Delimiting the Origin of a B Chromosome by FISH Mapping,Chromosome Painting and DNA Sequence Analysis in Astyanax paranae (Teleostei,Characiformes)

Supernumerary (B) chromosomes have been shown to contain a wide variety of repetitive sequences. For this reason, fluorescent in situ hybridisation (FISH) is a useful tool for ascertaining the origin of these genomic elements, especially when combined with painting from microdissected B chromosomes. In order to investigate the origin of B chromosomes in the fish species Astyanax paranae, these two approaches were used along with PCR amplification of specific DNA sequences obtained from the B chromosomes and its comparison with those residing in the A chromosomes. Remarkably, chromosome painting with the one-arm metacentric B chromosome probe showed hybridization signals on entire B chromosome, while FISH mapping revealed the presence of H1 histone and 18S rDNA genes symmetrically placed in both arms of the B chromosome. These results support the hypothesis that the B chromosome of A. paranae is an isochromosome. Additionally, the chromosome pairs Nos. 2 or 23 are considered the possible B chromosome ancestors since both contain syntenic H1 and 18S rRNA sequences. The analysis of DNA sequence fragments of the histone and rRNA genes obtained from the microdissected B chromosomes showed high similarity with those obtained from 0B individuals, which supports the intraspecific origin of B chromosomes in A. paranae. Finally, the population hereby analysed showed a female-biased B chromosome presence suggesting that B chromosomes in this species could influence sex determinism.     

Molecular Characterization of a Maize B Chromosome Centric Sequence

Supernumerary chromosomes are widespread in the plant kingdom but little is known of their molecular nature or mechanism of origin. We report here the initial cloning of sequences from the maize B chromosome. Our analysis suggests that many sequences are highly repetitive and shared with the normal A chromosomes. However, all clones selected for B-specificity contain at least one copy of a particular repeat. Cytological mapping using B chromosome derivatives and in situ hybridization show that the B specific repeats are derived from the centric region of the chromosome. Sequence analysis of this repeat shows homology to motifs mapped to various plant and animal centromeres and to the maize neocentromere. A precise localization of these sequences among breakpoints within the B centromere and an homology to a facultative centromere, suggest a role for this sequence in centromere function.     

Gene mapping on maize pachytene chromosomes by in situ hybridization

A sensitive in situ hybridization technique which was effective for mapping genes of low copy number on human metaphase chromosomes was used for gene mapping on maize pachytene chromosomes. A cloned genomic EcoR1 fragment of 10.8 kb, containing most or all of the sequence encoding the Waxy locus mRNA, was used as the probe. Southern DNA blotting analyses performed by Shure et al. (1983) indicated that the Waxy locus was a single copy sequence. In our in situ hybridization experiment, the probe hybridized to a specific site on chromosome 9. Labeling at this site was detected in 48.6% of 154 randomly selected copies of chromosome 9. To test the sensitivity of the method, subclones of the fragment with insert sizes of 6.6, 4.7, 3.5, 2.3, 1.9 and 0.8 kb were used for in situ hybridizations. Labeling efficiency for each probe was determined. The data showed that a single copy probe of 1.9 kb could be detected at the correct position in 18% of 183 randomly selected number 9 chromosomes.     

The genomic complexity of micro B chromosomes of Brachycome dichromosomatica

A major sequence component of the micro B chromosome of Brachycome dichromosomatica (2 n=4) is the tandem repeat Bdm29, which was found by in situ hybridisation to be distributed along the entire length of the chromosome. A high copy number of this sequence does not occur as a regular feature of the A chromosomes in this species but it was found in infrequent individuals in two wild populations that were analysed. In these instances Bdm29 is localised within heterochromatic, polymorphic segments on the long arm of chromosome 1. The origin of the micro B chromosomes was investigated by determining whether they are related to this A chromosome polymorphism by simple excision and/or integration. Results obtained by using Bdm29, together with a newly isolated repeat sequence, Bdm54, and a number of other sequences known to occur on the micro B chromosome, as probes in in situ hybridisation and Southern analysis demonstrated that the formation of micro B chromosomes is a complex multistep process. The observation that the genomic organisation of the micro B chromosome is unlike anything found on the A chromosomes precludes their origin by simple excision and also indicates that micro Bs do not integrate directly into the A complement to form polymorphic heterochomatic segments.     

