A novel repetitive sequence associated with the centromeric regions of Arabidopsis thaliana chromosomes

 The middle repetitive fraction of the Arabidopsis genome has been relatively poorly characterized. We describe here a novel repetitive sequence cloned in the plasmid mi167, and present in ∼90 copies in the genome of Arabidopsis thaliana ecotype Columbia. Hybridization analysis to physically mapped YAC clones representing Arabidopsis chromosome 4 revealed four mi167-hybridizing loci, all clustered near the centromere. No other loci were detected on YAC clones covering chromosome 4. In situ hybridisation experiments to Arabidopsis chromosome spreads showed that mi167-hybridizing sequences are clustered at the centromeric heterochromatin of all five chromosomes; on two chromosomes the hybridization appeared to be localised on one arm. Additional mi167-hybridizing loci were detected, one of which was adjacent to a non-centromeric, heterochromatic region. This work supports the idea that the majority of middle repetitive DNA in the Arabidopsis genome is clustered. It also adds to our understanding of the organization of the centromere of Arabidopsis chromosome 4. Received: 19 February 1996 / Accepted: 30 June 1996     

Analysis of clones carrying repeated DNA sequences in two YAC libraries of Arabidopsis thaliana DNA

YAC clones carrying repeated DNA sequences from the Arabidopsis thaliana genome have been characterized in two widely used Arabidopsis YAC libraries, the EG library and the EW library. Ribosomal, chloroplast and the paracentromeric repeat sequences are differentially represented in the two libraries. The coordinates of YAC clones hybridizing to these sequences are given. A high proportion of EG YAC clones were classified as containing chimaeric inserts because individual clones carried unique sequences and repetitive sequences originating from different locations in the genome. None of the EW YAC clones analysed were chimaeric in this way. YAC clones carrying tandemly repeated sequences, such as the paracentromeric or rDNA sequences, exhibited a high degree of instability. These observations need to be taken into account when using these libraries in the development of a physical map of the Arabidopsis genome and in chromosome walking experiments.     

Construction of a yeast artificial chromosome library of tomato and identification of cloned segments linked to two disease resistance loci

Summary We have constructed a yeast artificial chromosome (YAC) library of tomato for chromosome walking that contains the equivalent of three haploid genomes (22 000 clones). The source of high molecular weight DNA was leaf protoplasts from the tomato cultivars VFNT cherry and Rio Grande-PtoR, which together contain loci encoding resistance to six pathogens of tomato. Approximately 11 000 YACs have been screened with RFLP markers that cosegregate withTm-2a andPto — loci conferring resistance to tobacco mosaic virus andPseudomonas syringae pv.tomato, respectively. Five YACs were identified that hybridized to the markers and are therefore starting points for chromosome walks to these genes. A subset of the library was characterized for the presence of various repetitive sequences and YACs were identified that carried TGRI, a repeat clustered near the telomeres of most tomato chromosomes, TGRII, an interspersed repeat, and TGRIIl, a repeat that occurs primarily at centromeric sites. Evaluation of the library for organellar sequences revealed that approximately 10% of the clones contain chloroplast sequences. Many of these YAC clones appear to contain the entire 155 kb tomato chloroplast genome. The tomato cultivars used in the library construction, in addition to carrying various disease resistance genes, also contain the wild-type alleles corresponding to most recessive mutations that have been mapped by classical linkage analysis. Thus, in addition to its utility for physical mapping and genome studies, this library should be useful for chromosome walking to genes corresponding to virtually any phenotype that can be scored in a segregating population.     

