explains differences in the number of chromosomes (G. maculata), chromosomal polymorphism and chromosomal heterozygosity (G. helleri). Our molecular phylogeny based on the COI locus supported the monophyly of the genus Gonatoxia and separateness of the five examined species in accordance with their morphological features and chromosome numbers as well as the species’ distribution.

Analyses of the Updated "Animal rDNA Loci Database" with an Emphasis on Its New Features.

We report on a major update to the animal rDNA loci database, which now contains cytogenetic information for 45S and 5S rDNA loci in more than 2600 and 1000 species, respectively. The data analyses show the following: (i) A high variability in 5S and 45S loci numbers, with both showing 50-fold or higher variability. However, karyotypes with an extremely high number of loci were rare, and medians generally converged to two 5S sites and two 45S rDNA sites per diploid genome. No relationship was observed between the number of 5S and 45S loci. (ii) The position of 45S rDNA on sex chromosomes was relatively frequent in some groups, particularly in arthropods (14% of karyotypes). Furthermore, 45S rDNA was almost exclusively located in microchromosomes when these were present (in birds and reptiles). (iii) The proportion of active NORs (positively stained with silver staining methods) progressively decreased with an increasing number of 45S rDNA loci, and karyotypes with more than 12 loci showed, on average, less than 40% of active loci. In conclusion, the updated version of the database provides some new insights into the organization of rRNA genes in chromosomes. We expect that its updated content will be useful for taxonomists, comparative cytogeneticists, and evolutionary biologists.

The relevance of chromosome fissions for major ribosomal DNA dispersion in hymenopteran insects

Ribosomal DNA (rDNA) loci are essential for cellular metabolism due to their participation in ribosome biogenesis. Although these genes have been widely cytogenetically mapped, the evolutionary mechanisms behind their variability in number and chromosomal location remain elusive, even in well-known biological groups, such as ants, bees and wasps (Insecta: Hymenoptera). To address this question in Hymenoptera and therefore advance the understanding of rDNA evolution in insects in general, we integrated molecular cytogenetic data, a phylogenomic framework, model-based predictions and genome sequencing. Hence, we assessed the main evolutionary trends shaping the chromosomal distribution of rDNA loci in Hymenoptera. We noticed the conservation of one site of rDNA per haploid genome, suggesting that a single 45S rDNA locus is the putative ancestral pattern for aculeate Hymenoptera. Moreover, our results highlighted a nonrandom distribution of rDNA in Hymenoptera karyotypes, as well as a lineage-specific preferential location. The proximal location of rDNA is favoured in species with multiple loci and in the two families of Hymenoptera that show the highest range of chromosome numbers: Formicidae and Vespidae. We propose that chromosome fissions have played a crucial role in the distribution pattern of rDNA loci through the evolutionary diversification of Hymenoptera. Moreover, our genomic analysis of two species, one with a single locus of rDNA and one with multiple loci, supported that loci multiplication is followed by sequence divergence. Our results provide detailed information about the number and chromosomal position of rDNA in Hymenoptera and, therefore, broaden our knowledge regarding rDNA evolutionary dynamics in insects.

Karyotypic stability of Fragaria (strawberry) species revealed by cross-species chromosome painting.

Chromosome karyotyping analysis is particularly useful in determining species relationships and the origin of polyploid species. Identification of individual chromosomes is the foundation for karyotype development. For Fragaria (strawberry) species, definitive identification of the individual chromosomes is extremely difficult because of their small size and similar shape. Here, we identified all chromosomes for 11 representative Fragaria species with different ploidy using a set of oligonucleotide-based probes developed in Fragaria vesca. Comprehensive molecular cytogenetic karyotypes were established based on the individually identified chromosomes. In addition, we used oligo probes to assign the 5S and 45S rDNA loci to specific chromosomes in 16 Fragaria species. We found that these Fragaria species maintained a remarkably conserved karyotype. No inter-chromosomal structural rearrangements at the cytological level were observed in any of the chromosomes among these species. Despite karyotypic stability and similarity, variations in the signal intensity of oligo probes were observed among the homologous chromosomes in several polyploid species. Moreover, most Fragaria species also showed differences in the distribution patterns of 45S and 5S rDNA. These data provide new insights into the origins of several polyploid Fragaria species.

Primer reporte sobre identificación del locus 5s rADN en cromosomas de Physalis peruvianaL.