A single, recent origin of the accessory B chromosome of the grasshopper Eyprepocnemis plorans

B chromosomes are dispensable chromosomes found in >2000 eukaryotic species, usually behaving as genomic parasites. Most B chromosomes seem to be made up of the same kind of DNA sequences present in the A chromosomes. This sequence similarity makes it difficult to obtain specific molecular probes that may permit B-presence diagnosis without cytogenetic analysis. We have developed a sequence-characterized amplified region (SCAR) marker for B chromosomes in the grasshopper Eyprepocnemis plorans, which specifically amplifies a 1510-bp DNA fragment exclusively in B-carrying individuals. Fluorescent in situ hybridization and fiber FISH analyses showed that this marker is a tandemly repeated DNA sequence closely intermingled with 45S rDNA. PCR reactions showed the presence of SCAR-like sequences in the A chromosomes, but in two separate fragments, supporting the intraspecific origin of B chromosomes in this species. SCAR marker DNA sequence showed to be identical in B chromosome variants from several localities from Spain and Morocco, and it was very similar to those found in B chromosome variants from Greece and Armenia. This strongly suggests that this sequence was already present in the ancestral B chromosome of this species. In addition, the scarce sequence variation observed among several B variants from very distant populations suggests either a functional constraint or, more likely, a recent and unique origin for B chromosomes in this species.     

Organization and Mapping of a Sequence on the DROSOPHILA MELANOGASTER X and Y Chromosomes That Is Transcribed during Spermatogenesis

The D. melanogaster DNA segment in the recombinant phage lambda Dm2L1 contains at least eight copies of a tandemly repeated 1250-base pair (bp) sequence (henceforth called the 2L1 sequence). Testes from XO D. melanogaster males contain an abundant 800-base RNA species that is homologous to a 520-bp region of the 2L1 sequence. Blotting experiments show that the 2L1 sequence is repeated in the D. melanogaster genome and is present on both the X and Y chromosomes. With the use of X-Y translocations, the 2L1 sequence has been mapped to a region between kl-1 and kl-2 on the long arm of the Y chromosome. In Oregon-R wild type there are an estimated 200 copies of the 2L1 sequence on the X chromosome and probably at least 80 copies of the Y chromosome. In some other strains the repetition frequency on the Y chromosome is about the same, but the copy number on the X chromosome is much reduced. On the basis of the five strains investigated, there is a correlation between copy number of the 2L1 sequence on the X chromosome and the presence of a particular allele of the Stellate locus (Ste; 1-45.7). It seems that low copy number corresponds to Ste+ and high copy number corresponds to Ste. The Ste locus determines whether single or star-shaped crystals are observed in the spermatocytes of XO males. Studies using D. simulans and D. mauritiana DNA show that the 2L1 sequence is homologous to restriction fragments in male DNA but not female DNA, indicating that this sequence is present only on the Y chromosome in these two species. In DNA derived from D. erecta, D. teissieri and D. yakuba, there is very little, if any, hybridization with the 2L1 sequence probe.     

Mutation mechanisms that underlie turnover of a human telomere-adjacent segmental duplication containing an unstable minisatellite

Subterminal regions, juxtaposed to telomeres on human chromosomes, contain a high density of segmental duplications, but relatively little is known about the evolutionary processes that underlie sequence turnover in these regions. We have characterized a segmental duplication adjacent to the Xp/Yp telomere, each copy containing a hypervariable array of the DXYS14 minisatellite. Both DXYS14 repeat arrays mutate at a high rate (0.3 and 0.2% per gamete) but linkage disequilibrium analysis across 27 SNPs and a direct crossover assay show that recombination during meiosis is suppressed. Therefore instability at DXYS14a and b is dominated by intra-allelic processes or possibly conversion limited to the repeat arrays. Furthermore some chromosomes (14%) carry only one copy of the duplicon, including one DXYS14 repeat array that is also highly mutable (1.2% per gamete). To explain these and other observations, we propose there is another low-rate mutation process that causes copy number change in part or all of the duplicon.     