Cytogenetics for the model system Arabidopsis thaliana

A detailed karyotype of Arabidopsis thaliana is presented using meiotic pachytene cells in combination with fluorescence in situ hybridization. The lengths of the five pachytene bivalents varied between 50 and 80 μm, which is 20–25 times longer than mitotic metaphase chromosomes. The analysis confirms that the two longest chromosomes (1 and 5) are metacentric and the two shortest chromosomes (2 and 4) are acrocentric and carry NORs subterminally in their short arms, while chromosome 3 is submetacentric and medium sized. Detailed mapping of the centromere position further revealed that the length variation between the pachytene bivalents comes from the short arms. Individual chromosomes were unambiguously identified by their combinations of relative lengths, arm-ratios, presence of NOR knobs and FISH signals with a 5S rDNA probe and chromosome specific DNA probes. Polymorphisms were found among six ecotypes with respect to the number and map positions of 5S rDNA loci. All ecotypes contain 5S rDNA in the short arms of chromosomes 4 and 5. Three different patterns were observed regarding the presence and position of a 5S rDNA locus on chromosome 3. Repetitive DNA clones enabled us to subdivide the pericentromeric heterochromatin into a central domain, characterized by pAL1 and 106B repeats, which accommodate the functional centromere and two flanking domains, characterized by the 17 A20 repeat sequences. The upper flanking domains of chromosomes 4 and 5, and in some ecotypes also chromosome 3, contain a 5S rDNA locus. The detection of unique cosmids and YAC sequences demonstrates that detailed physical mapping of Arabidopsis chromosomes by cytogenetic techniques is feasible. Together with the presented karyotype this makes Arabidopsis a model system for detailed cytogenetic mapping.     

Rice genome organization: the centromere and genome interactions

Over the last decade, many varied resources have become available for genome studies in rice. These resources include over 4000 DNA markers, several bacterial artificial chromosome (BAC) libraries, P-1 derived artificial chromosome (PAC) libraries and yeast artificial chromosome (YAC) libraries (genomic DNA clones, filters and end-sequences), retrotransposon tagged lines, and many chemical and irradiated mutant lines. Based on these, high-density genetic maps, cereal comparative maps, YAC and BAC physical maps, and quantitative trait loci (QTL) maps have been constructed, and 93 % of the genome has also been sequenced. These data have revealed key features of the genetic and physical structure of the rice genome and of the evolution of cereal chromosomes. This Botanical Briefing examines aspects of how the rice genome is organized structurally, functionally and evolutionarily. Emphasis is placed on the rice centromere, which is composed of long arrays of centromere-specific repetitive sequences. Differences and similarities amongst various cereal centromeres are detailed. These indicate essential features of centromere function. Another view of various kinds of interactive relationships within and between genomes, which could play crucial roles in genome organization and evolution, is also introduced. Constructed genetic and physical maps indicate duplication of chromosomal segments and spatial association between specific chromosome regions. A genome-wide survey of interactive genetic loci has identified various reproductive barriers that may drive speciation of the rice genome. The significance of these findings in genome organization and evolution is discussed.     

Identification of a deletion hotspot on distal mouse chromosome 4 by YAC fingerprinting

Using repetitive elements as probes, genomic DNA fingerprints of four randomly selected yeast artificial chromosome (YAC) clones (two human and two mouse-derived YAC) were analyzed to determine the mutation level following X-ray exposure. Because the repetitive probes were derived from the mammalian host DNA, most of the fingerprint bands originated from the artificial chromosomes and not from the yeast genome. For none of the YAC clones was the mutation frequency elevated following X-ray exposure. However, for one mouse-derived YAC, the mutation level was unusually high (7%; 42 mutants of 607 clones analyzed), whereas for the other three YACs, the mutation level was nearly 0%. Surprisingly, 40 of the 42 mutations were deletions occurring only at three of the 20 mouse specific fingerprint bands. One of the frequently deleted fragments was cloned, sequenced and mapped to distal mouse chromosome 4, which has been repeatedly reported to be the most unstable region of the whole mouse genome, associated with various tumors. Deletion mapping of six YAC mutants revealed this fragment to be completely deleted in four YACs. In the other two mutants, recombination occurred within the fragment, in each case initiated at the same LINE-1 element. In conclusion, the presented YAC fingerprint is a useful tool for detecting and characterizing unstable regions in mammalian genomes.     

