Physical Mapping of Rice Chromosomes 8 and 9 with YAC Clones

First efforts for physical mapping of rice chromosomes 8 and9 were carried out by ordering YAC clones of a rice genomicDNA library covering six genome equivalents with mapped DNAmarkers. A total of 79 and 74 markers from chromosomes 8 and9, respectively, were analyzed by YAC colony and Southern hybridizationusing RFLP markers of cDNA and genomic clones, and by polymerasechain reaction (PCR) screening using PCR-derived and sequence-taggedsite (STS) markers. As a result, 252 YAC clones were confirmedto contain the mapped DNA fragments on both chromosomes. A contigmap was constructed by ordering these YAC clones and about 53%and 43% genome coverage was obtained for chromosomes 8 and 9,respectively, assuming a YAC clone size of 350 kb and overlapbetween neighboring YACs of 50%. A continuous array of YAC cloneswith minimum overlap gave a total size of 18.9 Mb for chromosome8 and 15.6 Mb for chromosome 9, which are close to previousestimates. These contig maps may provide valuable informationthat can be useful in understanding chromosome structure andisolating specific genes by map-based cloning.     

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.     

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.     

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.     

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.     

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.     

Comparative genetic maps of foxtail millet (Setaria italica) and rice (Oryza sativa)

 A foxtail millet-rice comparative genetic map was constructed using mapped rice RFLP markers and wheat genomic and cDNA clones with known map position in rice. About 74% and 37% of the cDNA and genomic clones, respectively, were transferable to foxtail millet, confirming that conservation at the DNA level is greatest in genic regions. A high degree of conserved colinearity was observed between the two genomes. Five entire foxtail millet chromosomes appear to be colinear with five entire rice chromosomes. The remaining four foxtail millet linkage groups each show colinearity with segments of two rice chromosomes. The rearrangements of rice chromosomes 3 and 10 to form foxtail millet chromosome IX, and 7 and 9 to form chromosome II are very similar to those required to form maize chromosomes 1 and 7 and sorghum linkage groups C and B, indicating Setaria’s clear taxonomic position within the subfamily of the Panicoideae. Received: 18 December 1996 / Accepted: 4 August 1997     

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.     

A 3-Mb Sequence-Ready Contig Map Encompassing the Multiple Disease Gene Cluster on Chromosome 11q13.1-q13.3

Despite the presence of several human disease genes on chromosome11q13, few of them have been molecularly cloned. Here, we reportthe construction of a contig map encompassing 11q13.1–q13.3using bacteriophage P1 (P1), bacterial artificial chromosome(BAC), and P1-derived artificial chromosome (PAC). The contigmap comprises 32 P1 clones, 27 BAC clones, 6 PAC clones, and1 YAC clone and spans a 3-Mb region from D11S480 to D11S913.The map encompasses all the candidate loci of Bardet-Biedlesyndrome type I (BBS1) and spinocerebellar ataxia type 5 (SCA5),one-third of the distal region for hereditary paraganglioma2 (PGL2), and one-third of the central region for insulin-dependentdiabetes mellitus 4 (IDDM4). In the process of map construction,61 new sequence-tagged site (STS) markers were developed fromthe Not I linking clones and the termini of clone inserts. Wehave also mapped 30 ESTs on this map. This contig map will facilitatethe isolation of polymorphic markers for a more re.ned analysisof the disease gene region and identi.cation of candidate genesby direct cDNA selection, as well as prediction of gene functionfrom sequence information of these bacterial clones.     

Detection and characterization of "chimeric" yeast artificial chromosome clones by fluorescent in situ suppression hybridization.

"Chimeric" yeast artificial chromosomes (YACs) are clones containing two or more noncontiguous segments of DNA and represent the most common artifact found in total genomic YAC libraries currently used for large-scale genome mapping. These YACs create spurious mapping information that complicates the construction of YAC contigs and leads to erroneous maps during chromosome walks. The presence of these artifactual clones necessitates laborious and time-consuming characterization of each isolated YAC clone, either by comparison of the physical map of the YAC with the corresponding source genomic DNA, or by demonstrating discrepant chromosomal origins for the two ends of the YAC by hybridization or polymerase chain reaction (PCR). Here, we describe a rapid and sensitive method for the assessment of YAC colinearity by fluorescence in situ suppression hybridization (FISSH) by utilizing fluorescein-12-dUTP for labeling YAC clones. We have analyzed 51 YACs and found that 43% (22 out of 51) are chimeric and significantly larger (302 kb) than colinear ones (228 kb). One of the 51 YAC clones (2%) examined contains portions of three chromosomes and 2 (4%) seem to map to a chromosome different than that of the identifying STS. FISSH analysis offers a straightforward visualization of the entire YAC insert on the chromosomes and can be used to examine many YACs simultaneously in few days.     

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.     

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.     

野败型水稻细胞质雄性不育恢复基因Rf-4的分子标记定位

为了用分子标记准确定位野败型水稻细胞质雄性不育恢复基因Rf-4,将日本水稻基因组项目（Rice Genome Program,RGP)构建的水稻遗传连锁图谱第10染色体分子遗传图上的分子标记R1877和G2155之间对应区域YAC物理图上的6个YAC克隆进行了亚克隆,获得119个片段,对这些探针进行多态性探查,获得了2个多态分子标记,用珍汕97A和恢复基因近等基因系的杂种F2分离群体中的117完全不育株进行连锁分析表明,从YAC4892获得的亚克隆Y3－8与Rf－4座位的连锁距离为0．9cM,从YAC4630获得的亚克隆Y1－10与Rf－4座位的连锁距离为3．2cM,根据以上结果把Rf－4座位定位于第10染色体的特定位置,为该基因的分子标记辅助选择和定位克隆打下了基础。     

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.     

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/.     

Application of restriction fragment fingerprinting with a rice microsatellite sequence to assembling rice YAC clones.

To refine the current physical map of rice, we have established a restriction fragment fingerprinting method for identifying overlap between pairs of rice yeast artificial chromosome (YAC) clones and defining the physical arrangement of YACs within contiguous fragments (contigs). In this method, Southern blots of rice YAC DNAs digested with a restriction endonuclease are probed with a rice microsatellite probe, (GGC)5. The probe produces a unique fingerprint profile characteristic of each YAC clone. The profile is then digitized, processed in a computer, and a statistic that represents the degree of overlap between two YACs is calculated. The statistics have been used to detect overlaps among YAC clones, thereby filling a gap between two neighbouring contigs and organizing overlapping rice YAC clones into contiguous fragments. We applied this method to rearranging YACs that had previously been assigned to rice chromosome 6 by anchoring with RFLP markers.     

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.