Genome size variation in Chenopodium quinoa (Chenopodiaceae)

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.     

Ribosomal DNA is an effective marker of Brassica chromosomes

Simultaneous fluorescence in situ hybridisation with 5S and 25S rDNA probes enables the discrimination of a substantial number of chromosomes of the complement of all diploid and tetraploid Brassica species of the ”U-triangle”, and provides new chromosomal landmarks for the identification of some chromosomes of this genus which were hitherto indistinguishable. Twelve out of 20 chromosomes can be easily identified in diploid Brassica campestris (AA genome), eight out of 16 in Brassica nigra (BB genome), and six out of 18 in Brassica oleracea (CC genome). Furthermore, just two rDNA markers permit 20 out of 36 chromosomes to be distinguished and assigned to either the A or B genomes of the allotetraploid Brassica juncea, and 18 out of 38 chromosomes identified and assigned to the A or C genomes of the allotetraploid Brassica napus. The number of chromosomes bearing rDNA sites in the tetraploids is not in all cases simply the sum of the numbers of sites in their diploid ancestors. This observation is discussed in terms of the phylogeny and variability within the genomes of the species of this group. Received: 13 September 2000 / Accepted: 1 February 2001     

Identification of individual chromosomes and parental genomes in Brassica juncea using GISH and FISH

The three diploid (B. nigra, B. oleracea, B. campestris) and three allotetraploid (B. carinata, B. juncea, B. napus) species of Brassica, known as the "U-triangle" are one of the best model systems for the study of polyploidy. Numerous molecular investigations have provided a wealth of new insights into the polyploid origin and changes during the evolution of Brassica, but there are still many controversial aspects of their relationship and evolution. Interpretation of genome changes during evolution requires individual chromosome identification within the genome and clear distinction of genomes within the allotetraploid. The aim of this study was to identify individual chromosomes of B. juncea (genome AABB; 2n = 4x = 36) and to determine their genomic origin. Fluorescence in situ hybridization with 5S and 45S rDNA probes enabled discrimination of a substantial number of chromosomes, providing chromosomal landmarks for 20 out of 36 chromosomes of B. juncea. Additionally, along with double target genomic in situ hybridization, it allowed assignment of all chromosomes to either the A or B genomes.     

Molecular cytogenetic analysis of Brassica rapa-Brassica oleracea var. alboglabra monosomic addition lines

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.This revised version was published online in April 2005 with corrections to Fig. 2.     

DNA damage induced by mutagens in plant and human cell nuclei in acellular comet assay

Higher plant cells have a long tradition of use in the studies on environmental mutagenesis in situ, especially in relation to human health risk determination. The studies on the response of plant and human cells to physical and chemical mutagens showed differences in their sensitivity. The differences in the presence of cell components in plants and humans could influence such response. Additionally, the level of the organization of the employed material could influence DNA-damaging effect: leukocytes are isolated cells and plant--an intact organism. To preclude these obstacles, the effects of direct treatment of isolated nuclei with genotoxic agents were determined to compare the sensitivity of plant and human cells. In the present study, we have determined the DNA-damaging effects of two chemical mutagens: maleic acid hydrazide (MH) and N-methyl-N-nitroso-urea (MNU) applied to isolated nuclei of both plant and human cells. In order to compare the sensitivity of the nuclei of Nicotiana tabacum var. xanthi and the nuclei of leukocytes, the acellular Comet assay was carried out. The results showed higher sensitivity of the nuclei of leukocytes as compared to the nuclei of plant cells to mutagenic treatment with the applied doses of MH and MNU.     

Comparative analysis of rDNA distribution in chromosomes of various species of Brassicaceae

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.     

In situ fluorescent nick translation procedure for plant chromosomes

Pectinase and cellulase, which are used to macerate plant material, always show traces of DNase activities that result in DNA nicking. Moreover, the DNA polymerase I usually applied in the in situ nick translation techniques shows both 5' to 3' and 3' to 5' exonuclease activities. As a result, significant nonspecific labeling appears in control preparations that are not digested by a restriction endonuclease. Our procedure includes blocking nonspecific nick labeling before incubation with restriction enzymes (HpaII and HaeIII). This is achieved by incorporation of ddGTP into DNA by the Taq polymerase which lacks 3' to 5' exonuclease activity. This method gives satisfactory results because it eliminates nonspecific nick translation signals that are present after applying the methods described for animal material.     

Transformed roots of Crepis capillaries — a sensitive system for the evaluation of the clastogenicity of abiotic agents

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.     

Multicolour FISH in an analysis of chromosome aberrations induced by N-nitroso-N-methylurea and maleic hydrazide in barley cells

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.     

Distribution of 5S and 35S rRNA gene sites in 34 Chenopodium species (Amaranthaceae)

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, ??, ??–??.