The species and chromosomal distribution of the centromeric alpha-satellite I sequence from sheep in the tribe Caprini and other Bovidae

The evolution of chromosomes in species in the family Bovidae includes fusion and fission of chromosome arms (giving different numbers of acrocentric and metacentric chromosomes with a relatively conserved total number of arms) and evolution in both DNA sequence and copy number of the pericentromeric alpha-satellite I repetitive DNA sequence. Here, a probe representing the sheep alpha-satellite I sequence was isolated and hybridized to genomic DNA digests and metaphase chromosomes from various Bovidae species. The probe was highly homologous to the centromeric sequence in all species in the tribe Caprini, including sheep (Ovis aries), goat (Capra hircus) and the aoudad or Barbary sheep (Amnotragus lervia), but showed no detectable hybridization to the alpha-satellite I sequence present in the tribe Bovini and at most very weak to species in the tribes Hippotragini, Alcelaphini or Aepycerotini. The sex chromosomes of sheep, goat and aoudad did not contain detectable alpha-satellite I sequence; in sheep, one of the three metacentric autosomal chromosomes does not carry the sequence, while in aoudad, it is essentially absent in three large autosomal pairs as well as the large metacentric chromosome pair. The satellite probes can be used as robust chromosome and karyotype markers of evolution among tribes and increase the resolution of the evolutionary tree at the base of the Artiodactyla.     

Conversion of a RAPD-generated PCR product,containing a novel dispersed repetitive element,into a fast and robust assay for the presence of rye chromatin in wheat

Bulk segregant analysis was used to obtain a random amplified polymorphic DNA (RAPD) marker specific for the rye chromosome arm of the 1BL.1RS translocation, which is common in many high-yielding bread wheat varieties. The RAPD-generated band was cloned and end-sequenced to allow the construction of a pair of oligonucleotide primers that PCR-amplify a DNA sequence only in the presence of rye chromatin. The amplified sequence shares a low level of homology to wheat and barley, as judged by the low strength of hybridization of the sequence to restriction digests of genomic DNA. Genetic analysis showed that the amplified sequence was present on every rye chromosome and not restricted to either the proximal or distal part of the 1RS arm. In situ hybridization studies using the amplified product as probe also showed that the sequence was dispersed throughout the rye genome, but that the copy number was greatly reduced, or the sequence was absent at both the centromere and the major sites of heterochromatin (telomere and nucleolar organizing region). The probe, using both Southern blot and in situ hybridization analyses, hybridized at a low level to wheat chromosomes, and no hybridizing restriction fragments could be located to individual wheat chromosomes from the restriction fragment length polymorphism (RFLP) profiles of wheat aneuploids. The disomic addition lines of rye chromosomes to wheat shared a similar RFLP profile to one another. The amplified sequence does not contain the RIS 1 sequence and therefore represents an as yet undescribed dispersed repetitive sequence. The specificity of the amplification primers is such that they will provide a useful tool for the rapid detection of rye chromatin in a wheat background. Additionally, the relatively low level of cross-hybridization to wheat chromatin should allow the sequence to be used to analyse the organization of rye euchromatin in interphase nuclei of wheat lines carrying chromosomes, chromosome segments or whole genomes derived from rye.     

Efficient combination of large DNA in vitro: in gel site specific recombination (IGSSR) of PAC fragments containing alpha satellite DNA and the human HPRT gene locus.

In an attempt to combine a cloned genomic copy of a selectable gene with different cloned centromeric sequences to develop mammalian artificial chromosomes (MAC) we used site specific recombination mediated by purified Cre recombinase acting on the loxP sequence in PAC vector DNA. A new method was required to purify highly concentrated, virtually 100% intact PAC DNA which could be stored for a long period. Here we show the efficient linking of linearized PACs containing alpha satellite DNA from chromosomes X and 17 with sizes of 125 and 140 kb, respectively, to a 95 kb restriction fragment derived from a 175 kb PAC containing the intact human HPRT gene locus.     

Localization of single copy DNA sequences on G-banded human chromosomes by in situ hybridization

Recombinant lambda bacteriophage clone H3 containing a human DNA segment of 14.9 kb present in one or two copies per haploid genome was isolated. In situ hybridization to human metaphase chromosomes of the ³H-labeled cloned DNA resulted in highly significant labeling (53% of cells) of band p36 of chromosome 1, such that 22% of all chromosomal grains were located on this region. Hybridization was dependent upon the presence of dextran sulfate in the hybridization mixture and was not affected by repetitive DNA competitor. These results demonstrate localization of a single copy sequence on human metaphase chromosomes.     

