A YAC contig map of Arabidopsis thaliana chromosome 3

We have constructed a YAC contig map of Arabidopsis thaliana chromosome 3. From an estimated total size of 25 Mb, about 21 Mb were covered by 148 clones arranged into nine YAC contigs, which represented most of the low-copy regions of the chromosome. YAC clones were anchored with 259 molecular markers, including 111 for which linkage information was previously available. Most of the genetic map was included in the YAC coverage, and more than 60% of the genetic markers from the reference recombinant inbred line map were anchored, giving a high level of integration between the genetic and physical maps. The submetacentric structure of the chromosome was confirmed by physical data; 3R (the top arm of the linkage map) was about 12 Mb, and 3L (the bottom arm of the linkage map) was about 9 Mb. This YAC physical map will aid in chromosome walking experiments and provide a framework for large-scale DNA sequencing of chromosome 3.     

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.     

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.     

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/.     

Identification and map position of YAC clones comprising one-third of the Arabidopsis genome.

YAC clones corresponding to 125 Arabidopsis thaliana RFLP markers have been identified. At least one YAC clone has been isolated for each of the RFLP markers tested. Based on CHEF gel analysis of 196 clones, the mean insert size of the available Arabidopsis YAC libraries is approximately 160 kb. The YACs of known genetic map location encompass about 30% of the Arabidopsis genome. The results presented here represent a first step towards assembly of an overlapping YAC library of the A. thaliana genome.     

Construction of YAC Contigs on Rice Chromosome 5

A physical map of rice chromosome 5 was constructed with yeastartificial chromosome (YAC) clones along a high-resolution molecularlinkage map carrying 118 DNA markers distributed over 123.7cM of genomic DNA. YAC clones have been identified by colonyand Southern hybridization for 105 restriction fragment lengthpolymorphism (RFLP) markers and by polymerase chain reaction(PCR) screening for 8 sequence-tagged site (STS) markers and5 randomly amplified polymorphic DNA (RAPD) markers. Of 458YACs, 235 individual YACs with an average insert length of 350kb were selected and ordered on chromosome 5 from the YAC library.Forty-eight contigs covering nearly 21 Mb were formed on thechromosome 5; the longest one was 6 cM and covered 1.5 Mb. Thelength covered with YAC clones corresponded to 62% of the totallength of chromosome 5. There were many multicopy sequencesof expressed genes on chromosome 5. The distribution of manycopies of these expressed gene sequences was determined by YACSouthern hybridization and is discussed. A physical map withthese characteristics provides a powerful tool for elucidationof genome structure and extraction of useful genetic informationin rice.     

Isolation of single-copy-sequence clones from a yeast artificial chromosome library of randomly-sheared Arabidopsis thaliana DNA

We describe the construction of a yeast artificial chromosome (YAC) library from the Arabidopsis thaliana genome. Randomly sheared high molecular weight source DNA was extracted from frozen, ground leaf tissue and blunt-end-ligated to the vector pYAC3. By size-fractionating the ligation products, we achieved an average clone size of 150 kb. Approximately 6% of the YACs contained inserts from the chloroplast genome. We screened clones equivalent to greater than four A. thaliana haploid nuclear genomes and isolated YACs homologous to five single-copy-sequence probes. The library should be useful chromosome walking and genome mapping experiments. In addition, the approach used for its construction should be applicable to other higher plant species.     

Construction of a YAC library from barley cultivar Franka and identification of YAC-derived markers linked to the Rh2 gene conferring resistance to scald (Rhynchosporium secalis).

The Rh2 resistance gene of barley (Hordeum vulgare) confers resistance against the scald pathogen (Rhynchosporium secalis). A high-resolution genetic map of the Rh2 region on chromosome I (7H) was established by the use of molecular markers. Tightly linked markers from this region were used to screen existing and a newly constructed yeast artificial chromosome (YAC) library of barley cv. Franka composed of 45,000 clones representing approximately two genome equivalents. Corresponding YAC clones were identified for most markers, indicating that the combined YAC library has good representation of the barley genome. The contiguous sets of YAC clones with the most tightly linked molecular markers represent entry points for map-based cloning of this resistance gene.     