Physalis peruviana (Golden berry) a native fruit of Peru, is an important Andean agricultural crop. Although classical cytogenetics research on this berry has been conducted there is no precise evidence based on molecular cytogenetic studies. The Fluorescent In Situ Hybridization (FISH) technique was applied to reveal 5S rDNA sites in chromosomes of San Pablo and Celendín populations, Cajamarca, Perú. This research was identified the 5S rDNA locus in the two populations of Golden berry, reporting six signals in 3 pairs of chromosomes which would indicate that the species is polyploid. Likewise, no differences were found in the number of 5S rDNA loci between both populations, so we could point out that they would not be different ecotypes.

Global gene expression perturbations in rapeseed due to the introduction of alien radish chromosomes

The aneuploidy chromosome addition lines with individual chromosomes of one species into the whole genome of another species developed from interspecific or intergeneric crosses are ideal for studying the gene expression regulation under asymmetrical genome interactions. Here, we chose two rapeseed-radish addition lines (2n=40, AACC+2R) with all chromosomes of rapeseed and one pair of radish chromosomes carrying 25S rDNA loci and both 5S and 25S rDNA loci, respectively. Transcriptome sequencing of these two rapeseed-radish addition lines, together with two parents (Brassica napus, 2n=38, AACC; Raphanus sativus, 2n=18, RR), was performed to assess gene expression changes due to the aneuploidy effect. Our results showed that the global gene expression perturbations in two addition lines showed asymmetric distributions between A and C subgenomes, for more downregulated genes located in the C subgenome. Moreover, several dysregulated domains occurred in the addition lines and majority of them were clustered in C subgenome, further revealing that C subgenome was more inclined to be repressed by the aneuploidy. Homoeolog expression was slightly biased toward C subgenome in the parent B. napus, but had no preference to either of A or C subgenome in the addition lines. The total number of biased genes increased sharply and most of them were shared only by two addition lines, indicating that aneuploidy could change the extent and direction of homoeolog expression. The triplicated subgenomes which were orthologs to the chromosomes of Arabidopsis thaliana also exhibited dramatic alternations in two additions. Together, our results revealed that the addition of individual radish chromosomes could induce dramatically transcriptomic disturbances and those expression changes gave asymmetric distributions between two subgenomes in B. napus. The possible mechanisms including the uniparental expression of rRNA genes in the allopolyploids and additions for the findings are discussed.

Molecular Evolution and Organization of Ribosomal DNA in the Hawkweed Tribe Hieraciinae (Cichorieae, Asteraceae).

Molecular evolution of ribosomal DNA can be highly dynamic. Hundreds to thousands of copies in the genome are subject to concerted evolution, which homogenizes sequence variants to different degrees. If well homogenized, sequences are suitable for phylogeny reconstruction; if not, sequence polymorphism has to be handled appropriately. Here we investigate non-coding rDNA sequences (ITS/ETS, 5S-NTS) along with the chromosomal organization of their respective loci (45S and 5S rDNA) in diploids of the Hieraciinae. The subtribe consists of genera Hieracium, Pilosella, Andryala, and Hispidella and has a complex evolutionary history characterized by ancient intergeneric hybridization, allele sharing among species, and incomplete lineage sorting. Direct or cloned Sanger sequences and phased alleles derived from Illumina genome sequencing were subjected to phylogenetic analyses. Patterns of homogenization and tree topologies based on the three regions were compared. In contrast to most other plant groups, 5S-NTS sequences were generally better homogenized than ITS and ETS sequences. A novel case of ancient intergeneric hybridization between Hispidella and Hieracium was inferred, and some further incongruences between the trees were found, suggesting independent evolution of these regions. In some species, homogenization of ITS/ETS and 5S-NTS sequences proceeded in different directions although the 5S rDNA locus always occurred on the same chromosome with one 45S rDNA locus. The ancestral rDNA organization in the Hieraciinae comprised 4 loci of 45S rDNA in terminal positions and 2 loci of 5S rDNA in interstitial positions per diploid genome. In Hieracium, some deviations from this general pattern were found (3, 6, or 7 loci of 45S rDNA; three loci of 5S rDNA). Some of these deviations concerned intraspecific variation, and most of them occurred at the tips of the tree or independently in different lineages. This indicates that the organization of rDNA loci is more dynamic than the evolution of sequences contained in them and that locus number is therefore largely unsuitable to inform about species relationships in Hieracium. No consistent differences in the degree of sequence homogenization and the number of 45S rDNA loci were found, suggesting interlocus concerted evolution.

