Chromosomal localization of two novel repetitive sequences isolated from the Chenopodium quinoa Willd. genome.

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.

Cytological events in explants of Arabidopsis thaliana during early callogenesis.

Leaf explants of diploid (2n = 2x = 10) and autotetraploid (2n = 4x = 20) plants of Arabidopsis thaliana ecotype Columbia were cytologically and cytogenetically analysed to determine the time and the mechanisms of the process of polyploidization. The first polyploid cells were observed after the third day of culture in both genotypes of explants. Polyploid cells were the result of pre-existing mixoploidy in explants of A. thaliana. Other factors such as endoreduplication, endomitosis, abnormal microtubules arrangement and DNA damage may have induced polyploidization during early stages of callogenesis.

Molecular and cytological characterization of ribosomal RNA genes in Chenopodium quinoa and Chenopodium berlandieri.

The nucleolus organizer region (NOR) and 5S ribosomal RNA (rRNA) genes are valuable as chromosome landmarks and in evolutionary studies. The NOR intergenic spacers (IGS) and 5S rRNA nontranscribed spacers (NTS) were PCR-amplified and sequenced from 5 cultivars of the Andean grain crop quinoa (Chenopodium quinoa Willd., 2n = 4x = 36) and a related wild ancestor (C. berlandieri Moq. subsp. zschackei (Murr) A. Zobel, 2n = 4x = 36). Length heterogeneity observed in the IGS resulted from copy number difference in subrepeat elements, small re arrangements, and species-specific indels, though the general sequence composition of the 2 species was highly similar. Fifteen of the 41 sequence polymorphisms identified among the C. quinoa lines were synapomorphic and clearly differentiated the highland and lowland ecotypes. Analysis of the NTS sequences revealed 2 basic NTS sequence classes that likely originated from the 2 allopolyploid subgenomes of C. quinoa. Fluorescence in situ hybridization (FISH) analysis showed that C. quinoa possesses an interstitial and a terminal pair of 5S rRNA loci and only 1 pair of NOR, suggesting a reduction in the number of rRNA loci during the evolution of this species. C. berlandieri exhibited variation in both NOR and 5S rRNA loci without changes in ploidy.

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.

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.

An improved nonfluorescent detection system for in situ hybridization in 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.

Nucleolar dominance does not occur in root tip cells of allotetraploid Brassica species.

Using in situ hybridization and silver staining methods, the numbers of active and inactive rDNA loci have been established for three allotetraploid species of Brassica (B. napus, B. carinata, and B. juncea) and their diploid ancestors (B. campestris, B. nigra, and B. oleracea). The allotetraploid species have chromosome numbers equal to the sum of the numbers in their diploid relatives, but have fewer rDNA loci. All species investigated have lower numbers of active NORs (AgNORs, nucleolar organizer regions) compared with the numbers of rDNA sites revealed by in situ hybridization. The number of active rDNA loci of the allotetraploid species is equal to the number of AgNORs in their diploid ancestors, indicating the absence of nucleolar dominance in amphidiploid Brassica species, at least in root meristematic cells.

Different rRNA gene expression in primary and adventitious roots of Allium cepa L.

Sequentially used silver staining and in situ hybridization allowed to estimate the number of rDNA loci and their activity in meristematic cells of Allium cepa roots. In primary roots, obtained from germinated seeds, the rDNA probe hybridized with four chromosomes and showed four strong sites of hybridization. All of them displayed very clear positive silver staining. In cells of adventitious roots, from bulbs, only one pair of rRNA gene loci was active and after in situ hybridization showed strong signals while two other sites were very weak. The results indicate different transcriptional rRNA gene activity in meristematic cells of roots of different developmental origin. The reduction of the number of active rRNA loci can be the result of DNA methylation but the reduction mechanism of in situ hybridization sites in adventitious roots of Allium cepa remains an open question.

Chromosomal rearrangement in autotetraploid plants of Arabidopsis thaliana.

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.