Assignment of YAC Clones Spanning Rice Chromosomes 10 and 12

Yeast artificial chromosome (YAC) clones were assigned on rice(Oryza sativa L. cv. Nipponbare) chromosomes 10 and 12 usingDNA markers from our high-density linkage map. Out of 1,383markers localized in this genetic map, 68 and 74 markers werelocated on chromosomes 10 and 12, respectively. Screening ofthe YAC genomic library was conducted by colony hybridizationand Southern hybridization using restriction fragment lengthpolymorphism (RFLP) markers or by polymerase chain reaction(PCR) using sequence-tagged site (STS) markers. We have completedthe screening of 68 markers on chromosome 10 and 74 markerson chromosome 12. A total of 134 and 103 YACs were assignedto chromosomes 10 and 12, respectively, with an estimated coverageof more than 60% for chromosome 10 and about 47% for chromosome12. As rice is considered a model plant for genome analysis,the ordered YAC clones on chromosomes 10 and 12 as well as otherchromosomes will certainly be helpful for isolation of agronomicallyand biologically important genes and for understanding the genomestructure of these chromosomes.     

Physical mapping of the rice genome with YAC clones

Construction of a rice physical map covered by YAC clones which have been arranged over half of the genome length is presented here. A total of 1285 RFLP and RAPD markers almost evenly distributed on the rice genetic map could select 2974 YAC clones and 2443 clones of them were located on their original positions. Rice YACs carrying 350 kb average insert fragments of 2443 clones could cover 222 megabase length of the rice genome, corresponding to 52% of the whole genome size (4.3 Mb). Chromosome landing with many YAC clones on the high-density genetic map loci efficiently integrated the genetic map with a physical map. This is the first step to generate a comprehensive genome map of rice. An integrated genome map should be an indispensable tool to figure out genome structure as well as to clone trait genes by map-based cloning.     

A Fine Physical Map of Arabidopsis thaliana Chromosome 5: Construction of a Sequence-ready Contig Map

A fine physical map of Arabidopsis thaliana chromosome 5 wasconstructed by ordering the clones from YAC, P1, TAC and BAClibraries of the genome using the sequences of a variety ofgenetic and EST markers and terminal sequences of clones. Themarkers used were 88 genetic markers, 13 EST markers, 87 YACend probes, 100 YAC subclone end probes, and 390 end probesof P1, TAC and BAC clones. The entire genome of chromosome 5,except for the centromeric and telomeric regions, was coveredby two large contigs 11.6 Mb and 14.2 Mb long separated by thecentromeric region. The minimum tiling path of the chromosomewas constituted by a total of 430 P1, TAC and BAC clones. Themap information is available at the Web site http://www.kazusa.or.jp/arabi/.     

A sequence-ready contig map of the top arm of Arabidopsis thaliana chromosome 3.

A fine physical map of the top arm of Arabidopsis thaliana chromosome 3 has been constructed by ordering P1, TAC and BAC clones using the sequences of a variety of DNA markers and end-sequences of clones. The marker sequences used in this study were derived from 58 DNA markers, 93 YAC end-sequences, and 807 end-sequences of P1, TAC and BAC clones. The entire top arm of chromosome 3, except for the centromeric and telomeric regions, was covered by a single contig 13.3 Mb long. This fine physical map will facilitate gene isolation by map-based cloning experiments as well as genome sequencing of the top arm of chromosome 3. The map and end-sequence information are available on the web site KAOS (Kazusa Arabidopsis data Opening Site) at [http://www.kazusa.or.jp/arabi/].     

Construction and characterization of a soybean yeast artificial chromosome library and identification of clones for the<Emphasis Type="Italic"> Rps6</Emphasis> region

We report the construction and characterization of the first soybean yeast artificial chromosome (YAC) library using high-molecular weight DNA isolated from leaf nuclei of the cultivar Conrad 94 that carries Phytophthora resistance genes Rps1-k and Rps6. The quality of this library has been evaluated through analysis of 393 randomly selected YAC clones. The library consists of 36,864 clones, of which 19,956 carry single soybean YACs with an average size of about 285 kb. The library represents approximately five soybean genome equivalents. The probability of finding any soybean sequences from this library is about 0.99. The library was screened for 43 SSR markers representing the whole soybean genome. We were able to identify positive YAC pools for 95% of the SSR markers. Two YAC clones carrying molecular markers linked to the Rps6 gene were identified. The YAC library reported here would be a useful resource for map-based cloning of agronomically important soybean genes and also to complement the effort towards construction of the physical map for the soybean genome.     

