Detection of alien chromatin conferring resistance to the beet cyst nematode (Heterodera schachtii Schm.) in cultivated beet (Beta vulgaris L.) using in situ hybridization

Chromatin originating from wild beets of the genus Beta, section Procumbentes, has been investigated in nematode-resistant hybrid-derived lines of sugar beet (Beta vulgaris L.) by in situ hybridization using satellite, telomeric and ribosomal DNA repeats, a yeast artificial chromosome (YAC) and total genomic DNA as probes. The alien chromosome was detected in three monosomic addition lines(2n=18+1) by genomic in situ hybridization. Fluorescence in situ hybridization with a genome-specific satellite repeat and YAC DNA enabled the visualization of Procumbentes chromosomes, and in double-target hybridization it was shown that they do not carry 18S–5.8S–25S rRNA and 5S rRNA genes. The wild beet-specific satellite repeat and the telomere sequence from Arabidopsis thaliana were used to perform a structural analysis of the wild beet chromosome fragments of two resistant fragment addition lines. It was shown that one physical end of the chromosome fragments consists of telomeric repeats. Comparison of fragment sizes indicated that the small chromosome fragments harbouring the resistance gene most likely resulted from the loss of one wild beet chromosome arm and an internal deletion of the remaining arm.This revised version was published online in November 2005 with corrections to the Cover Date.     

FISH to mitotic chromosomes and extended DNA fibres of Beta procumbens in a series of monosomic additions to beet (B. vulgaris)

The physical localization and organization of a Procumbentes-specific repetitive DNA sequence, PB6-4, on the chromosomes of Beta procumbens (2n=18) were studied, using FISH (fluorescence in situ hybridization) to mitotic chromosomes and extended DNA fibres. The chromosomes of B. procumbens were studied in metaphase complements of the species itself, as well as in preparations of a series of eight different B. procumbens-derived monosomic additions to B. vulgaris (2n=18). FISH to chromosome spreads of B. procumbens revealed that PB6-4 hybridizes to all chromosomes, predominantly in the pericentromeric regions, but with differences in size and brightness of the signals. Hybridization of PB6-4 to metaphase complements of B. vulgaris revealed no signals, indicating that cross-hybridization with the genome of this species was negligible. Consequently, hybridization of PB6-4 to metaphase complements of the monosomic additions yielded fluorescent signals on the alien chromosomes only. The previously observed differences in size and brightness of the fluorescent spots were confirmed using the single alien chromosomes. FISH of PB6-4 to extended DNA fibres of the monosomic additions indicated differences in the fluorescent track lengths between the alien chromosomes. Measurements of the fluorescent tracts allowed classification into discrete groups, varying from one to three groups per B. procumbens chromosome. The data revealed that the brightness or size of the signal at mitotic metaphase and the length of the fluorescent tracks on the DNA fibres were correlated. This revised version was published online in July 2006 with corrections to the Cover Date.     

Molecular organization of terminal repetitive DNA in Beta species

We have isolated families of subtelomeric satellite DNA sequences from species of four sections of the genus Beta and from spinach, a related Chenopodiaceae. Twenty-five clones were sequenced and representative repeats of each family were characterized by Southern blotting and FISH. The families of ApaI restriction satellite repeats were designated pAv34, pAc34, the families of RsaI repeats pRp34, pRn34 and pRs34. The repeating units are 344–362 bp long and 45.7–98.8% homologous with a clear species-specific divergence. Each satellite monomer consists of two subrepeats SR1 and SR2 of 165–184 bp, respectively. The repeats of each subrepeat group are highly identical across species, but share only a homology of 40.8–54.8% with members of the other subrepeat group. Two evolutionary steps could be supposed in the phylogeny of the subtelomeric satellite family: the diversification of an ancestor satellite into groups representing SR1 and SR2 in the progenitor of Beta and Spinacea species, followed by the dimerization and diversification of the resulting 360 bp repeats into section-specific satellite DNA families during species radiation. The chromosomal localization of telomeric, subtelomeric and rDNA tandem repeats was investigated by multi-colour FISH. High-resolution analysis by fibre FISH revealed a unique physical organization of B. vulgaris chromosome ends with telomeric DNA and subtelomeric satellites extending over a maximum of 63 kb and 125 kb, respectively.     