Molecular cytogenetical and biochemical studies on some Lupinus species

BackgroundLupins are cultivated as human consumption grains and forage legumes. The chromosomes of lupins are too small to be karyotyped by conventional techniques, because they reveal a general lack of distinctive cytological features. In the current study, Fluorescence in situ Hybridization (FISH) was used to locate 5S and 45S ribosomal gene sites on the chromosomes of Lupinus albus ssp albus, Lupinus albus ssp graecus, Lupnus termis (all with 2n = 50), and Lupinus polyphyllus lindl var. polyphyllus (2n = 48), FISH together with seed storage protein electrophoretic patterns were used to find out the relationship among these species.ResultsThe double-target FISH on the chromosomes of the studied species with rDNA probes revealed that the two types of rRNA genes are located on different chromosomes. The detected loci of rRNA genes partially reflected the taxonomical similarity among the two Lupinus albus subspecies and L. termis. Lupinus polyphyllus lindl var. polyphyllus was exception by having unique large chromosome mostly is covered by one signal of 45S rDNA, whereas its homologous chromosome seems to be normal-sized and have the other 45S rDNA locus. The similarity matrix among the Lupinus species as computed according to Jaccardʼs Coefficient from the SDS-PAGE, showed that L. albus ssp. Albus and L. albus ssp. Graecus are the most similar species (~ 97%), and then comes L. termis, and L. polyphyllus lindl var. polyphylus has been placed in separate clade and still the most related species to it among the studied species is L. termis (~ 70%).ConclusionIt could be postulated from FISH and seed storage protein electrophoretic patterns that the relationships among the studied species is as follows, Lupinus albus ssp albus, is the most related species to Lupinus albus ssp graecus then comes Lupnus termis and Lupinus polyphyllus lindl var. polyphyllus at a distal position.

Assessment of Thinopyrum ponticum (Podp.) Barkworth & D. R. Dewey accessions using universal rice primers and molecular cytogenetics

The aim of this study was to make morphological and molecular characterization of tall wheatgrass (Thinopyrum ponticum) accessions naturally found in Canakkale flora, Turkey. The seeds collected from 24 different locations in Canakkale vicinity were planted in the nursery field to determine thirteen morphological parameters. Twelve universal rice primers (URPs) were used to reveal genetic relationship among the accessions while ploidy analysis was done based on the nuclear DNA content of plants determined by Flow cytometer. Fluorescence in situ hybridization (FISH) method was used to determine 5S and 45S rDNA distributions on mitotic chromosomes. The agro-morphological data showed significant variation among the accessions for all parameters measured, except the number of nodes per plant. Twelve URP primers produced 73 alleles in total and 63 of those were polymorphic. The highest polymorphism information content value was obtained from URP 17R with 0.82. The first three components of Eigen values in PCA analysis explained 41.1% of total variation. The 2C nuclear DNA contents of the accessions ranged from 41.17 to 45.49 pg. All the accessions were determined as decaploid with 2n = 70 chromosomes. FISH analysis provided 18–20 interstial 5S rDNA and 12–14 terminal 45S rDNA loci. The results concluded that tall wheatgrass accessions used in this study contain a significant variation in morphological traits and PCR-based DNA polymorphism which could be used as a new genetic resource in breeding programs of tall wheatgrass and wheat improvement for intra- and intergeneric crosses.

Chromosome behavior with 45S rDNA loci at meiosis of Veronica persica

Chromosome behavior with 45S rDNA loci at meiosis of <i>Veronica persica</i>

Chromosome inheritance and meiotic stability in allopolyploid Brassica napus.

Homoeologous recombination, aneuploidy, and other genetic changes are common in resynthesized allopolyploid Brassica napus. In contrast, the chromosomes of cultivars have long been considered to be meiotically stable. To gain a better understanding of the underlying mechanisms leading to stabilization in the allopolyploid, the behavior of chromosomes during meiosis can be compared by unambiguous chromosome identification between resynthesized and natural B. napus. Compared with natural B. napus, resynthesized lines show high rates of nonhomologous centromere association, homoeologous recombination leading to translocation, homoeologous chromosome replacement, and association and breakage of 45S rDNA loci. In both natural and resynthesized B. napus, we observed low rates of univalents, A–C bivalents, and early sister chromatid separations. Reciprocal homoeologous chromosome exchanges and double reductions were photographed for the first time in meiotic telophase I. Meiotic errors were non-uniformly distributed across the genome in resynthesized B. napus, and in particular homoeologs sharing synteny along their entire length exhibited multivalents at diakinesis and polysomic inheritance at telophase I. Natural B. napus appeared to resolve meiotic errors mainly by suppressing homoeologous pairing, resolving nonhomologous centromere associations and 45S rDNA associations before diakinesis, and reducing homoeologous cross-overs.

