Survey of repetitive sequences in <Emphasis Type="Italic">Silene latifolia</Emphasis> with respect to their distribution on sex chromosomes

We carried out a global survey of all major types of transposable elements in Silene latifolia, a model species with sex chromosomes that are in the early stages of their evolution. A shotgun genomic library was screened with genomic DNA to isolate and characterize the most abundant elements. We found that the most common types of elements were the subtelomeric tandem repeat X-43.1 and Gypsy retrotransposons, followed by Copia retrotransposons and LINE non-LTR elements. SINE elements and DNA transposons were less abundant. We also amplified transposable elements with degenerate primers and used them to screen the library. The localization of elements by FISH revealed that most of the Copia elements were accumulated on the Y chromosome. Surprisingly, one type of Gypsy element, which was similar to Ogre elements known from legumes, was almost absent on the Y chromosome but otherwise uniformly distributed on all chromosomes. Other types of elements were ubiquitous on all chromosomes. Moreover, we isolated and characterized two new tandem repeats. One of them, STAR-C, was localized at the centromeres of all chromosomes except the Y chromosome, where it was present on the p-arm. Its variant, STAR-Y, carrying a small deletion, was specifically localized on the q-arm of the Y chromosome. The second tandem repeat, TR1, co-localized with the 45S rDNA cluster in the subtelomeres of five pairs of autosomes. FISH analysis of other Silene species revealed that some elements (e.g., Ogre-like elements) are confined to the section Elisanthe while others (e.g. Copia or Athila-like elements) are present also in more distant species. Similarly, the centromeric satellite STAR-C was conserved in the genus Silene whereas the subtelomeric satellite X-43.1 was specific for Elisanthe section. Altogether, our data provide an overview of the repetitive sequences in Silene latifolia and revealed that genomic distribution and evolutionary dynamics differ among various repetitive elements. The unique pattern of repeat distribution is found on the Y chromosome, where some elements are accumulated while other elements are conspicuously absent, which probably reflects different forces shaping the Y chromosome. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Distinct chromosomal distributions of highly repetitive sequences in maize

The majority of genomic DNA in most plant species is made up of repetitive elements including satellites and retrotransposons. The maize genome is intermediate in size and abundance of repetitive elements between small genomes such as Arabidopsis and rice and larger genomes such as wheat. Although repetitive elements are present throughout the maize genome, individual families are non-randomly distributed along chromosomes. In this work we use fluorescence in-situ hybridization (FISH) to examine the distribution of abundant LTR retroelement families and satellites contained in heterochromatic blocks called knobs. Different retroelement families have distinct patterns of hybridization. Prem1 and Tekay, two very closely related elements, both hybridize along the length of all chromosomes but do so with greater intensity near the centromeres, although subtle differences are detectable between the hybridization patterns. Opie, Prem2/Ji, and Huck are enriched away from the centromeres and Grande is distributed uniformly along the chromosomes. Double labeling with proximally and distally enriched elements on pachytene chromosomes produces alternating blocks of element enrichment. The maize elements hybridized in the same general patterns to chromosomes of maize relatives including Zea diploperennis and Tripsacum dactyloides. Additionally, abundant Tripsacum LTR retroelements are enriched in similar chromosomal regions among the different species. The 180 bp knob satellite is present in large blocks at interstitial locations on chromosome arms. With long exposures, smaller sites of hybridization are detected at the ends of chromosomes, adjacent to the telomere tract. This distal position for knob satellites is conserved among Zea and Tripsacum species. Electronic supplementary material Supplementary material to this paper is available in electronic form at and is accessible for authorized users.     

Divergence of satellite DNA and interspersion of dispersed repeats in the genome of the wild beet Beta procumbens