The chloroplast trnP-trnW-petG gene cluster in the mitochondrial genomes of Beta vulgaris,B. trigyna and B. webbiana: evolutionary aspects

The chloroplast trnP-trnW-petG gene cluster has been identified in the mitochondrial DNA (mtDNA) of sugar beet (Beta vulgaris). The chloroplast-derived trnW gene is transcribed in the mitochondria; the other two genes, however, do not seem to be transcribed. This gene cluster is also present in the mitochondrial genomes of two wild Beta species, B. trigyna and B. webbiana. Sugar beet and the two wild relatives share 100% sequence identity in the coding regions of both the mitochondrial trnP and trnW genes. On the other hand, the petG genes from the wild Beta mtDNAs were found to be disrupted either by a 5-bp duplication (B. trigyna) or by a deletion of the 5 region (B. webbiana). A data-base search revealed that a conserved sequence of 60 bp is present in the trnP-trnW intergenic region of the mitochondrial genomes of the three Beta species as well as in other higher plants, including wheat and maize, and that the conserved sequence is absent from the chloroplast counterpart. Our results thus favour the hypothesis of a monophyletic origin of the trnP-trnW-petG cluster found in the plant mitochondrial genomes examined.     

Mobilization and evolutionary history of miniature inverted-repeat transposable elements (MITEs) in Beta vulgaris L.

We have identified three families of miniature inverted-repeat transposable elements (VulMITEs) in the genome of sugar beet (Beta vulgaris L.), evidently derived from a member of the Vulmar family of mariner transposons. While VulMITEs I are typical stowaway-like MITEs, VulMITEs II and VulMITEs III are rearranged stowaway elements of increased size. The integration of divergent moderately and highly repetitive sequences into VulMITEs II and, in particular in VulMITEs III, respectively, shows that amplification of repetitive DNA by MITEs contribute to the increase of genome size with possible implications for plant genome evolution. Fluorescent in-situ hybridization (FISH), for the first time visualizing stowaway MITE distribution on plant chromosomes, revealed a dispersed localization of VulMITEs along all B. vulgaris chromosomes. Analysis of the flanking sequences identified a dispersed repeat as target site for the integration of the stowaway element VulMITE I. Recent transposition of VulMITE I, which most likely occurred during the domestication of cultivated beets, was concluded from insertional polymorphisms between different B. vulgaris cultivars and species. ^†Sequence data from this article have been deposited in the EMBL/GenBank Data Library under the accession nos. AM231630-AM231653 and AM259123-AM259125.     

Complex satellite DNA reshuffling in the polymorphic t(1;29) Robertsonian translocation and evolutionarily derived chromosomes in cattle

We have analysed and mapped physically the satellite I, III (subunits pvu and sau) and IV DNA sequences in cattle using in-situ hybridization. Four breeds were analysed including individuals with a chromosome number of 2n=60 and individuals with the widespread t(1;29) in the homozygous (2n=58) and heterozygous state (2n=59). All three satellite DNA families were present at the centromeres of the many but not all of the autosomal acrocentric chromosomes, and essentially absent from the sex chromosomes. In the translocated t(1;29) chromosome, the satellite DNA families showed a different pattern from that simply derived by fusion of the acrocentric autosomes and loss of satellite sequences, with no variation between breeds. A model of centromeric evolution is presented involving two independent events. Knowledge of mechanisms of translocation formation within cattle is important for a functional understanding of centromere and satellites, investigation of chromosomal abnormalities, and for understanding chromosomal fusion during evolution of other bovids and genome evolution in general. This revised version was published online in July 2006 with corrections to the Cover Date.     

Chromosomal localization of six repeated DNA sequences among species of Microtus (Rodentia)

C-banding and fluorescence in situ hybridization (FISH) document the distribution of constitutive heterochromatin and six highly repeated DNA families (MSAT2570, MSAT21, MSAT160, MS2, MS4 and STR47) in the chromosomes of nine species of Microtus (M. chrotorrhinus, M. rossiaemeridionalis, M. arvalis, M. ilaeus, M. transcaspicus, M. cabrerae, M. pennsylvanicus, M. miurus and M. ochrogaster). Autosomal heterochromatin is largely centromeric and contains different repeated families in different species. Similarly, large C-band positive blocks on the sex chromosomes of four species contain different repeated DNAs. This interspecific variation in the chromosomal distribution and copy number of the repeats suggests that a common ancestor to modern species contained most of the repetitive families, and that descendant species selectively amplified or deleted different repeats on different chromosomes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Autosomal location of a new subtype of 1.688 satellite DNA ofDrosophila melanogaster

