Ordered YAC Clone Contigs Assigned to Rice Chromosomes 3 and 11

Yeast artificial chromosome (YAC) clones were arranged on thepositions of restriction fragment length polymorphism (RFLP)and sequence-tagged site (STS) markers already mapped on thehigh-resolution genetic maps of rice chromosomes 3 and 11. Froma total of 416 and 242 YAC clones selected by colony/Southernhybridization and polymerase chain reaction (PCR) analysis,238 and 135 YAC clones were located on chromosomes 3 and 11,respectively. For chromosomes 3 and 11, 24 YAC contigs and islandswith total coverage of about 46% and 12 contigs and islandswith coverage of about 40%, respectively, were assigned. Althoughmany DNA fragments of multiple copy marker sequences could notbe mapped to their original locations on the genetic map bySouthern hybridization because of a lack of RFLP, the physicalmapping of YAC clones could often assign specific locationsof such multiple copy sequences on the genome. The informationprovided here on contig formation and similar sequence distributionrevealed by ordering YAC clones will help to unravel the genomeorganization of rice as well as being useful in isolation ofgenes by map-based cloning.     

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.     

Physical Mapping of Rice Chromosomes 4 and 7 Using YAC Clones

Physical maps of rice chromosomes 4 and 7 were constructed bylanding yeast artificial chromosomes (YACs) along our high-densitymolecular linkage map. Using 114 DNA markers, 258 individualYACs were located on chromosome 4. Sixty-two out of 258 YACscarried two or more DNA marker positions and formed 16 contigswhich covered a total length of 17.1 cM. The other YACs werearranged to 23 positions. On chromosome 7, 203 individual YACswere landed on 109 DNA markers. Sixty-four out of 203 YACs formed15 contigs which covered a total length of 21.8 cM and 139 YACswere localized to 26 positions. Chromosomes 4 and 7 were coveredwith minimum tiling paths of 45 and 48 YACs, respectively. Takingthe average size of YAC insert DNA to be 350 kb and the entiregenome size to be 430 Mb, about 16–18 Mb of each chromosomeor an estimated 50% of their total lengths have been coveredwith YACs. Physical maps of these 2 chromosomes should be ofgreat help in identifying useful trait genes and unravelinggenetic and biological characteristics in rice.     

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.     

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.     

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.     

A physical map with yeast artificial chromosome (YAC) clones covering 63% of the 12 rice chromosomes.

A new YAC (yeast artificial chromosome) physical map of the 12 rice chromosomes was constructed utilizing the latest molecular linkage map. The 1439 DNA markers on the rice genetic map selected a total of 1892 YACs from a YAC library. A total of 675 distinct YACs were assigned to specific chromosomal locations. In all chromosomes, 297 YAC contigs and 142 YAC islands were formed. The total physical length of these contigs and islands was estimated to 270 Mb which corresponds to approximately 63% of the entire rice genome (430 Mb). Because the physical length of each YAC contig has been measured, we could then estimate the physical distance between genetic markers more precisely than previously. In the course of constructing the new physical map, the DNA markers mapped at 0.0-cM intervals were ordered accurately and the presence of potentially duplicated regions among the chromosomes was detected. The physical map combined with the genetic map will form the basis for elucidation of the rice genome structure, map-based cloning of agronomically important genes, and genome sequencing.     

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/.     

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.     

Mapping of quantitative trait locus related to submergence tolerance in rice with aid of chromosome walking.

The major QTL for submergence tolerance was locate in the 5.9 cM interval between flanking RFLP markers. To narrow down this region, a physical map was constructed using YAC and BAC clones. A 400-kb YAC was identified in this region and later its end fragments were used to screen a rice BAC library. Through chromosome walking, 24 positive BAC clones formed two contigs around linked-RFLP markers, R1164 and RZ698. Using one YAC end, six BAC ends and three RFLP markers, a fine-scale map was constructed of the 6.8-cM interval of S10709-RZ698 on rice chromosome 9. The submergence tolerance and related trait were located in a small, well-defined region around BAC-end marker 180D1R and RFLP marker R1164. The physical-to-map distance ratio in this region is as small as 172.5 kb/cM, showing that this region is a hot spot for recombination in the rice genome.     

Physical map-based sizes of the centromeric regions of Arabidopsis thaliana chromosomes 1, 2, and 3.