High macro-collinearity between lima bean (Phaseolus lunatus L.) and the common bean (P. vulgaris L.) as revealed by comparative cytogenetic mapping

Common bean (P. vulgaris) and lima bean (P. lunatus) are the most important crop species from the genus Phaseolus. Both species have the same chromosome number (2n = 22) and previous cytogenetic mapping of BAC clones suggested conserved synteny. Nevertheless, karyotype differences were observed, suggesting structural rearrangements. In this study, comparative cytogenetic maps for chromosomes 3, 4 and 7 were built and the collinearity between the common bean and lima bean chromosomes was investigated. Thirty-two markers (30 BACs and 2 bacteriophages) from P. vulgaris were hybridized in situ on mitotic chromosomes from P. lunatus. Nine BACs revealed a repetitive DNA pattern with pericentromeric distribution and 23 markers showed unique signals. Nine of these markers were mapped on chromosome 3, eight on chromosome 4 and six on chromosome 7. The order and position of all analyzed BACs were similar between the two species, indicating a high level of macro-collinearity. Thus, although few inversions have probably altered centromere position in other chromosomes, the main karyotypic differences were associated with the repetitive DNA fraction.     

Genes and genomes: Towards construction of an overlapping YAC library of the Arabidopsis thaliana genome

Arabidopsis thaliana (Thale cress, Arabidopsis) is an ideal model organism for the molecular genetic analysis of many plant processes. The availability of a complete physical map would greatly facilitate the gene cloning steps in these studies. The small genome size of Arabidopsis makes the construction of such a map a feasible goal. One of the approaches to construct an overlapping library of the Arabidopsis genome takes advantage of the many mapped markers and the availability of Arabidopsis yeast artificial chromosome (YAC) libraries. Mapped molecular markers are used to identify corresponding YAC clones and thereby place them on the genetic map. Subsequently, these YAC clones provide the framework for directed walking experiments aimed at closing the gaps between the YAC contigs. Adopting this strategy, YAC clones comprising about 10% of the genome have been assigned to the top halves of Arabidopsis chromosomes 4 and 5. Extensive walking experiments in a 10 cM interval of chromosome 4 have resulted in two contiguous regions in the megabase size range.     

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.     

Extended physical maps and a consensus physical map of the homoeologous group-6 chromosomes of wheat (Triticum aestivum L. em Thell.)

Extended physical maps of chromosomes 6A, 6B and 6D of common wheat (Triticum aestivum L. em Thell., 2n=6x=42, AABBDD) were constructed with 107 DNA clones and 45 homoeologous group-6 deletion lines. Two-hundred and ten RFLP loci were mapped, including three orthologous loci with each of 34 clones, two orthologous loci with each of 31 clones, one locus with 40 clones, two paralogous loci with one clone, and four loci, including three orthologs and one paralog, with one clone. Fifty five, 74 and 81 loci were mapped in 6A, 6B and 6D, respectively. The linear orders of the mapped orthologous loci in 6A, 6B and 6D appear to be identical and 65 loci were placed on a group-6 consensus physical map. Comparison of the consensus physical map with eight linkage maps of homoeologous group-6 chromosomes from six Triticeaespecies disclosed that the linear orders of the loci on the maps are largely, if not entirely, conserved. The relative distributions of loci on the physical and linkage maps differ markedly, however. On most of the linkage maps, the loci are either distributed relatively evenly or clustered around the centromere. In contrast, approximately 90% of the loci on the three physical maps are located either in the distal one-half or the distal two-thirds of the six chromosome arms and most of the loci are clustered in two or three segments in each chromosome. Received: 19 April 1999 / Accepted: 28 July 1999     

A Physical Map of Arabidopsis thaliana Chromosome 3 Represented by Two Contigs of CIC YAC, P1, TAC and BAC Clones

We have constructed a physical map of Arabidopsis thaliana chromosome3 by ordering the clones from CIC YAC, P1, TAC and BAC librariesusing the sequences of a variety of genetic and EST markersand terminal sequences of clones. The markers used were 112DNA markers, 145 YAC end sequences, and 156 end sequences ofP1, TAC and BAC clones. The entire genome of chromosome 3, exceptfor the centromeric and telomeric regions, was covered by twolarge contigs, 13.6 Mb and 9.2 Mb long. This physical map willfacilitate map-based cloning experiments as well as genome sequencingof chromosome 3. The map and end sequence information are availableon the KAOS (Kazusa Arabidopsis data Opening Site) web siteat http://www.kazusa.or.jp/arabi/.     