Random amplified polymorphic DNA analysis, genome size, and genomic in situ hybridization of triploid viviparous onions

Triploid viviparous onions (Allium cepa L. var. viviparum Metzg. (ALEF.), auct.), (2n = 3x = 24), are known in some countries only as a rare relic crop, while in other parts of the world they are still traditionally or even commercially cultivated. Results indicating an identical random amplified polymorphic DNA (RAPD) banding pattern and the same DNA content (2C = 43.4 pg) establish the high genetic similarity and the unique origin of the Croatian clone Ljutika and the Indian clone Pran. In order to determine the parental Allium species of these natural triploid hybrids, genomic fluorescent in situ hybridization (GISH) was applied. Biotinylated genomic DNAs from six diploid Allium species (A. cepa L., A. fistulosum L., A. roylei Stearn, A. vavilovii M. Pop. et Vved., A. galanthum Kar. et Kir., A. oschaninii O. Fedtsch.) were used as probes in this study. While probes obtained from genomic DNA of A. cepa, A. vavilovii, and A. roylei hybridized to somatic chromosomes of Ljutika probes from A. fistulosum, A. galanthum, and A. oschaninii did not. The DNA probes of A. cepa and A. roylei each completely or predominantly labelled one genome (eight chromosomes). A few chromosomes, the markers of the triploid karyotype, were not completely labelled by any probe applied. Our GISH results indicate that triploid viviparous onions might possess a complex triparental genome organization.

A change of ploidy can modify epigenetic silencing.

A silent transgene in Arabidopsis thaliana was reactivated in an outcross but not upon selfing of hemizygous plants. This result could only be explained by assuming a genetic difference between the transgene-free gametes of the wild-type and hemizygous transgenic plants, respectively, and led to the discovery of ploidy differences between the parental plants. To investigate whether a change of ploidy by itself can indeed influence gene expression, we performed crosses of diploid or tetraploid plants with a strain containing a single copy of a transgenic resistance gene in an active state. We observed reduced gene expression of the transgene in triploid compared with diploid hybrids. This led to loss of the resistant phenotype at various stages of seedling development in part of the population. The gene inactivation was reversible. Thus, an increased number of chromosomes can result in a new type of epigenetic gene inactivation, creating differences in gene expression patterns. We discuss the possible impact of this finding for genetic diploidization in the light of widespread, naturally occurring polyploidy and polysomaty in plants.

Physical mapping of rDNA loci in Brassica species.

The number of major rDNA loci (the genes coding for 18S-5.8S-26S rRNA) was investigated in the economically important Brassica species and their wild relatives by in situ hybridization of an rDNA probe to metaphase chromosomes and interphase nuclei. The diploid species B. nigra (B genome) has two major pairs of rDNA loci, B. oleracea (C genome) has two major pairs and one minor pair of loci, while B. campestris (A genome) has five pairs of loci. Among the three tetraploid species arising from these three diploid ancestors, B. carinata (BBCC genomes) has four loci, B. juncea (AABB genomes) has five major pairs and one minor pair of loci, and B. napus (AACC genomes) has six pairs of loci, indicating that the number of loci has been reduced during evolution. The complexity of the known rDNA restriction fragment length polymorphism patterns gave little indication of number of rDNA loci. It is probable that chromosome rearrangements have occurred during evolution of the amphidiploid species. The data will be useful for physical mapping of genes relative to rDNA loci, micro- and macro-evolutionary studies and analysis of aneuploids including addition and substitution lines used in Brassica breeding programs.

Ribosomal RNA genes in B chromosomes of Crepis capillaris detected by non-radioactive in situ hybridization.

A non-radioactive in situ hybridization method using biotin-labelled rDNA has made it possible to localize rRNA genes not only at the secondary constriction in both homologous chromosomes No. 3 of Crepis capillaris but also in the B chromosomes occurring in the plants employed. Very clear dot-like rDNA signals at the telomeres of both arms were observed in all B chromosomes. Histochemical silver staining, which is indicative of transcriptional activity of rRNA gene clusters, resulted in both darkly-staining nucleolar constrictions of chromosomes No. 3 and silver deposits at the telomeres of Bs. We conclude that the B chromosomes of C. capillaris are isochromosomes with active rRNA genes located near both telomeres.