The sizes of the centromeric regions of Arabidopsis thaliana chromosomes 1, 2, and 3 were determined by construction of their physical maps on the basis of restriction analysis. As the reported centromeric regions contain large gaps in the middle due to highly repetitive sequences, appropriate probes for Southern hybridization were prepared from the sequences reported for the flanking regions and from the sequences of BAC and YAC clones newly isolated in this work, and restriction analysis was performed using DNA of a hypomethylated strain (ddm1). The sizes of the genetically defined centromeric regions were deduced to be 9 megabases (Mb), 4.2 Mb and 4.1 Mb, respectively (chromosome 1, from markers T22C23-t7 to T3P8-sp6; chromosome 2, from F5J15-sp6 to T15D9; chromosome 3, from T9G9-sp6 to T15M14; G. P. Copenhaver et al. Science, 286, 2468-2479, 1999). By combining the sizes of the centromeric regions previously estimated for chromosomes 4 and 5 and the sequence data reported for the A. thaliana genome, the total genome size of A. thaliana was estimated to be approximately 146.0 Mb.     

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.     

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/.     

Towards map-based cloning of the barley stem rust resistance genes Rpg1 and rpg4 using rice as an intergenomic cloning vehicle

The barley stem rust resistance genes Rpg1 and rpg4 were mapped in barley on chromosomes 1P and 7M, respectively and the syntenous rice chromosomes identified as 6P and 3P by mapping common probes in barley and rice. Rice yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) and cosmid clones were used to isolate probes mapping to the barley Rpg1 region. The rice BAC isolated with the pM13 probe was a particularly excellent source of probes. A high-resolution map of the Rpg1 region was established with 1400 gametes yielding a map density of 3.6 markers per 0.1 cM. A detailed physical map was established for the rice BAC fragment containing the Rpg1-flanking markers pM13 and B24. This fragment covers a barley genetic distance of 0.6 cM and a rice DNA physical distance of ca. 70 kb. The distribution of barley cross-overs in relation to the rice DNA physical distances was extremely uneven. The barley genetic distance between the pM13 marker and Rpg1 was 0.1 cM per ca. 55 kb, while on the proximal side it was 0.5 cm per ca. 15 kb. Three probes from the distal end of the pM13 BAC mapped 3.0 cm proximal of Rpg1 and out of synteny with rice. These experiments confirm the validity of using large insert rice clones as probe sources to saturate small barley (and other large genome cereals) genome regions with markers. They also establish a note of caution that even in regions of high microsynteny, there may be small DNA fragments that have transposed and are no longer in syntenous positions.     

Isolation and FISH mapping of Yeast Artificial Chromosomes (YACs) encompassing an allele of the Gm2 gene for gall midge resistance in rice

 Ten yeast artificial chromosomes (YACs) spanning the Gm2 locus have been isolated by screening high-density filters containing a total of approximately 7000 YAC (representing six genome equivalents) clones derived from a japonica rice, Nipponbare. The screening was done with five RFLP markers flanking a gall midge resistance gene, Gm2, which was previously mapped onto chromosome 4 of rice. This gene confers resistance to biotype 1 and 2 of gall midge (Orseolia oryzae), a major insect pest of rice in South and Southeast Asia. The RFLP markers RG214, RG329 and F8 hybridized with YAC Y2165. Two overlapping YAC clones (Y5212 and Y2165) were identified by Southern hybridization, with Gm2-flanking RFLP markers, and their inserts isolated. The purified YACs and RFLP markers flanking Gm2 were labeled and physically mapped by the fluorescence in situ hybridization (FISH) technique. All of them mapped to the long arm of chromosome 4 of the resistant variety of rice, ‘Phalguna’, confirming the previous RFLP mapping data. Received: 15 December 1997 / Accepted: 5 March 1998     

Construction of a potato YAC library and identification of clones linked to the disease resistance loci R1 and Gro1

 A yeast artificial chromosome (YAC) library was constructed from high-molecular-weight DNA of potato (Solanum tuberosum). Potato DNA fragments obtained after complete digestion with four different rare-cutter restriction enzymes were cloned using the pYAC-RC vector. The library consists of 21 408 YAC clones with an average insertion size of 140 kb. The frequency of YAC clones having insertions of chloroplast or mitochondrial DNA was estimated to be 0.5% and 0.3%, respectively. The YAC library was screened by PCR with 11 DNA markers detecting single genes or small gene families in the potato genome. YACs for 8 of the 11 markers were detected in the library. Using 2 markers that are linked to the resistance genes R1 and Gro1 of potato, we isolated two individual YAC clones. One of these YAC clones was found to harbour one member of a small family of candidate genes for the nematode resistance gene Gro1. Received : 5 May 1997 / Accepted : 20 May 1997     

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/].     

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.     

通过染色体步查建立水稻G200座位的重叠群

分别以G200及其旁侧区的Y2587L、Y4073L和L688片段为探针筛选水稻基因组YAC文库,共得到4个阳性克隆,均与G200、Y2587L和Y4073L座位重叠,插入片段的大小为240～650kb。用反向PCR法分离YAC克隆插入片段的末端序列,并利用这些末端序列确定克隆的方向以及进行染色体步查,共筛选到7个YAC克隆,建立了一个8厘摩(cM)的G200 YAC重叠群。