Angeles Cuadrado

Mapping and organization of highly-repeated DNA sequences by means of simultaneous and sequential FISH and C-banding in 6×-triticale

Three families of highly repeated sequences from rye and the rRNA multigenes (NOR and 5S) have been mapped by FISH and C-banding, in chromosomes of triticale. The pSc119.2 probe showed interstitial hybridization in chromosome arms 1RS, 1RL, 4RL, 5RL, 6RS, 6RL, 7RS and 7RL, and is very effective for chromosome identification of rye chromosomes in triticale. This sequence also hybridizes to the 4A, 5A and the seven B-genome wheat chromosomes. Simultaneous hybridization with the pSc119.2 and pTa794 (5S rRNA) is very useful to distinguish the metacentric chromosomes 2R and 3R. The pSc74 probe appears at interstitial sites in the long arm of the most heterobrachial chromosomes (5R and 6R). The three repetitive sequences of 120 bp, 480 bp, and 610 bp hybridize to telomeric regions in rye chromosomes. Different arrangements and complex organizations consisting of arrays of three or more family sequences were found. The results demonstrate a great variation in the relative arrangement of the repetitive sequences in the telomeres of the rye chromosomes. There were quantitative differences in each cytological marker between triticale lines in bothin situ labelling and C-banding, probably as the result of differences in the number and/or kind of repeat sequence.

Identification of different chromatin classes in wheat using in situ hybridization with simple sequence repeat oligonucleotides.

Abstract Clusters of four simple sequence repeats (SSRs), AAC, AAG, AG and CAT, have been mapped physically to hexaploid wheat chromosomes; 15—24-bp synthetic oligonucleotides were labelled by random-primer labelling and used as probes for fluorescent in situ hybridization with standard formamide and low-salt conditions. AAC hybridized strongly to the pericentromeric regions and several intercalary sites of all seven chromosomes of the B-genome corresponding to N bands and enabling their identification. Most of the AAC sites also co-localize with AAG, although the strength of the AAC and AAG signal was often different at the same location. Not all heterochromatic bands showed AAC signals and a few AAC sites were detected that are neither AAG nor N band positive, revealing the complex and heterogeneous genome organization of wheat and identifying the four most frequent classes of banded chromatin. Clusters characterised by a high concentration of AG repeats were detected on chromosome arms 3BS, 4BL, 5BS and 5BL, adjacent to AAG sites. The only detectable CAT cluster was found on chromosome arm 3BL, making this oligonucleotide valuable in identifying this particular chromosome. SSR in situ hybridization is useful as a diagnostic tool in cytogenetics and for understanding genome organization in wheat.

Physical mapping of repetitive DNA sequences and 5S and 18S-26S rDNA in five wild species of the genus Hordeum.

The genetic relationships between several wild species and subspecies of the genusHordeum were assessed using fluorescencein situ hybridization (FISH). Plant material included natural populations of wild barley growing in Spain of the annual species,H. marinum ssp.marinum (2n=14) andgussoneanum (2n=14), andH. murinum ssp.murinum (2n=28), andleporinum (2n=28) and the perennial speciesH. bulbosum (2n=14) andH. secalinum (2n=28), plus the South American perennial speciesH. chilense (2n=14). FISH was used to locate the chromosomal sites of two rDNA multigene families 5S and 18S–26S (pTa71 and pTa794) and three repetitive DNA sequences (pSc119.2, pAs1 and pHch950) isolated from different species and genera. The seven chromosomes of the diploid species were readily distinguished by their external morphology and hybridization patterns to pTa71, pTa794, pSc119.2 and pAs1. These DNA probes were also useful for the identification of homologous chromosomes and in differentiating these from unidentified chromosomes in the tetraploid taxa. The use of the probe pHch950 permitted intergenomic differentiation in tetraploids and supports the diphyletic origin ofH. murinum andH. secalinum. Thein situ experiments yielded the following conclusions: (1) differences between the subspeciesmarinum andgussoneanum; (2) close relationships between the subspeciesmurinum andLeporinum; and (3) major differences in physical mapping betweenH. bulbosum and the remaining taxa. The genomic and phylogenetic relationships between taxa, as inferred from the results, are discussed.