Molecular Characterization of Novel Cryptosporidium Fish Genotypes in Edible Marine Fish

Current knowledge of Cryptosporidium species/genotypes in marine fish is limited. Following phylogenetic analysis at the 18S rDNA locus, a recent study identified six new genotypes of Cryptosporidium colonizing edible fish found in European seas. Of these, five grouped in a clade together (#Cryptofish 1–5) and one grouped separately (#Cryptofish 7). In the present study, after phylogenetic analyses of #Cryptofish1, #Cryptofish2, #Cryptofish4, #Cryptofish5 and #Cryptofish7 at the actin locus, the presence of two major clades was confirmed. In addition, when possible, longer 18S amplicons were generated. In conclusion, the small genetic distances between these genotypes designated as a novel marine genotype I (#Cryptofish 1-5) suggest that they may be genetic variants of the same species, while the designated novel marine genotype 2 (#Cryptofish 7) is clearly representative of a separate species.

Global DNA methylation predicts epigenetic reprogramming and transgenerational plasticity in Poa annua L

AbstractPoa annua L. (annual bluegrass) is a remarkably versatile grass species with biotypes ranging from perennial to annual. Despite its recent evolutionary origins, Poa annua can be found as an invasive weed on all seven continents of the globe including extremely inhospitable locations such as Mount Kilimanjaro and the Antarctic mainland. The molecular mechanisms underlying Poa annua versatility are unresolved but thought to relate to its neoallopolyploid origin and influence diverse molecular responses ranging from DNA methylation to chromosomal architecture. Fluorescence in situ hybridization (FISH) karyotyping of 5S rDNA loci added further evidence for Poa infirma Kunth. (weak bluegrass) and Poa supina Schrad. (supina bluegrass) as the progenitor species of Poa annua while results of 45S rDNA were less clear and possibly suggest that large‐scale variation in chromosomal architecture might have occur between individual Poa annua genotypes. At the methylation level, abiotic stress was observed to induce global DNA methylation, and methylation in the progeny of stressed plants remained slightly elevated, suggesting partial heritability of the methylation landscape acquired through abiotic stress on the parents. Further, the progeny of mowed clones had 37% reduced shoot area when compared with the progeny of unmowed clones across eight genotypes, suggesting that the altered phenotype is non‐Mendelian inherited. Poa annua plasticity and transgenerational inheritance is correlated with increased DNA methylation at the global level but causality needs experimental validation. These results suggest that Poa annua plastic phenotype is matched by genetic and epigenetic plasticity.

Molecular cytogenetic characterization of common bean (Phaseolusvulgaris L.) accessions

With an annual global production of approximately 25 million tons, the common bean (Phaseolus vulgaris L), a member of the genus Phaseolus, is one of the major protein sources used as food for humans. In this study, it was aimed to investigate the genome size of the common bean genetic resource collection (154 common bean accessions) in Turkey by flow cytometry (FCM) and determine whether geographical variables affected the genome size. In addition, the number and distribution of 5S and 45S ribosomal DNA loci were designated by performing a fluorescence in situ hybridization (FISH) analysis in some of the accessions. The FCM analyses revealed that the mean nuclear DNA content of the accessions varied from 1.28 pg2C-1 to 1.55 pg2C-1 (mean 1.35 pg2C-1), and the differences between these accessions were statistically significant (P &lt; 0.01). Intraspecific variation in the genome size was determined, and a positive correlation was found between the altitude and genome size. However, latitude and longitude did not have any statistically significant effect on the genome size. In the principal coordinate analysis, the accessions were divided into 3groups. Based on the results of the FISH analysis performed on 5 different accessions with varying genome sizes, using 5S and 45S rDNA genes as probes, the number of 5S rDNA loci was 4 in the common bean and stable among the common bean accessions, while the number of 45S rDNA loci was highly polymorphic, varying between 6 and 16. Consequently, it was determined in the present study that the genetic resource collection of common bean had a wide variation in terms of genome size and genome organization.

