Physical mapping of rDNA and heterochromatin in chromosomes of 16 Coffea species: A revised view of species differentiation

The chromosome organization among 15 wild diploid Coffea species and cultivated tetraploid C. arabica was determined by fluorochrome banding (CMA, DAPI) and double fluorescence in-situ hybridization (FISH) of 5S and 18S rDNA achieved on the same chromosome plates. Two to five chromosome pairs (plus one putative chromosome B) are marked. Overall, there are two SAT-chromosome pairs for East African species and one for the Malagasy and the West and Central African species. 18S rDNA loci are telomeric and strongly marked the SAT-chromosome pairs. Generally, only one pericentromeric 5S rDNA locus characterized East African species, while an additional minor locus co-localized with the 18S rDNA-SAT locus for the Malagasy species and West and Central African species. A combination of rDNA FISH plus CMA and DAPI banding patterns enables identification of almost all the species, even those for which the genetic or botanical status is still being discussed. C. arabica clearly appears to be an allotetraploid species, including one genome from East Africa and one from West and Central Africa. However, since the minor 5S rDNA-SAT locus present in West/Central African genomes is not detected, two evolutionary hypotheses could be put forward for C. arabica. Considering only the diploid species, global trends are obvious in rDNA signal patterns, genome size variations, and geographic distribution of the species, but there are no clear evolutionary trends. However, complex interactions between these factors and environmental growing conditions exist, which have resulted in loss and gain of rDNA loci and probably also in copy repeat number variations in each rDNA family.     

Physical mapping of repetitive DNA sequences and 5S and 18S–26S rDNA in five wild species of the genusHordeum

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.accepted by J.S. (Pat) Heslop-Harrison     

Surface spreading of synaptonemal complexes in the clam <Emphasis Type="Italic">Dosinia exoleta</Emphasis> (Mollusca,Bivalvia)

There are only a few reports on the chromosomal location of DNA sequences in bivalve species, none of them using meiotic chromosomes. Mitotic chromosomes of the clam Dosinia exoleta were analysed by means of Giemsa, silver and fluorochrome staining and fluorescent in situ hybridization (FISH) with 18S + 28S rDNA and telomeric probes. A technique for surface spreading of synaptonemal complexes (SCs) of Dosinia exoleta was developed for the first time in a bivalve species. Silver and DAPI/PI staining and SC-FISH were also applied to the study of the meiotic chromosomes of this clam. The diploid chromosome number in this species is 38 and the karyotype is composed of 11 pairs of metacentric and eight pairs of submetacentric chromosomes. 18S + 28S rDNA clusters map to the subtelomeric region of the short arm of one metacentric chromosome pair whereas telomeric signals appear at both ends of every chromosome.     

Detection of rDNA sites in sugarcane by FISH

Hybridization sites of an rDNA probe coding for the 5.8S, 18S and 26S genes were detected on the chromosomes of sugarcane and a related genus,Erianthus, using fluorescencein situ hybridization. One unpaired and five paired hybridization sites were detected in aSaccharum spp. hybrid. A first introgression hybrid (I₁) betweenSaccharum officinarum andSaccharum spontaneum had seven pairs of hybridization sites. A clone ofErianthus arundinaceus showed six hybridization sites in somatic tissue.     

Triploid origin of the gibel carp as revealed by 5S rDNA localization and chromosome painting

5S ribosomal DNA (rDNA) was isolated and sequenced from the gibel carp Carassius auratus gibelio with 162 chromosomes and crucian carp Carassius auratus with 100 chromosomes, and fluorescent probes for chromosome localization were prepared to ascertain the ploidy origin and evolutionary relationship between the two species. Using fluorescence in-situ hybridization (FISH), major 5S rDNA signals were localized to the short arms of three subtelocentric chromosomes in the gibel carp and to the short arms of two subtelocentrics in the crucian carp. In addition, some minor signals were detected on other chromosomes of both species. Simultaneously, six chromosomes were microdissected from the gibel carp metaphase spreads using glass needles, and the isolated chromosomes were amplified in vitro by degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR). Significantly, when the DOP-PCR-generated probes prepared from each single chromosome were hybridized, three same-sized chromosomes were painted in each gibel carp metaphase, whereas only two painted chromosomes were observed in each crucian carp metaphase spread. The data indicate that gibel carp is of triploid origin in comparison with diploid crucian carp.     

Activity of the En/Spm-like transposons in meiosis as a base for chromosome repatterning in a small, isolated, peripheral population of Aegilops speltoides Tausch.