Construction and characterization of a YAC library with a lowfrequency of chimeric clones from flow-sorted human chromosome 9

Human chromosome 9 DNA, flow-sorted from somaticcell hybrid PK-87-9, has been used to construct two complete digest YAC libraries. The combined representation of chromosome 9 in these libraries, estimated by hybridization of chromosome 9-specific sequences to YAC colony grids, is 95%. The frequency of chimeric clones, analyzed by fluorescence in situ hybridization of chromosome 9 YACs to human metaphase chromosomes, was estimated to be 4%. These libraries provide a resource for physical mapping and for moving from genetic markers to disease loci on chromosome 9.     

Identification of clones carrying minisatellite-like loci in an Arabidopsis thaliana YAC library

Minisatellite-like DNA elements occur in the Arabidopsis thalianagenome in low copy and are weakly polymorphic between ecotypes.YAC clones from the EG-Arabidopsis library were identified withhomology to minisatellite 33.15 and bacteriophage M13 repeatelements. Other highly repeated A. thaliana DNA elements tendnot to be found in YAC clones carrying the minisatellite elementssuggesting that the elements are dispersed in the Arabidopsisgenome in regions of low complexity. The minisatellite elementsare represented at low copy in the EG-YAC library reflectingtheir frequency in the Arabidopsis genome. Key words: Minisatellite elements, Arabidopsis thaliana, YAC library screening     

Towards a Drosophila genome map.

A physical map of the genome of Drosophila melanogaster has been created using 965 yeast artificial chromosome (YAC) clones assigned to locations in the cytogenetic map by in situ hybridization with the polytene salivary gland chromosomes. Clones with insert sizes averaging about 200 kb, totaling 1.7 genome equivalents, have been mapped. More than 80% of the euchromatic genome is included in the mapped clones, and 75% of the euchromatic genome is included in 161 cytological contigs ranging in size up to 2.5 Mb (average size 510 kb). On the other hand, YAC coverage of the one-third of the genome constituting the heterochromatin is incomplete, and clones containing long tracts of highly repetitive simple satellite DNA sequences have not been recovered.     

Map-based cloning of the tomato genomic region that spans the Sw-5 tospovirus resistance gene in tomato

Two yeast artificial chromosomes (YACs) containing genomic DNA from tomato have been isolated using CT220, an RFLP marker which is tightly linked to the tomato spotted wilt virus resistance gene, Sw-5. High-resolution mapping of the YAC ends and internal YAC probes demonstrated that one of the YAC clones, TY257 (400?kb), spans Sw-5. By chromosome walking in a cosmid library, the position of Sw-5 has been delimited within the YAC to a maximal chromosomal segment of 100?kb, spanned by nine overlapping cosmid clones.     

In silico integration of quantitative trait loci for seed yield and yield-related traits in Brassica napus

Mapping quantitative trait loci (QTLs) is a foundation for molecular marker-assisted selection and map-based gene cloning. During the past decade, numerous QTLs for seed yield (SY) and yield-related traits in Brassica napus L. have been identified. However, integration of these results in order to compare QTLs from different mapping populations has not been undertaken, due to the lack of common molecular markers between studies. Using previously reported Brassica rapa and Brassica oleracea genome sequences, we carried out in silico integration of 1,960 QTLs associated with 13 SY and yield-related traits from 15 B. napus mapping experiments over the last decade. A total of 736 SY and yield-related QTLs were mapped onto 283 loci in the A and C genomes of B. napus. These QTLs were unevenly distributed across the 19 B. napus chromosomes, with the most on chromosome A3 and the least on chromosome C6. Our integrated QTL map identified 142 loci where the conserved QTLs were detected and 25 multifunctional loci, mostly for the traits of flowering time (FT), plant height, 1,000-seed weight, maturity time and SY. These conserved QTLs and multifunctional loci may result from pleiotropism or clustered genes. At the same time, a total of 146 genes underlying the QTLs for FT and other yield-related traits were identified by comparative mapping with the Arabidopsis genome. These results facilitate the retrieval of B. napus SY and yield-related QTLs for research communities, increase the density of targeted QTL-linked markers, validate the existence of QTLs across different populations, and advance the fine mapping of genes.     