Several repetitive sequences of the genome of Beta procumbens Chr. Sm., a wild beet species of the section Procumbentes of the genus Beta have been isolated. According to their genomic organization, the repeats were assigned to satellite DNA and families of dispersed DNA sequences.The tandem repeats are 229–246 bp long and belong to an AluI restriction satellite designated pAp11. Monomers of this satellite DNA form subfamilies which can be distinguished by the divergence or methylation of an internal restriction site. The satellite is amplified in the section Procumbentes, but is also found in species of the section Beta including cultivated beet (Beta vulgaris). The existence of the pAp11 satellite in distantly related species suggests that the AluI sequence family is an ancient component of Beta genomes and the ancestor of the diverged satellite subfamily pEV4 in B. vulgaris. Comparative fluorescent in-situ hybridization revealed remarkable differences in the chromosomal position between B. procumbens and B. vulgaris, indicating that the pAp11 and pEV4 satellites were most likely involved in the expansion or rearrangement of the intercalary B. vulgaris heterochromatin.Furthermore, we describe the molecular structure, and genomic and chromosomal organization of two repetitive DNA families which were designated pAp4 and pAp22 and are 1354 and 582 bp long, respectively. The families consist of sequence elements which are widely dispersed along B. procumbens chromosomes with local clustering and exclusion from distal euchromatic regions. FISH on meiotic chromosomes showed that both dispersed repeats are colocalized in some chromosomal regions. The interspersion of repeats of the pAp4 and pAp22 family was studied by PCR and enabled the determination of repeat flanking sequences. Sequence analysis revealed that pAp22 is either derived from or part of a long terminal repeat (LTR) of an Athila-like retrotransposon. Southern analysis and FISH with pAp4 and pAp22 showed that both dispersed repeats are species-specific and can be used as DNA probes to discriminate parental genomes in interspecific hybrids. This was tested in the sugar beet hybrid PRO1 which contains a small B. procumbens chromosome fragment.     

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.     

Characterisation of <Emphasis Type="Italic">Zygosaccharomyces rouxii</Emphasis> centromeres and construction of first <Emphasis Type="Italic">Z. rouxii</Emphasis> centromeric vectors

Zygosaccharomyces rouxii is a hemiascomycetous yeast known for its high osmotolerance, the basis of which still remains unknown. By exploring the Génolevures I database, four Z. rouxii fragments homologous to Saccharomyces cerevisiae centromeres were identified. Two of them were subjected to further analysis. Their function as centromeres in Z. rouxii was proved, and they were localized to Z. rouxii chromosomes II and VII, respectively. The species-specificity of centromeres was observed; plasmids with a Z. rouxii centromere were not recognized as centromeric in S. cerevisiae, and a S. cerevisiae centromere did not function as a centromere in Z. rouxii. Constructed plasmids bearing Z. rouxii centromeres serve as the first specific centromeric plasmids, and thus contribute to the so-far limited set of genetic tools needed to study the Z. rouxii specific features.     

The large-scale organization of the centromeric region in Beta species

In higher eukaryotes, the DNA composition of centromeres displays a high degree of variation, even between chromosomes of a single species. However, the long-range organization of centromeric DNA apparently follows similar structural rules. In our study, a comparative analysis of the DNA at centromeric regions of Beta species, including cultivated and wild beets, was performed using a set of repetitive DNA sequences. Our results show that these regions in Beta genomes have a complex structure and consist of variable repetitive sequences, including satellite DNA, Ty3-gypsy-like retrotransposons, and microsatellites. Based on their molecular characterization and chromosomal distribution determined by fluorescent in situ hybridization (FISH), centromeric repeated DNA sequences were grouped into three classes. By high-resolution multicolor-FISH on pachytene chromosomes and extended DNA fibers we analyzed the long-range organization of centromeric DNA sequences, leading to a structural model of a centromeric region of the wild beet species Beta procumbens. The chromosomal mutants PRO1 and PAT2 contain a single wild beet minichromosome with centromere activity and provide, together with cloned centromeric DNA sequences, an experimental system toward the molecular isolation of individual plant centromeres. In particular, FISH to extended DNA fibers of the PRO1 minichromosome and pulsed-field gel electrophoresis of large restriction fragments enabled estimations of the array size, interspersion patterns, and higher order organization of these centromere-associated satellite families. Regarding the overall structure, Beta centromeric regions show similarities to their counterparts in the few animal and plant species in which centromeres have been analyzed in detail.     

Coexistence of NtCENH3 and two retrotransposons in tobacco centromeres

Although a centromeric DNA fragment of tobacco (Nicotiana tabacum), Nt2-7, has been reported, the overall structure of the centromeres remains unknown. To characterize the centromeric DNA sequences, we conducted a chromatin immunoprecipitation assay using anti-NtCENH3 antibody and chromatins isolated from two ancestral diploid species (Nicotiana sylvestris and Nicotiana tomentosiformis) of N. tabacum and isolated a 178-pb fragment, Nto1 from N. tomentosiformis, as a novel centromeric DNA. Fluorescence in situ hybridization (FISH) showed that Nto1 localizes on 24 out of 48 chromosomes in some cells of a BY-2 cell line. To identify the origins of the Nt2-7 and Nto1, a tobacco bacterial artificial chromosome (BAC) library was constructed from N. tabacum, and then screened by polymerase chain reaction (PCR) with primer sets designed from the Nt2-7 and Not1 DNA sequences. Twelve BAC clones were found to localize on the centromeric regions by FISH. We selected three BAC clones for sequencing and identified two centromeric retrotransposons, NtCR and NtoCR, the DNA sequences of which are similar to that of Nt2-7 and Nto1, respectively. Quantitative PCR analysis using coprecipitated DNA with anti-NtCENH3 clearly showed coexistence of NtCENH3 with both retrotransposons. These results indicate the possibility that these two retrotransposons act as centromeric DNA sequences in tobacco. NtoCR was found to be specific to N. tomentosiformis and T genome of N. tabacum, and a NtCR-like centromeric retrotransposon (TGRIV) exists in tomato. This specificity suggests that the times of amplification of these centromeric retrotransposons were different.     