Physical organisation of simple sequence repeats (SSRs) in Triticeae: structural, functional and evolutionary implications

A significant fraction of the nuclear DNA of all eukaryotes is occupied by simple sequence repeats (SSRs) or microsatellites. This type of sequence has sparked great interest as a means of studying genetic variation, linkage mapping, gene tagging and evolution. Although SSRs at different positions in a gene help determine the regulation of expression and the function of the protein produced, little attention has been paid to the chromosomal organisation and distribution of these sequences, even in model species. This review discusses the main achievements in the characterisation of long-range SSR organisation in the chromosomes of Triticum aestivum L., Secale cereale L., and Hordeum vulgare L. (all members of Triticeae). We have detected SSRs using an improved FISH technique based on the random primer labelling of synthetic oligonucleotides (15–24 bases) in multi-colour experiments. Detailed information on the presence and distribution of AC, AG and all the possible classes of trinucleotide repeats has been acquired. These data have revealed the motif-dependent and non-random chromosome distributions of SSRs in the different genomes, and allowed the correlation of particular SSRs with chromosome areas characterised by specific features (e.g., heterochromatin, euchromatin and centromeres) in all three species. The present review provides a detailed comparative study of the distribution of these SSRs in each of the seven chromosomes of the genomes A, B and D of wheat, H of barley and R of rye. The importance of SSRs in plant breeding and their possible role in chromosome structure, function and evolution is discussed.

Fluorescence in situ hybridization with multiple repeated DNA probes applied to the analysis of wheat-rye chromosome pairing

Abstract Fluorescence in situ hybridization (FISH) with multiple probes has been applied to meiotic chromosome spreads derived from ph1b common wheat x rye hybrid plants. The probes used included pSc74 and pSc 119.2 from rye (the latter also hybridizes on wheat, mainly B genome chromosomes), the Ae. squarrosa pAs1 probe, which hybridizes almost exclusively on D genome chromosomes, and wheat rDNA probes pTa71 and pTa794. Simultaneous and sequential FISH with a two-by-two combination of these probes allowed unequivocal identification of all of the rye (R) and most of the wheat (W) chromosomes, either unpaired or involved in pairing. Thus not only could wheat-wheat and wheat-rye associations be easily discriminated, which was already feasible by the sole use of the rye-specific pSc74 probe, but the individual pairing partners could also be identified. Of the wheat-rye pairing observed, which averaged from about 7% to 11% of the total pairing detected in six hybrid plants of the same cross combination, most involved B genome chromosomes (about 70%), and to a much lesser degree, those of the D (almost 17%) and A (14%) genomes. Rye arms 1RL and 5RL showed the highest pairing frequency (over 30%), followed by 2RL (11%) and 4RL (about 8%), with much lower values for all the other arms. 2RS and 5RS were never observed to pair in the sample analysed. Chromosome arms 1RL, 1RS, 2RL, 3RS, 4RS and 6RS were observed to be exclusively bound to wheat chromosomes of the same homoeologous group. The opposite was true for 4RL (paired with 6BS and 7BS) and 6RL (paired with 7BL). 5RL, on the other hand, paired with 4WL arms or segments of them in more than 80% of the cases and with 5WL in the remaining ones. Additional cases of pairing involving wheat chromosomes belonging to more than one homoeologous group occurred with 3RL, 7RS and 7RL. These results, while adding support to previous evidence about the existence of several translocations in the rye genome relative to that of wheat, show that FISH with multiple probes is an efficient method by which to study fundamental aspects of chromosome behaviour at meiosis, such as interspecific pairing. The type of knowledge attainable from this approach is expected to have a significant impact on both theoretical and applied research concerning wheat and related Triticeae.

The nonrandom distribution of long clusters of all possible classes of trinucleotide repeats in barley chromosomes

This paper is the first to report the long-range organization of all possible classes of trinucleotide motifs in a higher plant genome. Fluorescent in situ hybridization (FISH), employing the synthetic oligonucleotides (AAC)5, (AAG)5, (AAT)5, (AGG)5, (CAC)5, (CAT)5, (CAG)5, (ACT)5, (ACG)5 and (GCC)5, was used to characterize the nonrandom and motif-dependent distribution of tandem arrays of trinucleotide repeats in the metaphase chromosomes and interphase nuclei of barley (Hordeum vulgare L.). This provided detailed information on the sequence content of barley chromatin and allowed the saturation of the physical map of all barley chromosomes. The following conclusions were also drawn: (1) Except for (AAT)5 and (GCC)5, the studied repetitive motifs have a characteristic pattern of distribution in terms of their in situ FISH signals. Some permit the accurate identification of individual chromosomes. (2) (CAG)5, (CAT)5 and (ACT)5 are not found in all barley chromosomes. (3) With the exception of (ACT)5, the remaining trinucleotide repeats occur predominantly in the heterochromatin and are largely absent from the euchromatic regions. Moreover, (CAC)5, (ACG)5 and (CAG)5 are exclusively concentrated in the centromeres. The employment of simple synthetic probes for the identification of chromosomes and genomic characterization, and their importance in studies on genome organization, function and evolution, are discussed.