Chromosomal repatterning is considered to be one of the main mechanisms for plant genome evolution. Here, we report the first cytogenetic evidence for the involvement of En/Spm transposons in ongoing chromosomal repatterning leading to the rise of new fertile genomic forms in a small, isolated, peripheral plant population. Cytogenetical screening of original individual plants of Aegilops speltoides Tausch. with different phenotypes revealed a wide spectrum of chromosomal abnormalities including extra chromosomes, chromosomal rearrangements, and variability in chromosomal position/number of 45S and 5S rDNA sites. Cytogenetic analysis of the dynamics of En/Spm transposons in meiosis indicates that: (i) this type of transposon is active during male gametogenesis; (ii) separately or in conjunction with rDNA they form clusters in the hot spots of large chromosomal rearrangements; (iii) appearance of at least part of the mobile rDNA sites in genome of Ae. speltoides are connected with meiotic activity of En/Spm transposons. PCR screening for the site-selected transposon insertions confirm the presence of combined fragments that consist partly of the sequence of En/Spm transposon and partly of 5S rDNA sequence.     

Physical mapping, expression patterns and interphase organisation of rDNA loci in Portuguese endemic Silene cintrana and Silene rothmaleri

Double target in situ hybridization to root tip metaphase and interphase cells of Silene cintrana and Silene rothmaleri was used to allocate the position of 18S-5.8S-25S and 5S rRNA genes. In both species, the 18S-5.8S-25S rDNA probe labelled four sites located on the short arms of two submetacentric chromosomes. Only one locus for 5S rDNA was mapped adjacent to 18S-5.8S-25S genes in a subterminal position on the centromere side: in S. rothmaleri the 5S rDNA locus was adjacent to the small 18S-5.8S-25S locus while in S. cintrana it was near the large one. The NOR activity analysed by Ag-staining in metaphase cells revealed proportionality between in situ labelling dimensions and Ag-NORs. In both species all rDNA loci were potentially active, although in S. rothmaleri a tendency for the expression of only one locus was observed. Interphase organisation analysis of rDNA showed some differences between both species that were correlated with NOR activity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Telomeric and interstitial telomeric sequences in holokinetic chromosomes of Lepidoptera: Telomeric DNA mediates association between postpachytene bivalents in achiasmatic meiosis of females

Telomeres, besides their main role in the protection and maintenance of chromosome ends, have several other vital functions in the cell cycle. We studied their role in the achiasmatic meiosis of female Lepidoptera, insects with holokinetic chromosomes. By fluorescence in-situ hybridization (FISH) with the insect telomeric probe, (TTAGG) _n , we mapped the distribution of telomeric and interstitial telomeric sequences (ITS) in female meiotic chromosomes of two species, Orgyia antiqua with a reduced chromosome number (2n=28) and Ephestia kuehniella mutants, possessing a radiation-induced chromosome fusion in the genome (2n=59). In addition to the strong typical telomeric signals, O. antiqua displayed weaker hybridization signals in interstitial sites of pachytene bivalents. The observed ITS most probably reflect remnants of chromosomal rearrangements and support the hypothesis that the Orgyia karyotype had arisen by multiple fusions of ancestral chromosomes. On the other hand, the absence of ITS in the chromosome fusion of Ephestia indicated the loss of telomeres before the two original chromosomes fused. When the telomeric probe was amplified by enzymatic reaction with tyramid, the number of ITS observed increased in Orgyia, and a few ITS were also observed in several chromosomes of Ephestia but not in the fused chromosome. This suggests that the genomes of both species also contain ITS other than those originating from chromosome fusions. The analysis of female meiotic prophase I revealed non-homologous associations of postpachytene bivalents mediated by telomeric DNA, which were not observed in the pachytene stage. Surprisingly, in early postpachytene nuclei the telomeric associations also involved ITS, whereas later postpachytene nuclei displayed chains of bivalents interconnected only by true telomeres. This finding favours a hypothesis that telomeric associations between bivalents play a role in chromosome segregation in the achiasmatic meiosis of female Lepidoptera. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cytogenetics of the bleak (Alburnus alburnus), with special emphasis on the B chromosomes