Sequence organization and evolutionary dynamics of Brachypodium-specific centromere retrotransposons

Brachypodium distachyon is a wild annual grass belonging to the Pooideae, more closely related to wheat, barley, and forage grasses than rice and maize. As an experimental model, the completed genome sequence of B. distachyon provides a unique opportunity to study centromere evolution during the speciation of grasses. Centromeric satellite sequences have been identified in B. distachyon, but little is known about centromeric retrotransposons in this species. In the present study, bacterial artificial chromosome (BAC)-fluorescence in situ hybridization was conducted in maize, rice, barley, wheat, and rye using B. distachyon (Bd) centromere-specific BAC clones. Eight Bd centromeric BAC clones gave no detectable fluorescence in situ hybridization (FISH) signals on the chromosomes of rice and maize, and three of them also did not yield any FISH signals in barley, wheat, and rye. In addition, four of five Triticeae centromeric BAC clones did not hybridize to the B. distachyon centromeres, implying certain unique features of Brachypodium centromeres. Analysis of Brachypodium centromeric BAC sequences identified a long terminal repeat (LTR)-centromere retrotransposon of B. distachyon (CRBd1). This element was found in high copy number accounting for 1.6 % of the B. distachyon genome, and is enriched in Brachypodium centromeric regions. CRBd1 accumulated in active centromeres, but was lost from inactive ones. The LTR of CRBd1 appears to be specific to B. distachyon centromeres. These results reveal different evolutionary events of this retrotransposon family across grass species.     

A tandem repetitive sequence located in the centromeric region of common wheat (Triticum aestivum) chromosomes

Although TaiI-family sequences are present in the subtelomeric region of Leymus racemosus, it became apparent in the present study that such sequences are also present in the centromeric region of common wheat (Triticum aestivum). These sequences hybridized to all chromosomes with various degrees of signal strength. FISH using TaiI and Ty3/gypsy, a conservative sequence in cereal centromeres, revealed a complicated arrangement of both sequences in all wheat chromosomes at once. Unlike the Arabidopsis centromeres characterized by massive tandem arrays of 180-bp family with flanking paracentromeric retrotransposons in all chromosomes, wheat chromosomes showed various arrangement patterns of TaiI and Ty3/gypsy sequences depending on the chromosome; TaiI-family sequences were scattered in many wheat centromeres as isolated colonies instead of forming uninterrupted solid tandem arrays. This pattern may have resulted from retrotransposon insertion within pre-existing TaiI-tandem arrays or a two-step amplification mechanism of the TaiI family where each TaiI colony was amplified to form arrays independently after the insertion of TaiI-family sequences along the entire centromere. Although sequence analysis of centromeric TaiI repeats in wheat and subtelomeric TaiI repeats in L. racemosus showed variable and conservative regions between the two repeats, they did not show a distinctive difference phylogenically. The widespread presence of tandem repetitive sequences in the eucaryotic centromere suggests a significant role for them in centromeric formation.     

Centromeric heterochromatin in the cattle rob(1;29) translocation: α-satellite I sequences, in-situ MspI digestion patterns, chromomycin staining and C-bands

The centromeric regions and -satellite I sequence were studied on chromosomes 1, 29 and the rob(1;29) translocation in a Portuguese breed of cattle, Barrosã, carrying the translocation. Rob(1;29) centromeric regions showed heterochromatic bands with propidium iodide but, unlike the acrocentric autosomes, no strong centromeric bands were revealed with chromomycin A3. An -satellite I sequence was not found at the centromeres of the X, Y and rob(1;29) chromosomes in the breed, although it was present at the centromeres of all acrocentric chromosomes including 1 and 29. Restriction enzyme banding with MspI revealed polymorphisms between different rob(1;29) chromosomes in both centromeric and intercalary regions. The data show that the centromeric region of the rob(1;29) chromosome has lost the -satellite I sequences, while retaining other heterochromatin, and suggest that this important and widespread translocation has occurred multiple times.     