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.     

Identification of a YAC clone carrying the Xa-1 allele, a bacterial blight resistance gene in rice

Map-based cloning methods have been applied for isolation of Xa-1, one of the bacterial blight resistance genes in rice.Xa-1 was previously mapped on chromosome 4 using molecular markers. For positional cloning of Xa-1, a high-resolution genetic map was made for theXa-1 region using an F₂ population of 402 plants and additional molecular markers. Three restriction fragment length polymorphism (RFLP) markers, XNpb235, XNpb264 and C600 were found to be linked tightly to Xa-1, with no recombinants, and U08 ₇₅₀ was mapped 1.5 cM from Xa-1. The screening of a yeast artificial chromosome (YAC) library using theseXa-1-linked RFLP markers resulted in the identification of ten contiguous YAC clones. Among these, one YAC clone, designated Y5212, with an insert of 340 kb, hybridized with all three tightly linked markers. This YAC was confirmed to possess the Xa-1 allele by mapping the Xa-1 gene between both end clones of this YAC (Y5212R and Y5212L).     

A high-resolution cytogenetic map of 168 cosmid DNA markers for human chromosome 11.

We have constructed a high-resolution cytogenetic map with 168 DNA markers, including 90 RFLP markers for human chromosome 11. The cosmid clones were mapped by fluorescence in situ suppression hybridization, in which discrete fluorescent signals can be detected directly on prometaphase R-banded chromosomes. Although these cosmid clones were distributed throughout the chromosome, they had some tendency to localize in the regions of R-positive band, such as 11p15, 11p11.2, 11q13, 11q23, and 11q25. Since these regions of chromosome 11 are considered to contain genes responsible for certain genetic diseases, cancer breakpoints involved in chromosome rearrangements, and tumor-suppressor genes, this high-resolution cytogenetic map will contribute to the molecular characterization of such genes. This map will also provide many landmarks essential for construction of the complete physical map with contigs of cosmid and YAC clones.     

Yeast Artificial Chromosome Clones of Rice Chromosome 2 Ordered Using DNA Markers

Yeast artificial chromosome (YAC) clones were ordered for thephysical mapping of rice chromosome 2, the last of the 12 ricechromosomes to be assigned YACs by the Rice Genome ResearchProgram. A total of 128 restriction fragment length polymorphismmarkers and 4 sequence-tagged site (STS) markers located onour high-density genetic map were used for YAC clone landing.By colony/Southern hybridization and polymerase chain reactionscreening, a total of 239 individual YACs were selected fromour YAC library of 6934 clones covering six genome equivalents.The YACs located on the corresponding marker positions in thelinkage map formed 43 contigs and islands and were estimatedto encompass about 50% of the length of rice chromosome 2.     

Construction and characterization of a rice YAC library for physical mapping

Genomic libraries of rice,Oryza sativa L. cv. Nipponbare, in yeast artificial chromosomes were prepared for construction of a rice physical map. High-molecular-weight genomic DNA was extracted from cultured suspension cells embedded in agarose plugs. After size fractionation of theEco RI- andNot I-digested DNA fragments, they were ligated with pYAC4 and pYAC55, respectively, and used to transformSaccharomyces cerevisiae AB1380. A total of 6932 clones were obtained containing on average ca. 350 kb DNA. The YAC library was estimated to contain six haploid genome equivalents. The YACs were examined for their chimerism by mapping both ends on an RFLP linkage map. Most YACs withEco RI fragments below 400 kb were intact colinear clones. About 40% of clones were chimeric. Genetic mapping of end clones from large size YACs revealed that the physical distance corresponding to 1 cM genetic distance varies from 120 to 1000 kb, depending on the chromosome region. To select and order YAC clones for making contig maps, high-density colony hybridization using ECL was applied. With several probes, at least one and at most ten YAC clones could be selected in this library. The library size and clone insert size indicate that this YAC library is suitable for physical map construction and map-based cloning.