Multiple locations of the rDNA sites in holocentric chromosomes of Rhynchospora (Cyperaceae)

Several cytogenetic studies have shown that representatives of the family Cyperaceae have holocentric chromosomes. Despite their interesting chromosome morphology, the chromosome organization has not been studied. This paper reports on the number and distribution of 18S–5.8S–26S ribosomal RNA sites by fluorescence in situ hybridization in eight Brazilian species of Rhynchospora. The signal of the rDNA probe was always localized in the telomeric regions. A high degree of variation was observed in the number of labelled sites, ranging from 4–8 in karyotypes with 2n = 10 to 30 sites in a karyotype with 50 chromosomes. It is possible that the same mechanism involved in the multiplication of these regions in organisms with monocentric chromosomes also plays a role in the polymorphism observed in holocentric chromosomes of Rhynchospora. An interesting feature of most hybridization sites was their diffuse state observed through to early metaphase. The decondensed state probably reflects the later transcription of this region during the cell cycle.

Localization of 45S rDNA and telomeric sites on holocentric chromosomes of Rhynchospora tenuis Link (Cyperaceae)

Rhynchospora tenuis Link (Cyperaceae) is a weed widely distributed in Brazil that presents a small number of holocentric chromosomes (2n = 4) with some autopolyploid populations (2n = 8). The haploid number n = 2 is considered as a derivative of the base number x = 5. 45S rDNA probes and telomeric DNA were hybridized in both chromosome races of R. tenuis, looking for indications of chromosome fusions. The results showed that hybridization sites of the telomeric probe were restricted to end chromosome regions whereas rDNA sites were terminally located. The chromosome race with n = 4 exhibited a doubled number of sites, with similar size and location to the hybridized sequences, confirming its autopolyploid origin. Furthermore, the terminal location of the single 45S rDNA site in the 2n = 4 race suggested that disploid reduction in Rhynchospora, from n = 5 to n = 2, was followed by elimination or reorganization events, keeping the terminal distribution of these sites, as in an others species of the genus.

Chromosome characterization in Thinopyrum ponticum (Triticeae, Poaceae) using in situ hybridization with different DNA sequences

Thinopyrum ponticum (2n = 10x = 70, JJJJsJs) belongs to the Triticeae tribe, and is currently used as a source of pathogen resistance genes in wheat breeding. In order to characterize its chromosomes, the number and position of 45S and 5S rDNA sites, as well as the distribution of the repetitive DNA sequences pAs1 and pSc119.2, were identified by fluorescent in situ hybridization. The number of nucleoli and NORs was also recorded after silver nitrate staining. Seventeen 45S and twenty 5S rDNA sites were observed on the short arms of 17 chromosomes, the 45S rDNA was always located terminally. On three other chromosomes, only the 5S rDNA site was observed. Silver staining revealed a high number of Ag-NORs (14 to 17) on metaphase chromosomes, whereas on interphase nuclei there was a large variation in number of nucleoli (one to 15), most of them (82.8%) ranging between four and nine. The pAs1 probe hybridized to the terminal region of both arms of all 70 chromosomes. In addition, a disperse labeling was observed throughout the chromosomes, except in centromeric and most pericentromeric regions. When the pSc119.2 sequence was used as a probe, terminal labeling was observed on the short arms of 17 chromosomes and on the long arms of five others. The relative position of 45S and 5S rDNA sites, together with the hybridization pattern of pAs1 and pSc119.2 probes, should allow whole chromosomes or chromosome segments of Th. ponticum to be identified in inbred lines of wheat x Th. ponticum.

Chromosome markers in the tetraploid wheat Aegilops ventricosa analysed by in situ hybridization

Abstract Three lines of the tetraploid wheat Aegilops ventricosa Tausch (2n=4x=28), which contains good resistance to eyespot, were analysed using fluorescent in situ hybridization. Probes used included rDNA, cloned repeated sequences from wheat and rye, simple-sequence repeats (SSRs) and total genomic DNA. The banding patterns produced could be used to distinguish most chromosome arms and will aid in the identification of Ae. ventricosa chromosomes or chromosome segments in breeding programmes. All lines had a single major 18S-25S rDNA site, the nucleolar organizing region (NOR) in chromosome 5N and several minor sites of 18S-25S rDNA and 5S rDNA. A 1NL.3DL, 1NS.3DS translocation was identified, and other minor differences were found between the lines.