During the screening of aDrosophila melanogaster YAC library with DNA from the minichromosomeDp(1;f)1187 we isolated a clone, yw20D5, which contains a new subtype of 1.688 satellite DNA. Although the sequences of several monomers subcloned from the YAC show a considerable variation in length, the derived consensus sequence is 356-bp long. This new subtype and the one constituted by the 353-bp repeats are both located on the left arm heterochromatin of chromosome 3, arranged in separate arrays. Despite their autosomal location, phylogenetic relationships among 1.688 satellite sequences suggest that they may have originated from the 359-bp repeats of the X chromosome heterochromatin. We have used the new 356-bp repeats to investigate whether sequences related to the 1.688 satellite are dispersed along the euchromatic arms of the autosomes in a similar way to that in which they are found along the X chromosome euchromatin.accepted for publication by D. Ward     

Characterization and evolutionary dynamics of a complex family of satellite DNA in the leaf beetle <Emphasis Type="Italic">Chrysolina carnifex</Emphasis> (Coleoptera,Chrysomelidae)

The present study characterizes the complex satellite DNA from the specialized phytophagous beetle species Chrysolina carnifex. The satellite DNA is formed by six monomer types, partially homologous but having diverged enough to be separate on the phylogenetic trees, since each monomer type is located on a different branch, having statistically significant bootstrap values. Its analysis suggests a common evolutionary origin of all monomers from the same 211-bp sequence mainly by means of base-substitution mutations evolutionarily fixed to each monomer type and duplications and/or deletions of pre-existing segments in the 211-bp sequence. The analysis of the sequences and Southern hybridizations suggest that the monomers are organized in three types of repeats: monomers (211-bp) and higher-order repeats in the form of dimers (477-bp) or even trimers (633-bp). These repetitive units are not isolated from others, and do not present the pattern characteristic for the regular tandem arrangement of satellite DNA. In-situ hybridization with biotinylated probes corresponding to the three types of repeats showed the pericentromeric location of these sequences in all meiotic bivalents, coinciding with the heterochromatic blocks revealed by C-banding, indicating in addition that each type of repeat is neither isolated from others nor located in specific chromosomes but rather that they are intermixed in the heterochromatic regions. The presence of this repetitive DNA in C. haemoptera, C. bankii and C. americana was also tested by Southern analysis. The results show that this satellite DNA sequence is specific to the C. carnifex genome but has not been found in three other species of Chrysolina occupying similar or different host plants.     

Molecular cloning and characterization of the repetitive DNA sequences that comprise the constitutive heterochromatin of the A and B chromosomes of the Korean field mouse (<Emphasis Type="Italic">Apodemus peninsulae</Emphasis>, Muridae,Rodentia)

Three novel families of repetitive DNA sequences were molecularly cloned from the Korean field mouse (Apodemus peninsulae) and characterized by chromosome in-situ hybridization and filter hybridization. They were all localized to the centromeric regions of all autosomes and categorized into major satellite DNA, type I minor, and type II minor repetitive sequences. The type II minor repetitive sequence also hybridized interspersedly in the non-centromeric regions. The major satellite DNA sequence, which consisted of 30 bp elements, was organized in tandem arrays and constituted the majority of centromeric heterochromatin. Three families of repetitive sequences hybridized with B chromosomes in different patterns, suggesting that the B chromosomes of A. peninsulae were derived from A chromosomes and that the three repetitive sequences were amplified independently on each B chromosome. The minor repetitive sequences are present in the genomes of the other seven Apodemus species. In contrast, the major satellite DNA sequences that had a low sequence homology are present only in a few species. These results suggest that the major satellite DNA was amplified with base substitution in A. peninsulae after the divergence of the genus Apodemus from the common ancestor and that the B chromosomes of A. peninsulae might have a species-specific origin.     

Sequence analysis, chromosomal distribution and long-range organization show that rapid turnover of new and old pBuM satellite DNA repeats leads to different patterns of variation in seven species of the Drosophila buzzatii cluster

We aimed to study patterns of variation and factors influencing the evolutionary dynamics of a satellite DNA, pBuM, in all seven Drosophila species from the buzzatii cluster (repleta group). We analyzed 117 alpha pBuM-1 (monomer length 190 bp) and 119 composite alpha/beta (370 bp) pBuM-2 repeats and determined the chromosome location and long-range organization on DNA fibers of major sequence variants. Such combined methodologies in the study of satDNAs have been used in very few organisms. In most species, concerted evolution is linked to high copy number of pBuM repeats. Species presenting low-abundance and scattered distributed pBuM repeats did not undergo concerted evolution and maintained part of the ancestral inter-repeat variability. The alpha and alpha/beta repeats colocalized in heterochromatic regions and were distributed on multiple chromosomes, with notable differences between species. High-resolution FISH revealed array sizes of a few kilobases to over 0.7 Mb and mutual arrangements of alpha and alpha/beta repeats along the same DNA fibers, but with considerable changes in the amount of each variant across species. From sequence, chromosomal and phylogenetic data, we could infer that homogenization and amplification events involved both new and ancestral pBuM variants. Altogether, the data on the structure and organization of the pBuM satDNA give insights into genome evolution including mechanisms that contribute to concerted evolution and diversification. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