Some of the largest B chromosomes so far discovered in vertebrates are present in the cyprinid fish Alburnus alburnus. Previous cytogenetic analyses revealed a diploid chromosome number of 2n = 50. In addition, in some individuals one or two unusually large B chromosomes are present. Two morphologically different types of B chromosomes were observed. The frequency of animals bearing a supernumerary chromosome was found to vary considerably between different populations. A more detailed analysis of the A and B chromosomes of A. alburnus by conventional banding techniques, as well as fluorescence in-situ hybridization (FISH) with the telomeric DNA repeats (GGGTTA)₇/(TAACCC)₇, 18S + 28S rDNA and 5S rDNA were performed in the present study. Furthermore, a B chromosome-specific DNA probe obtained by amplified length polymorphism (AFLP) was hybridized on metaphases of A. alburnus carrying supernumerary B chromosomes. The banding analyses showed that the B chromosomes are completely heterochromatic, consist of GC-rich DNA sequences, replicate their DNA in the very late S-phase of the cell cycle and are composed mainly of a specific retrotransposable DNA element. Finally, blood probes from A. alburnus were collected for DNA-flow cytometric measurements. It could be shown that the huge supernumerary chromosomes represent nearly 10% of the total genome size of A. alburnus.     

Ribosomal RNA location in the Antarctic fishChampsocephalus gunnari (Notothenioidei,Channichthyidae) using banding and fluorescencein situ hybridization

A biotinylated 28S rDNA probe was prepared from the genomic DNA of the Antarctic ice-fishChampsocephalus gunnari and hybridized to metaphase chromosomes of the same species by fluorescencein situ hybridization (FISH). The hybridization signal appeared over the whole heterochromatic arm of the submetacentric chromosomes bearing the nucleolar organizer regions. The results of rDNA/FISH are compared with those coming from classical cytogenetic (C, Q, Ag-NOR, chromomycin A3) banding techniques. Thein situ detection of a specific DNA sequence offers a new more precise perspective for understanding the evolving process in chromosomes of Antarctic fish and will provide an interesting contribution to comparative cytogenetics of lower vertebrates.accepted for publication by M. Schmid     

Additional locus of rDNA sequence specific to the X chromosome of the liverwort, Marchantia polymorpha

The molecular cytogenetic organization of 17S ribosomal RNA genes (17S rDNA), a part of the 45S rDNA repeat, was investigated on the chromosomes of the liverwort Marchantia polymorpha using fluorescence in-situ hybridization (FISH). The numbers of 17S rDNA loci visualized in female and male chromosomes were ten and nine, respectively. This heterogeneous localization was due to the presence of an additional 17S rDNA locus on the X chromosome and its absence on the Y chromosome. The signal on the X chromosome covered almost the entire region of its long arm. The other nine signals were observed on the same loci of respective autosomes in both sexes. Southern hybridization analysis revealed an additional band including 17S rDNA exclusively on EcoRI digested female genomic DNA supporting the existence of an additional 17S rDNA locus on the X chromosome.     

Unusual distribution pattern of telomeric repeats in the shrews <Emphasis Type="Italic">Sorex araneus</Emphasis> and <Emphasis Type="Italic">Sorex granarius</Emphasis>

Sorex araneus and Sorex granarius are sibling species within the Sorex araneus group with karyotypes composed of almost identical chromosome arms. S. granarius has a largely acrocentric karyotype, while, in S. araneus, various of these acrocentrics have combined together by Robertsonian (Rb) fusions to form metacentrics, with the numbers and types of metacentrics differing between chromosomal races. Our studies on telomeric sequences in S. araneus and S. granarius revealed differences between chromosomes and between species. In S. araneus (the Novosibirsk race), hybridization signals were present on the telomeres of all the chromosomes after FISH with a PCR-generated telomeric probe. In addition, hybridization signals were observed at high frequencies in the pericentric regions of some but not all metacentrics formed by Rb fusion. There were fewer signals on those metacentrics formed earlier in the evolution of S. araneus. This suggests that S. araneus chromosomes retain at least some telomeric repeats during Rb fusion, but that these repeats are lost or modified over time. These results are critical for the interpretation of the well-studied hybrid zones between chromosomal races of S. araneus, given that Rb fission has been postulated in such hybrid zones and that the likelihood of Rb fission will relate to presence/absence of telomeric sequences at the centromeres of metacentrics. In S. granarius, there were strong signals at the proximal (centromeric) telomeres of the acrocentrics after FISH with a DNA telomeric probe. FISH with a PNA telomeric probe on S. granarius acrocentrics showed that the proximal telomeres were 213 kb on average, while the length of the distal telomeres was 3.8 kb on average. Two-colour FISH, using a telomeric DNA probe and a microdissected probe generated from the pericentric regions of the S. granarius chromosomes a and b, revealed regions on distinct chromatin fibres where telomeric and microdissected probes were colocalized or localized sequentially. The proximal telomeres of S. granarius are highly unusual both in their large size and their heterogeneous structure relative to the telomeres of other mammals.     