The position of repetitive DNA sequence in the southern cattle tick genome permits chromosome identification

Fluorescent in-situ hybridization (FISH) using meiotic chromosome preparations and highly repetitive DNA from the southern cattle tick, Rhipicephalus microplus, was undertaken to investigate genome organization. Several classes of highly repetitive DNA elements were identified by screening a R. microplus bacterial artificial chromosome (BAC) library. A repeat unit of approximately 149 bp, RMR-1 was localized to the subtelomeric regions of R. microplus autosomes 1–6 and 8–10. A second repeat unit, RMR-2 was localized to the subtelomeric regions of all autosomes and the X chromosome. RMR-2 was composed of three distinct repeat populations, RMR-2a, RMR-2b and RMR-2c of 178, 177 and 216 bp in length, respectively. Localization of an rDNA probe identified a single nucleolar organizing region on one autosome. Using a combination of labeled probes, we developed a preliminary karyotype for R. microplus. We present evidence that R. microplus has holocentric chromosomes and explore the implications of these findings for tick chromosome biology and genomic research. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular characterization of a centromeric satellite DNA in the hemiclonal hybrid frogRana esculenta and its parental species

Hybrid water frogsRana esculenta reproduce by hybridogenesis: one parental genome (ofRana lessonae) is excluded in the germ line, the other (ofRana ridibunda) is clonally transmitted to haploid gametes. The two parental species differ in that the amount of centromeric heterochromatin revealed by differential staining is much higher inRana ridibunda. An abundant, tandemly arrayed, centromeric satellite DNA, designated RrS1, is revealed inRana ridibunda genomes by the restriction endonucleaseStul, which generates a major repetitive sequence fragment of 300 and a minor one of 200 bp. This AT-rich (68%) satellite family is located at the centromeres of the five largest chromosomes (1–5) and of a medium to small heterobrachial one (8 or 9); it thus constitutes only part of the centromeric heterochromatin that characterizes allRana ridibunda chromosomes. RrS1 represents about 2.5% of the genome ofRana ridibunda; it may represent as little as 0.2% of the genome ofRana lessonae, and cannot be detected inXenopus laevis frogs orSalamandra salamandra andTriturus carnifex salamanders. Segments of the satellite sequence are similar to sequences of yeast centromeric DNA element CDEIII and of the mammalian CENP-B box. A role for RrS1 and other centromeric satellite DNAs in the germ line genome exclusion of the hybridogenetic frog hybrids, although suggested, has not yet been demonstrated.     

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.     

Chromosomal location and evolution of a satellite DNA family in seven sturgeon species

The Hind III satellite DNA family, isolated from the Acipenser naccarii genome, was used as a probe for fluorescent in-situ hybridization (FISH) on the karyotype of seven sturgeon species, six belonging to the genus Acipenser and one to Huso. All species except one (A. sturio) exhibit from 8 to 80 chromosome hybridization signals, mainly localized at the pericentromeric regions. Eight chromosomes with weak hybridization signals are present in H. huso and A. ruthenus, which are characterized by a karyotype with about 120 chromosomes. The species with 240–260 chromosomes, A. transmontanus, A naccarii, A. gueldenstaedtii, and A. baerii, show from 50 to 80 signals, prevalently localized around centromeres. Moreover, A. transmontanus and A. gueldenstaedtii show from 4 to 8 chromosomes with a double signal. The phylogenetic and evolutionary relationships among sturgeon species are discussed on the basis of number and morphology of signal-bearing chromosomes and on the localization of signals.     

Molecular structures of centromeric heterochromatin and karyotypic evolution in the Siamese crocodile (Crocodylus siamensis) (Crocodylidae, Crocodylia)