FISH mapping and molecular organization of the major repetitive sequences of tomato

This paper presents a bird’s-eye view of the major repeats and chromatin types of tomato. Using fluorescence in-situ hybridization (FISH) with Cot-1, Cot-10 and Cot-100 DNA as probes we mapped repetitive sequences of different complexity on pachytene complements. Cot-100 was found to cover all heterochromatin regions, and could be used to identify repeat-rich clones in BAC filter hybridization. Next we established the chromosomal locations of the tandem and dispersed repeats with respect to euchromatin, nucleolar organizer regions (NORs), heterochromatin, and centromeres. The tomato genomic repeats TGRII and TGRIII appeared to be major components of the pericentromeres, whereas the newly discovered TGRIV repeat was found mainly in the structural centromeres. The highly methylated NOR of chromosome 2 is rich in [GACA]₄, a microsatellite that also forms part of the pericentromeres, together with [GA]₈, [GATA]₄ and Ty1-copia. Based on the morphology of pachytene chromosomes and the distribution of repeats studied so far, we now propose six different chromatin classes for tomato: (1) euchromatin, (2) chromomeres, (3) distal heterochromatin and interstitial heterochromatic knobs, (4) pericentromere heterochromatin, (5) functional centromere heterochromatin and (6) nucleolar organizer region.     

Molecular characterization and physical localization of highly repetitive DNA sequences from Brazilian Alstroemeria species

Highly repetitive DNA sequences were isolated from genomic DNA libraries of Alstroemeria psittacina and A. inodora. Among the repetitive sequences that were isolated, tandem repeats as well as dispersed repeats could be discerned. The tandem repeats belonged to a family of interlinked Sau3A subfragments with sizes varying from 68–127 bp, and constituted a larger HinfI repeat of approximately 400 bp. Southern hybridization showed a similar molecular organization of the tandem repeats in each of the Brazilian Alstroemeria species tested. None of the repeats hybridized with DNA from Chilean Alstroemeria species, which indicates that they are specific for the Brazilian species. In-situ localization studies revealed the tandem repeats to be localized in clusters on the chromosomes of A. inodora and A. psittacina: distal hybridization sites were found on chromosome arms 2PS, 6PL, 7PS, 7PL and 8PL, interstitial sites on chromosome arms 2PL, 3PL, 4PL and 5PL. The applicability of the tandem repeats for cytogenetic analysis of interspecific hybrids and their role in heterochromatin organization are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Comparative genomic in situ hybridization (cGISH) analysis on plant chromosomes revealed by labelled Arabidopsis DNA

A new approach for comparative cytogenetic banding analysis of plant chromosomes has been established. The comparative GISH (cGISH) technique is universally applicable to various complex genomes of Monocotyledonae (Triticum aestivum, Agropyron elongatum, Secale cereale, Hordeum vulgare, Allium cepa, Muscari armenaticum and Lilium longiflorum) and Dicotyledonae (Vicia faba, Beta vulgaris, Arabidopsis thaliana). Labelled total genomic DNA of A. thaliana generates signals at conserved chromosome regions. The nucleolus organizing regions (NORs) containing the majority of tandemly repeated rDNA sequences, N-band regions containing satellite DNA, conserved homologous sequences at telomeres and additional chromosome-characteristic markers were detected in heterologous FISH experiments. Multicolour FISH analysis with repetitive DNA probes simultaneously revealed the chromosome assignment of 56 cGISH signals in rye and 61 cGISH signals in barley. Further advantages of this technique are: (1) the fast and straightforward preparation of the probe; (2) the generation of signals with high intensity and reproducibility even without signal amplification; and (3) no requirement of species-specific sequences suitable for molecular karyotype analysis. Hybridization can be performed without competitive DNA. Signal detection without significant background is possible under low stringency conditions. The universal application of this fast and simple one-step fluorescence banding technique for plant cytogenetic and plant genome evolution is discussed.     