Chromosomal Localization of 5S rDNA Genes in Leporinus Fish (Anostomidae, Characiformes)

The large 45S rDNA chromosome sites have often been analyzed in fish. In contrast, little is known about the 5S genes in this animal group. In the genus Leporinus, the NOR chromosomal location has been shown to be very diverse. In the present work, chromosome mapping of 5S rDNA in three anostomids, Leporinus elongatus, L. obtusidens and L. friderici, is investigated using fluorescence in-situ hybridization (FISH) with PCR-obtained 5S probes and primed in-situ labeling (PRINS). Major 5S rDNA chromosomal sites were found to be subterminally located in a small metacentric pair, while minor ones were detected near the centromeric region of a medium-sized submetacentric pair in all studied species. The 5S rDNA genes were not associated with the NORs or sex chromosomes. A highly conserved chromosomal location of these genes appears to characterize the karyotype evolution of this fish group.     

Complex rearrangements are involved in <Emphasis Type="Italic">Cephalanthera</Emphasis> (Orchidaceae) chromosome evolution

The genus Cephalanthera is an excellent plant group for karyotype evolution studies because it exhibits a dysploid series and bimodal karyotypes. With the aim of understanding their chromosomal and phylogenetic relationships, rRNA genes and the Arabidopsis-type telomeric sequence were mapped by fluorescence in-situ hybridization (FISH), and the rDNA intergenic spacer (ITS) was sequenced for the first time in three European species: C. longifolia (2n = 4x = 32), C. damasonium (2n = 4x = 36) and C. rubra (2n = 4x = 44). One 45S and three 5S rDNA sites are observed in C. longifolia, one 45S and two 5S sites in C. damasonium, and two 45S and one 5S site in C. rubra. Telomeric signals were observed at every chromosome end in all three species and C. damasonium also displays interstitial signals on three chromosome pairs. In agreement with chromosome data, molecular analyses support C. longifolia and C. damasonium as closely related taxa, while C. rubra stands apart. Possible pathways of karyotype evolution are discussed in reference to a previous hypothesis. The results indicate that complex chromosomal rearrangements, possibly involving Robertsonian fusions and fissions, loss of telomeric repeats, gain or loss of rDNA sites and other heterochromatic sequences and inversions, may have contributed to generating the present-day karyotypes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Heterogeneity of rDNA distribution and genome size in Silene spp.

Genus Silene L. (Caryophyllaceae) contains about 700 species divided into 44 sections. According to recent taxonomic classification this genus also includes taxa previously classified in genera Lychnis and Melandrium. In this work, four Silene species belonging to different sections were studied: S. latifolia (syn. Melandrium album, Section Elisanthe), S. vulgaris (Inflatae), S. pendula (Erectorefractae), and S. chalcedonica (syn. Lychnis chalcedonica, Lychnidiformes). Flow cytometric analysis revealed a genome size of 2.25 and 2.35 pg/2C for S. vulgaris and S. pendula and of 5.73 and 6.59 pg/2C for S. latifolia and S. chalcedonica. All four species have the same chromosome number including the pair of sex chromosomes of the dioecious S. latifolia (2n = 2x = 24). Double target fluorescence in-situ hybridization revealed the chromosomal locations of 25S rDNA and 5S rDNA. A marked variation in number and localization of rDNA loci but no correlation between the numbers of rDNA clusters and genome size was found. FISH and genome size data indicate that nuclear genomes of Silene species are highly diversified as a result of numerous DNA amplifications and translocations.     

Loss of telomeric sites in the chromosomes ofMus musculus domesticus (Rodentia: Muridae) during Robertsonian rearrangements

Mouse chromosomes possessing multiple Robertsonian rearrangements (Rb chromosomes) have been examined using fluorescencein situ hybridization with the telomeric consensus sequence (TTAGGG)_n. No hybridization signals were detected at the primary constriction of Rb chromosomes. This observation leads us to conclude that the formation of Rb chromosomes in the mouse is invariably associated with the loss of telomeric regions. More significantly, a further alteration in regions flanking the primary constrictions was observed after hybridizing with a minor satellite DNA probe to Rb chromosomes. It seems likely that the breakpoints required for a Robertsonian process do not include telomeric sites exclusively but extend to the adjacent pericentromeric regions of the original acrocentric chromosomes. In contrast to previous reports, these observations demonstrate the elimination of substantial amounts of chromosomal DNA during the formation of mouse Rb chromosomes.     

Chromosomal location of endogenous geminivirus-related DNA sequences inNicotiana tabacum L.