Crocodilians have several unique karyotypic features, such as small diploid chromosome numbers (30–42) and the absence of dot-shaped microchromosomes. Of the extant crocodilian species, the Siamese crocodile (Crocodylus siamensis) has no more than 2n = 30, comprising mostly bi-armed chromosomes with large centromeric heterochromatin blocks. To investigate the molecular structures of C-heterochromatin and genomic compartmentalization in the karyotype, characterized by the disappearance of tiny microchromosomes and reduced chromosome number, we performed molecular cloning of centromeric repetitive sequences and chromosome mapping of the 18S-28S rDNA and telomeric (TTAGGG) _n sequences. The centromeric heterochromatin was composed mainly of two repetitive sequence families whose characteristics were quite different. Two types of GC-rich CSI-HindIII family sequences, the 305 bp CSI-HindIII-S (G+C content, 61.3%) and 424 bp CSI-HindIII-M (63.1%), were localized to the intensely PI-stained centric regions of all chromosomes, except for chromosome 2 with PI-negative heterochromatin. The 94 bp CSI-DraI (G+C content, 48.9%) was tandem-arrayed satellite DNA and localized to chromosome 2 and four pairs of small-sized chromosomes. The chromosomal size-dependent genomic compartmentalization that is supposedly unique to the Archosauromorpha was probably lost in the crocodilian lineage with the disappearance of microchromosomes followed by the homogenization of centromeric repetitive sequences between chromosomes, except for chromosome 2.     

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.     

Parental genome separation and asynchronous centromere division in interspecific F1 hybrids inLathyrus

Chromosomes were studied in root-tip metaphase cells of several F₁ interspecificLathyrus hybrids including:L. hirsutus × L. cassius (H × C),L. cassius × L. hirsutus (C × H),L. cassius × L. odoratus (C × O), and their parents, all with 2n = 2x = 14. Two types of morphologically distinct centromeres were identified in the hybrids on the basis of the degree of contraction of the primary constriction. At least 12 well-defined centromeres were seen in all cells ofL. hirsutus, L. cassius andL. odoratus, and about 80% of cells had 14. The hybrids were more variable than the species. H × O contained between six and 14 welldefined centromeres, while cells of H × C, C × H and C × O all had seven well-defined and seven weakly defined centromeres. These were used as markers to plot their spatial disposition in two dimensions on metaphase spreads. In H × C, C × H and C × O the two types of centromeres showed a significant tendency to occupy two spatially distinct and concentrically arranged domains on the metaphase plate (P < 0.005). Owing to shortage of material subsequent work was restricted to H × C and C × H. Six or seven chromosomes of one parental genome were selectively labelled byin situ hybridization using biotinylated total genomic DNA from either parent as a probe. Moreover, there was a very strong correlation between centromere type and genomic origin (P < 0.001). Clearly the tendency for spatial segregation of parental genomes into concentrically arranged domains previously noted in several F₁ interspecific sexual hybrids between grasses can occur strongly in dicots. The present work onLathyrus also included the first comparative study of the spatial arrangement of parental genomes in reciprocal hybrids. Significantly,L. cassius chromosomes tended to surroundL. hirsutus chromosomes in both H × C and C × H. Thus, the polarity of concentric parental genome separation was not determined as a maternal effect.     

Chromosomal study of a lamprey (<Emphasis Type="Italic">Lampetra zanandreai</Emphasis> Vladykov, 1955) (Petromyzonida: Petromyzontiformes): conventional and FISH analysis

Karyotype and other chromosomal characteristics in the Adriatic brook lamprey Lampetra zanandreai, representative of one of the most ancestral group of vertebrates, were examined using conventional (Ag-staining, C-banding as well as CMA₃ and DAPI fluorescence) and molecular (FISH with 18/28S rDNA and EcoRI satDNA as probes) protocols with metaphase chromosomes derived from whole blood cultures. The chromosome complement had a modal diploid chromosome number of 2n = 164, as in other petromyzontid lamprey species. Ag-staining and CMA₃ fluorescence, as well as FISH with 18/28S rDNA probes, detected nucleolar organizer regions (NORs) close to the centromeres of the biarmed chromosomes of pairs 1 and 2, the largest chromosome pairs of the complement. In addition to NORs, CMA₃ fluorescence revealed positive signals in approximately 40 other chromosomes. DAPI stained mostly centromeric regions of many chromosomes as well as conspicuously massive blocks overlapping NOR sites. C-banding evidenced a large amount of constitutive heterochromatin in somatic chromosomes, with approximately 40 C-positive acrocentric elements completely heterochromatic, corresponding with the 40 CMA₃+ chromosomes and positive heterochromatic blocks in pericentromeric regions of chromosome pairs 1 and 2. Polymerase chain reaction (PCR)-based cloning of satellite DNA with primers derived from Petromyzon marinus centromeric sequences was successful for L. zanandreai genomic DNA. The sequence was AT-rich (59%) and characterized by short consensus motifs similar to other centromeric satellite motifs. FISH using satDNA clones as a probe produced a fluorescent signal on a single pair of small chromosomes. This sequence was PCR-amplified also in L. planeri and P. marinus genomic DNA, and the evolution of this repetitive element in the above species was analysed.