Ribosomal DNA localization on Lathyrus species chromosomes by FISH

BackgroundFluorescence In Situ Hybridization (FISH) played an essential role to locate the ribosomal RNA genes on the chromosomes that offered a new tool to study the chromosome structure and evolution in plant. The 45S and 5S rRNA genes are independent and localized at one or more loci per the chromosome complement, their positions along chromosomes offer useful markers for chromosome discriminations. In the current study FISH has been performed to locate 45S and 5S rRNA genes on the chromosomes of nine Lathyrus species belong to five different sections, all have chromosome number 2n=14, Lathyrus gorgoni Parl, Lathyrus hirsutus L., Lathyrus amphicarpos L., Lathyrus odoratus L., Lathyrus sphaericus Retz, Lathyrus incospicuus L, Lathyrus paranensis Burkart, Lathyrus nissolia L., and Lathyrus articulates L. ResultsThe revealed loci of 45S and 5S rDNA by FISH on metaphase chromosomes of the examined species were as follow: all of the studied species have one 45S rDNA locus and one 5S rDNA locus except L. odoratus L., L. amphicarpos L. and L. sphaericus Retz L. have two loci of 5S rDNA. Three out of the nine examined species have the loci of 45S and 5S rRNA genes on the opposite arms of the same chromosome (L. nissolia L., L. amphicarpos L., and L. incospicuus L.), while L. hirsutus L. has both loci on the same chromosome arm. The other five species showed the loci of the two types of rDNA on different chromosomes. ConclusionThe detected 5S and 45S rDNA loci in Lathyrus could be used as chromosomal markers to discriminate the chromosome pairs of the examined species. FISH could discriminate only one chromosome pair out of the seven pairs in three species, in L. hirsutus L., L. nissolia L. and L. incospicuus L., and two chromosome pairs in five species, in L. paranensis Burkart, L. odoratus L., L. amphicarpos L., L. gorgoni Parl. and L. articulatus L., while it could discriminate three chromosome pairs in L. sphaericus Retz. these results could contribute into the physical genome mapping of Lathyrus species and the evolution of rDNA patterns by FISH in the coming studies in future.

Cytological Distinctions Between Timun Suri and Cucumber Discovered by Fluorescence In Situ Hybridization (FISH) Using 45S Ribosomal DNA Gene

The genus Cucumis including timun suri, melon and cucumber, is an important horticultural crop of flowering plants. The dispute of timun suri terminology is leading to false-positive results in the nomenclature of timun suri in Indonesia. Although molecular research on these species has been widely conducted, detailed information and precise evidence based on the molecular cytogenetic approach are poorly investigated. The FISH technique was applied to reveal the cytological distinctions among these species. Here, this research conducted physical mapping of 45S ribosomal DNA (rDNA) on six accessions of Cucumis. The chromosome number of cucumber is 14 chromosomes, while timun suri and melon are 24 chromosomes. The number of 45S rDNA loci in cucumber, melon and timun suri was highly conserved. All cucumber accessions in this study had nine to ten 45S rDNA loci with strong and weak signal intensities located at proximal regions of the short arms. In melon and timun suri the signals of two pairs of 45S rDNA were located at the terminal and interstitial regions of the short arms. These were clear shreds of evidence to confirm that timun suri was cytogenetically closed to melon instead of cucumber. It confirmed that timun suri cannot be classified as a cucumber

Rapid chromosomal evolution in the bush-cricket Gonatoxia helleri Hemp, 2016 (Orthoptera, Phaneropterinae).

Gonatoxiahelleri Hemp, 2016 is one of the most widespread bush-crickets of the genus Gonatoxia Karsch, 1889 in East Africa. This species with seven large chromosomes (2n♂ = 7) differs from other representatives of the genus Gonatoxia drastically by its reduced chromosome number, the asymmetrical karyotype including karyomorphs rarely found in tettigoniids, as well as in irregularities in the course of meiosis. To better understand the origin of such an exceptional karyotype, chromosomes of 29 specimens from four populations/localities were studied using classical techniques, such as C-banding, silver impregnation, fluorochrome double staining and fluorescence in situ hybridization (FISH) technique with 18S rDNA and (TTAGG)n telomeric probes. FISH showed many 18S rDNA loci as well as interstitial telomeric sequences, where chromosome morphology varied in these components in terms of quantity and distribution. The 18S rDNA loci coincided with active NORs and C-banding patterns. We suggest that a combination of Robertsonian rearrangements and/or multiple common tandem fusions involving the same chromosomes contributed to the formation of this karyotype/karyomorphs. The results are the first step towards a better understanding of chromosomal reorganization and evolution within the genus Gonatoxia. Low chromosome number, together with the incidence of chromosomal polymorphism that is higher in G.helleri than previously reported in bush-crickets, implies that this species can be a valuable new model for cytogenetic and speciation studies. Our findings suggest that chromosomal translocations lead to diversification and speciation in this species and could be the driving force of adaptive radiation.