The effects of genotype-dependent recombination, and transmission asymmetry, on linkage disequilibrium

A recent sperm-typing study by Jeffreys and Neumann suggested that recombination rates in different individuals at the DNA2 recombination hotspot appeared to be highly dependent on their genotype at a particular A/G SNP, FG11. Specifically, individuals who carried at least one copy of the A allele at this SNP exhibited rates of crossover considerably higher than those of individuals with no copies. Further, recombinant sperm from heterozygous individuals showed a preferential tendency to carry the G allele. We consider the effects of these phenomena on patterns of linkage disequilibrium and find them to be more subtle than might have been expected. In particular, our analysis suggests that, perhaps surprisingly, patterns of LD among chromosomes carrying the "hot" allele (in this case, A) will typically be similar to those among chromosomes carrying the "cold" allele (G).     

Linkage Analysis Reveals the Independent Origin of Poeciliid Sex Chromosomes and a Case of Atypical Sex Inheritance in the Guppy (Poecilia reticulata)

Among different teleost fish species, diverse sex-determining mechanisms exist, including environmental and genetic sex determination, yet chromosomal sex determination with male heterogamety (XY) prevails. Different pairs of autosomes have evolved as sex chromosomes among species in the same genus without evidence for a master sex-determining locus being identical. Models for evolution of Y chromosomes predict that male-advantageous genes become linked to a sex-determining locus and suppressed recombination ensures their co-inheritance. In the guppy, Poecilia reticulata, a set of genes responsible for adult male ornaments are linked to the sex-determining locus on the incipient Y chromosome. We have identified >60 sex-linked molecular markers to generate a detailed map for the sex linkage group of the guppy and compared it with the syntenic autosome 12 of medaka. We mapped the sex-determining locus to the distal end of the sex chromosome. We report a sex-biased distribution of recombination events in female and male meiosis on sex chromosomes. In one mapping cross, we observed sex ratio and male phenotype deviations and propose an atypical mode of genetic sex inheritance as its basis.     

A molecular and cytogenetic survey of major repeated DNA sequences in tomato (Lycopersicon esculentum)

Summary The major families of repeated DNA sequences in the genome of tomato (Lycopersicon esculentum) were isolated from a sheared DNA library. One thousand clones, representing one million base pairs, or 0.15% of the genome, were surveyed for repeated DNA sequences by hybridization to total nuclear DNA. Four major repeat classes were identified and characterized with respect to copy number, chromosomal localization by in situ hybridization, and evolution in the family Solanaceae. The most highly repeated sequence, with approximately 77000 copies, consists of a 162 bp tandemly repeated satellite DNA. This repeat is clustered at or near the telomeres of most chromosomes and also at the centromeres and interstitial sites of a few chromosomes. Another family of tandemly repeated sequences consists of the genes coding for the 45 S ribosomal RNA. The 9.1 kb repeating unit in L. esculentum was estimated to be present in approximately 2300 copies. The single locus, previously mapped using restriction fragment length polymorphisms, was shown by in situ hybridization as a very intense signal at the end of chromosome 2. The third family of repeated sequences was interspersed throughout nearly all chromosomes with an average of 133 kb between elements. The total copy number in the genome is approximately 4200. The fourth class consists of another interspersed repeat showing clustering at or near the centromeres in several chromosomes. This repeat had a copy number of approximately 2100. Sequences homologous to the 45 S ribosomal DNA showed cross-hybridization to DNA from all solanaceous species examined including potato, Datura, Petunia, tobacco and pepper. In contrast, with the exception of one class of interspersed repeats which is present in potato, all other repetitive sequences appear to be limited to the crossing-range of tomato. These results, along with those from a companion paper (Zamir and Tanksley 1988), indicate that tomato possesses few highly repetitive DNA sequences and those that do exist are evolving at a rate higher than most other genomic sequences.     