Cloning of human centromeres by transformation-associated recombination in yeast and generation of functional human artificial chromosomes

Human centromeres remain poorly characterized regions of the human genome despite their importance for the maintenance of chromosomes. In part this is due to the difficulty of cloning of highly repetitive DNA fragments and distinguishing chromosome-specific clones in a genomic library. In this work we report the highly selective isolation of human centromeric DNA using transformation-associated recombination (TAR) cloning. A TAR vector with alphoid DNA monomers as targeting sequences was used to isolate large centromeric regions of human chromosomes 2, 5, 8, 11, 15, 19, 21 and 22 from human cells as well as monochromosomal hybrid cells. The alphoid DNA array was also isolated from the 12 Mb human mini-chromosome ΔYq74 that contained the minimum amount of alphoid DNA required for proper chromosome segregation. Preliminary results of the structural analyses of different centromeres are reported in this paper. The ability of the cloned human centromeric regions to support human artificial chromosome (HAC) formation was assessed by transfection into human HT1080 cells. Centromeric clones from ΔYq74 did not support the formation of HACs, indicating that the requirements for the existence of a functional centromere on an endogenous chromosome and those for forming a de novo centromere may be distinct. A construct with an alphoid DNA array from chromosome 22 with no detectable CENP-B motifs formed mitotically stable HACs in the absence of drug selection without detectable acquisition of host DNAs. In summary, our results demonstrated that TAR cloning is a useful tool for investigating human centromere organization and the structural requirements for formation of HAC vectors that might have a potential for therapeutic applications.     

Molecular genetic analysis of theripening-inhibitor andnon-ripening loci of tomato: A first step in genetic map-based cloning of fruit ripening genes

Ripening represents a complex developmental process unique to plants. We are using tomato fruit ripening mutants as tools to understand the regulatory components that control and coordinate the physiological and biochemical changes which collectively confer the ripe phenotype. We have genetically characterized two loci which result in significant inhibition of the ripening process in tomato,ripening-inhibitor (rin), andnon-ripening (nor), as a first step toward isolating genes likely to encode key regulators of this developmental process. A combination of pooled-sample mapping as well as classical restriction fragment length polymorphism (RFLP) analysis has permitted the construction of high-density genetic maps for the regions of chromosomes 5 and 10 spanning therin andnor loci, respectively. To assess the feasibility of initiating a chromosome walk, physical mapping of high molecular weight genomic DNA has been employed to estimate the relationship between physical distance (in kb) and genetic distance (in cM) around the targeted loci. Based on this analysis, the relationship in the region spanning therin locus is estimated to be 200–300 kb/cM, while thenor locus region ratio is approximately 200 kb/1 cM. Using RFLP markers tightly linked torin andnor, chromosome walks have been initiated to both loci in a yeast artificial chromosome (YAC) library of tomato genomic DNA. We have isolated and characterized several YAC clones linked to each of the targeted ripening loci and present genetic evidence that at least one YAC clone contains thenot locus.     

Description of 31 YAC contigs spanning the majority of Arabidopsis thaliana chromosome 5

In order to generate a physical map of Arabidopsis thaliana chromosome 5, 142 molecular markers mapping to chromosome 5 have been used in colony hybridization experiments with four Arabidopsis, ecotype Columbia, yeast artificial chromosome (YAC) libraries. This resulted in 634 YAC clones being anchored on chromosome 5. Southern blot analysis confirmed their positioning and provided data, which along with knowledge of the sizes of all the YAC clones, enabled the clones to be arranged into 31 contigs. Genetic mapping of markers located within 29 of these contigs on the Landsberg erecta/Columbia recombinant inbred lines allowed positioning of the contigs along the chromosome. A high proportion of the YAC clones were found to contain chimaeric inserts. The availability of this YAC contig map will accelerate chromosome-walking experiments, provide substrates for large-scale genomic sequencing projects and facilitate the mapping of new probes to this chromosome.