Heterochromatin banding and rDNA physical mapping in 22Epimediumspecies and twoVancouveriaspecies: implications for evolution inEpimedium

AbstractHeterochromatin banding patterns and the physical locations of 45S and 5S rDNA loci in mitotic metaphase chromosomes in 22 Epimedium spp. and two Vancouveria spp. species were studied using orcein staining and double-colour fluorescent in situ hybridization, respectively. Four types of chromosomal heterochromatin bands (satellite, centromeric, intercalary and terminal) were detected in all species, and there was clear variation in the number of bands and total length. In all species examined, at least one pair each of 45S and 5S rDNA signals was detected. The position of 5S rDNA is more conserved than that of 45S rDNA. The chromosomal distribution of 45S rDNA loci detected in Epimedium can be divided into three types. The chromosomal distributions of heterochromatin bands and 45S and 5S rDNA loci were closely related to the geographical distribution and may provide important evidence concerning the classification and systematics of Epimedium. Finally, based on results of the present study and pervious research, the centre of origin and formation of modern geographical distribution of Epimedium are discussed.

Chromosome polymorphism in species of the genus Hedysarum L. (Fabaceae) originated from Southern Sibеria

For the first time, chromosomal polymorphism in karyotypes of three species from the section Hedysarum (= syn. Gamotion) of the genus Hedysarum L. (Fabaceae) grown in Southern Siberia has been studied with the useof molecular cytogenetic markers. This comparative molecular cytogenetic analysis revealed high similarity in morphology of chromosomes in H. alpinum L., H. austrosibiricum B. Fedtsch. and H. theinum Krasnob. as well as in patterns ofdistribution of 45S and 5S rDNA loci in their karyotypes confirming their close relationships. Considerable intra-specificpolymorphism on 45S rDNA chromosome localization was detected in H. theinum. In karyotype of H. alpinum, unlikethe other two species, two chromosome pairs bearing 5S rDNA locus were observed which could be used as additionalspecies-specific markers.

Artificial Chromosome Doubling in Allotetraploid Calendula officinalis.

Calendula officinalis L. is known as an ornamental plant as well as a source of biochemical compounds used in cosmetics and industry. C. officinalis has a complex karyotype. Published chromosome numbers differ between 2n = 4x = 28 or 32. We have estimated genome sizes in nine commercial cultivars and evaluated the ploidy level by karyotyping and fluorescent in situ hybridization (FISH) using 5S and 45S rDNA loci. The detection of chromosome sets of two rather than four homologues would suggest that C. officinalis has an allotetraploid background. In addition, four signals for 45S but only two for 5S were found by using FISH. Artificial chromosome doubling is a common technique in plant breeding, as polyploidization results in several consequences for plant growth and development. Especially the suggested allotetraploid background in C. officinalis is interesting when examining the effect of chromosome doubling on the plant phenotype. Here we describe chromosome doubling of three allotetraploid cultivars of C. officinalis, ‘Nova,’ ‘WUR 1553-7’ and ‘Orange Beauty’. Three antimitotic agents – colchicine, oryzalin and trifluralin - were used in different concentrations to find the combination of the best agent and the best dosage to obtain octaploids. For all three cultivars a few octaploids were obtained. A concentration of 200 and 400 ppm of colchicine was most efficient for chromosome doubling in ‘Nova’ and ‘Orange Beauty,’ respectively. For ‘WUR 1553-7’ the treatment with 20 ppm oryzalin was also effective. Cell numbers and first observations of the phenotype in the chromosome doubled plants show thicker leaves and bigger cells, as commonly observed after ploidy doubling. Due to the low number of chromosome doubled plants obtained more elaborate phenotyping will be performed on following generations cultivated under field conditions.