The ins and outs of population relationships in west-Mediterranean islands: data from autosomal <Emphasis Type="Italic">Alu</Emphasis> polymorphisms and <Emphasis Type="Italic">Alu</Emphasis>/STR compound systems

The islands of the West Mediterranean have played a central role in numerous archaeological, historical and anthropological studies due to their active participation in the history of main Mediterranean civilisations. However, genetic data failed to fit in both their degree of internal differentiation and relationships. A set of 18 Alu markers and three short tandem repeats (STRs) closely linked to the CD4, F13B and DM Alu have been analysed in seven samples from Majorca, Corsica, Sardinia and Sicily to explore some of these issues. Our samples show a high genetic heterogeneity inside and among islands for the Alu data. Global differentiation among islands (F_ST 2.2%) is slightly higher than that described for Europeans and North Africans. Both the estimated divergence times among samples and the high population heterogeneity revealed by Alu data are compatible with population differences since the first islands’ settlement in the Paleolithic period. However, the high within-population diversities and the remarkable homogeneity observed in both STR and Alu/STR haplotype variation indicated that, at least since Neolithic times, gene flow has been acting in west Mediterranean. Genetic drift in west-coast Sardinia and gene flow in west Sicily have contributed to their general differentiation, whereas Corsica, Majorca and east Sicily seem to reflect more recent historical relationships from continental south Europe. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

Evidence for chromosome and Pst I satellite DNA family evolutionary stasis in the Bufo viridis group (Amphibia, Anura)

The West Palearctic green toads, Bufo viridis, represent a species complex. Apart from tetraploid populations, which form at least one separate species, evidence exists for relevant differentiation among diploid populations. We present the results of a chromosomal (C-, Ag-NOR-, Replication pattern, DAPI and CMA₃ banding) and molecular study (isolation and characterization of a satellite DNA family) carried out on a number of Central Asian, European and North African populations. For comparative purposes, our molecular analysis was also extended to specimens of three additional Bufo species (B. bufo, B. mauritanicus and B. cf. regularis), as well as two rare African bufonids (Werneria mertensis and Wolterstoffina sp.). Our results demonstrate a remarkable karyological and molecular evolutionary stasis in the Bufo viridis complex. In fact, all chromatinic markers showed the same pattern and/or composition in all specimens, independently of their origin and ploidy levels. Even the NOR loci were invariably two and located on the telomeric regions of two chromosomes of the sixth pair, or quartet. Furthermore, very similar patterns of genomic hybridization of a monomeric unit of the Pst I satellite DNA family (named pBv) were observed in all diploid and tetraploid populations, as well as in B. bufo and B. mauritanicus. Finally, pBv hybridizes with monomeric units of Pst I satellite DNA in all species studied, including Werneria and Wolterstorffina, which are thought to have separated from Bufo as early as in the Mesozoic. This revised version was published online in July 2006 with corrections to the Cover Date.     

The location and possible evolutionary significance of small dispersed repeats in wheat ctDNA

Summary Low-stringency hybridisation between recombinant plasmids representing the complete T. aestivum chloroplast genome has revealed small repeated DNA segments dispersed through the molecule. Thirty-two repeated DNA segments were detected, and they could be divided into 12 unrelated sets; no repeat was detected as multiple copies. The longest of the small repeats mapped just within the large inverted repeat in spinach and mung-bean ctDNAs. It was found to have been duplicated after the divergence of a cereal progenitor to generate a third, dispensible copy, 0.2 kbp downstream of rbcL. In maize at least, this copy has also become integrated, with rbcL, in the mitochondrial genome. Another of the repeats is thought to have mediated a chloroplast DNA inversion (Howe 1985). Thus the diverse collection of small repeats probably represents some consequences and causes of past recombination events as well as a mechanism for further intramolecular ctDNA recombination. Their possible significance in the restructuring and evolution of chloroplast genomes is discussed.     

Comparison of the lignin-degrading white rot fungi Phanerochaete chrysosporium and Sporotrichum pulverulentum at the DNA level

Summary DNA-hybridisation studies showed a close relationship between Phanerochaete chrysosporium ME446, most used in lignin degradation studies, and Sporotrichum pulverulentum Novobranova, the other standard lignin degrading strain. Two other strains of P. chrysosporium were both less related. We show that P. chrysosporium ME446 and S. pulverulentum Novobranova both have a GC-content of 59% for chromosomal DNA with the rRNA genes present as an AT-rich satellite; the mitochondrial DNA has a GC-content of 33%. The genome size estimated for P. chrysosporium ME446 is about 4–5 × 10⁷ bp.