TheN. tabacum (tobacco) nuclear genome carries approximately 25 multiple direct repeats of a geminivirus-related DNA (GRD) sequence that probably arose by illegitimate recombination, following geminivrus infection, duringNicotiana evolution. Each GRD repeat carries sequences similar to the geminiviralAL1 gene of the tomato golden mosaic virus (TGMV), encoding a protein required for viral DNA replication, plus thecis-essential replication origin. Using a cloned 14-kb GRD repeat sequence as a probe for fluorescencein situ hybridization (FISH), we identified a unique tobacco chromosome carrying GRD. Translocations between chromosomes of the tobacco S and T genomes were used as physical markers by sequentially hybridizing chromosomes with labelled GRD and total genomic DNA fromN. sylvestris (equivalent to the S genome). The 25S, 18S and 5.8S ribosomal gene clusters were detected in double-labelling experiments for use as additional markers to identify the chromosomal location of GRD. GRD occupies one site on a homologous pair of small submetacentrics from the T genome characterized by a lack of either translocated segments from the S genome or ribosomal genes. GRD provides an additional marker for the small chromosomes of the T genome and a useful phylogenetic tool.     

Isolation, characterization and chromosome localization of repetitive DNA sequences in bananas (Musa spp.)

Partial genomic DNA libraries were constructed in Musa acuminata and M. balbisiana and screened for clones carrying repeated sequences, and sequences carrying rDNA. Isolated clones were characterized in terms of copy number, genomic distribution in M. acuminata and M. balbisiana, and sequence similarity to known DNA sequences. Ribosomal RNA genes have been the most abundant sequences recovered. FISH with probes for DNA clones Radka1 and Radka7, which carry different fragments of Musa 26S rDNA, and Radka14, for which no homology with known DNA sequences has been found, resulted in clear signals at secondary constrictions. Only one clone carrying 5S rDNA, named Radka2, has been recovered. All remaining DNA clones exhibited more or less pronounced clustering at centromeric regions. The study revealed small differences in genomic distribution of repetitive DNA sequences between M. acuminata and M. balbisiana, the only exception being the 5S rDNA where the two Musa clones under study differed in the number of sites. All repetitive sequences were more abundant in M. acuminata whose genome is about 12% larger than that of M. balbisiana. While, for some sequences, the differences in copy number between the species were relatively small, for some of them, e.g. Radka5, the difference was almost thirty-fold. These observations suggest that repetitive DNA sequences contribute to the difference in genome size between both species, albeit to different extents. Isolation and characterization of new repetitive DNA sequences improves the knowledge of long-range organization of chromosomes in Musa. This revised version was published online in July 2006 with corrections to the Cover Date.     

The 5S rDNA of mussels Mytilus galloprovincialis and M. edulis: sequence variation and chromosomal location

The 5S ribosomal DNA of the mussels Mytilus galloprovincialis and M. edulis was amplified by PCR using contiguous primers. The most general 5S rDNA amplification pattern consisted of several products in both mussels. Two main PCR products of about 250 bp and 760 bp were cloned and sequenced, revealing two classes of 5S rDNA units. These were characterized as containing an identical coding region of 119 bp but with highly divergent spacers. Clones of each unit type exhibited minimal differences except those of the large unit of M. edulis. The sequences analysed of the two mussels possess the same coding region and only six fixed base changes on the spacers. FISH, carried out with specific probes, consistently showed hybridization signals on the largest metacentric pair (two differentiated sites) and with variable frequency on two other metacentric pairs (one site on each pair). Differences in the 5S rDNA distribution between both mussels were not found. In M. edulis, chromosomes carrying 18S-28S rDNA were also identified by FISH. These correspond to two submetacentric-subtelocentric pairs, as was previously reported in M. galloprovincialis, demonstrating that the two rDNA multigene families are located on different chromosome pairs in these mussels.     

Trigenomic origin of the hexaploid Psammopyrum athericum (Triticeae: Poaceae) revealed by in-situ hybridization

The genomic constitution of the hexaploid Psammopyrum athericum was studied with in-situ DNA hybridization using both genomic DNA and isolated cloned sequences as probes. A genomic probe from Thinopyrum bessarabicum (E genome) hybridized successfully to 14 chromosomes of Ps. athericum and a probe from Festucopsis serpentinii (L genome) hybridized to another 14 chromosomes. The remaining chromosomes did not hybridize, apart from in the centromeric region, to any of the genomic probes used. It is thus proposed that Ps. athericum contains the genomes E, L and X where X stands for a so-far unknown genome. Psammopyrum athericum has three pairs of pTa71 sites and approximately 30 pSc119:2 sites. The origin of the third genome will be a matter for further research using genomic and genome-specific probes.