The FSHD-Associated Repeat, D4Z4, Is a Member of a Dispersed Family of Homeobox-Containing Repeats, Subsets of Which Are Clustered on the Short Arms of the Acrocentric Chromosomes

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder that maps to human chromosome 4q35. FSHD is tightly linked to a polymorphic 3.3-kb tandem repeat locus, D4Z4. D4Z4 is a complex repeat: it contains a novel homeobox sequence and two other repetitive sequence motifs. In most sporadic FSHD cases, a specific DNA rearrangement, deletion of copies of the repeat at D4Z4, is associated with development of the disease. However, no expressed sequences from D4Z4 have been identified. We have previously shown that there are other loci similar to D4Z4 within the genome. In this paper we describe the isolation of two YAC clones that map to chromosome 14 and that contain multiple copies of a D4Z4-like repeat. Isolation of cDNA clones that map to the acrocentric chromosomes and Southern blot analysis of somatic cell hybrids show that there are similar loci on all of the acrocentric chromosomes. D4Z4 is a member of a complex repeat family, and PCR analysis of somatic cell hybrids shows an organization into distinct subfamilies. The implications of this work in relation to the molecular mechanism of FSHD pathogenesis is discussed. We propose the name 3.3-kb repeat for this family of repetitive sequence elements.     

Molecular and cytological characterization of genomic variability in hexaploid wheat 'Lindstr?m'.

'Lindstr?m' wheat (AABBDD+rye B chromosomes) was used to study the effects of alien chromatin introgressed into a wheat genetic background, subjecting the wheat genome to a new and transient allopolyploidisation episode. Using this experimental material, we have previously demonstrated that no large-scale chromosomal translocations occurred as a result of the genomic constitution of the addition line. However, we have shown that the presence of a number of rye B chromosomes is associated with changes in the interphase organization and expression patterns of wheat rDNA loci. We have now extended our studies to focus on a further characterization of 'Lindstr?m' 5S rDNA loci and also on high molecular weight glutenin subunit (HMW-GS) patterns. In the process, we have uncovered an unusually large variant of the 5S rDNA locus on wheat chromosome 1B (not to be confused with rye B chromosomes) and 2 novel HMW glutenin y-type alleles. These changes are not directly related to variation in rye B chromosome number in the present material, but the fact that a new, and still segregating, 1Dy HMW-GS gene was identified indicates a recent timescale for its origin. Strikingly, the 'Lindstr?m' 5S rDNA 1B locus integrates a unit sharing 94% homology with a rye 5S rDNA sequence, suggesting the possibility that the wheat locus was colonized by highly homologous rye sequences during the breeding of 'Lindstr?m', when the rye and wheat genomes were together, albeit briefly, in the same nucleus.     

The role of predation in variation in body shape in guppies Poecilia reticulata: a comparison of field and common garden phenotypes

The body shapes of both wild-caught and laboratory-reared male and female Trinidadian guppies Poecilia reticulata from two low-predation and two high-predation populations were studied, but predation regime did not seem to be the most important factor affecting body shape. Instead, complicated patterns of plasticity in body shape among populations and the sexes were found. In particular, populations differed in the depth of the caudal peduncle, which is the muscular region just anterior to the tail fin rays and from which most swimming power is generated. Strikingly, the direction of population differences in caudal peduncle depth observed in wild-caught individuals was reversed when P. reticulata were raised in a common laboratory environment.     

A note on the presence of B chromosome in the small Japanese field mouse,Apodemus argenteus,in central Honshu,Japan

Previously, many studies have revealed the presence of B chromosomes in wild mouse taxa of the genus Apodemus (Rodentia, Muridae). In one of the Apodemus species, A. argenteus, which is endemic to Japan, it is known that B chromosomes were confirmed only in individuals (2n = 46 + B chromosome) from Hokkaido, Japan. There is no report of the presence of B chromosomes from other localities in the Japanese Islands. In this study, we analyzed the chromosomal constitutions of 43 individuals of A. argenteus from three localities in Honshu, Japan. A total of three individuals from central Honshu showed 2n = 47, and each individual carried a dot-like B chromosome. In addition, these B chromosome features were analyzed by differential staining methods, and the C- and QM-banding patterns of the B chromosomes were identical to those of the X chromosomal heterochromatic region showing the delayed-fluorescent response. Thus, it is considered that these B chromosomes would be derived from the heterochromatin of the X chromosomes, as reported in previously published papers.