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.     

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 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.     

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 the rice genome with BACs

The development of genetics in this century has been catapulted forward by several milestones: rediscovery of Mendel's laws, determination of DNA as the genetic material, discovery of the double-helix structure of DNA and its implications for genetic behavior, and most recently, analysis of restriction fragment length polymorphisms (RFLPs). Each of these milestones has generated a huge wave of progress in genetics. Consequently, our understanding of organismal genetics now extends from phenotypes to their molecular genetic basis. It is now clear that the next wave of progress in genetics will hinge on genome molecular physical mapping, since a genome physical map will provide an invaluable, readily accessible system for many detailed genetic studies and isolation of many genes of economic or biological importance. Recent development of large-DNA fragment (>100 kb) manipulation and cloning technologies, such as pulsed-field gel electrophoresis (PFGE), and yeast artificial chromosome (YAC) and bacterial artificial chromosome (BAC) cloning, has provided the powerful tools needed to generate molecular physical maps for higher-organism genomes. This chapter will discuss (1) an ideal physical map of plant genome and its applications in plant genetic and biological studies, (2) reviews on physical mapping of the genomes of Caenorhabditis elegans, Arabidopsis thaliana, and man, (3) large-insert DNA libraries: cosmid, YAC and BAC, and genome physical mapping, (4) physical mapping of the rice genome with BACs, and (5) perspectives on the physical mapping of the rice genome with BACs.     

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.     

人Xp11.2区4.3MbYAC重叠群：大尺度限制图与CpG岛分析

人Ｘｐ１１．２区域具有重要的医学遗传学和基础遗传学价值,它包含很多遗传疾病基因,且至少包含一个逃避Ｘ染色体失活的位点,非常规的基化多态也有发现。我们利用这一区域已知的一系列ＤＮＡ位标,从我们构建的ＹＡＣ库中筛选出一系列ＹＡＣ克隆。     

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/.     

Isolation of lambda and YAC clones from defined regions of the rye genome

A dispersed, rye-specific element has been used to isolate clones of rye origin from wheat plants containing only a single rye chromosome arm or segment. In this way a set of 23 YAC clones has been isolated from the short arm of rye chromosome 1 (1RS). This technique was extended to isolate clones from a small region of 1RS that contains a large number of agronomically important genes. The targeted cloning method allowed the isolation of 26 classes of lambda clones representing about 5% of the region. Ten of the lambda clones could be mapped to segments within this region. A third example of the application of this technique involved the isolation of clones from a very small but fully functional rye chromosome, the midget chromosome. These clones have allowed the confirmation of the origin of the midget from 1RL, and may provide a tool for the isolation of structural elements of cereal chromosomes. This technique allows the identification of clone libraries for any rye chromosome or chromosome arm, since substitution, addition and translocation lines are available for all rye chromosomes. Furthermore, the technique allows isolation of clones derived from segments of the rye genome recombined into wheat. The method is technically simple and both lambda and YAC libraries can be constructed. Synteny between the genomes of the cereals allows region-specific libraries from rye to be used to target regions of the wheat and barley genomes. Received: 25 June 1997 / Accepted: 11 November 1997     

Physical mapping of the mitochondrial genome of Arabidopsis thaliana by cosmid and YAC clones

As part of the worldwide efforts at molecular analysis of Arabidopsis thaliana as a model plant the complete structure of the mitochondrial genome has been determined. The mitochondrial DNA molecules were mapped by restriction fragment analysis of more than 300 cosmid clones and purified mitochondrial DNA. The entire genome of 372 kb is contained in three different configurations of circular molecules and is split into two additional subgenomic molecules of 234 kb and 138 kb, respectively. These arrangements result from recombinations of the two sets of repeats present in combinations of inverted and/or direct orientation. Alignment of YAC clones confirms the in vivo presence of continuous DNA molecules of more than 300 kb in A. thaliana mitochondria. The presence of this comparatively large mitochondrial genome in a plant with one of the smallest nuclear genomes shows that different size constraints act upon the different genomes in plant cells.     

Sequence-ready 1-Mb YAC, BAC and cosmid contigs covering the distal imprinted region of mouse chromosome 7.

We have constructed approximately 1-Mb contigs of yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) and cosmid clones covering the imprinted region in mouse chromosome band 7F4/F5. This region is syntenic to human chromosome 11p15.5, which is associated with Beckwith-Wiedemann syndrome (BWS) and certain childhood and adult tumors. These contigs provide the basis for genomic sequencing, identification of genes and their regulatory elements, and functional studies in transgenic and knockout mice, which should be of help to understand not only the mechanisms of imprinting but also the molecular events involved in the genesis of BWS and tumors.     

Rapid identification of polymorphic CA-repeats in YAC clones

Positional cloning of rare disease genes depends on the availability of highly polymorphic markers near the disease loci. The most abundant class of polymorphic markers in the human genome is CA-repeats. We have developed a strategy for the rapid isolation of highly polymorphic CA-repeats from YAC clones. Total DNA of yeast clones containing partly overlapping YACs is digested with frequent cutter restriction enzymes, blotted and hybridized with a poly(CA/GT) probe under high stringency conditions that enable preferential detection of long CA-repeats. The repeats detected in this way are isolated by PCR using vectorette linkers, sequenced, and appropriate flanking markers are constructed for genotyping. All of the CA-repeats identified using this approach were highly polymorphic. This simple and rapid approach should allow the development of highly polymorphic markers at any genomic region cloned in YACs.     

A rapid and accurate strategy for rice contig map construction by combination of fingerprinting and hybridization

A rapid and accurate strategy for rice contig map construction was described. Rice BAC library with average insert of 120 kb in length was used as building materials in contig mapping. The contigs of varied lengths ranging from 500 kb to several megabases with sufficient redundancy to ensure the accuracy of the joining between individual BACs were formed by fingerprinting. The contigs were then assigned to and ordered along the chromosomes by various molecular markers through their hybridization against the whole rice genomic library. The accuracy of clone overlaps in contig was further confirmed by the existence in contigs of well fit stacks of marker-lodged clones. He contigs thus obtained covered nearly the rice genome.     

Physical mapping of unique nucleotide sequences on identified rice chromosomes

A physical mapping method for unique nucleotide sequences on specific chromosomal regions was developed combining objective chromosome identification and highly sensitive fluorescence in situ hybridisation (FISH). Four unique nucleotide sequences cloned from rice genomic DNAs, varying in size from 1.3 to 400 kb, were mapped on a rice chromosome map. A yeast artificial chromosome (YAC) clone with a 399 kb insert of rice genomic DNA was localised at the distal end of the long arm of rice chromosome (1q2.1) and a bacterial artificial chromosome (BAC) clone (180 kb) containing the rice leaf blast-resistant gene (Pi-b) was shown to occur at the distal end of the long arm of chromosome 2 (2q2.1). A cosmid (35 kb) with the resistance gene (Xa-21) against bacterial leaf blight was mapped on the interstitial region of the long arm on chromosome 11 (11q1.3). Furthermore a single RFLP marker, 1.29 kb in size, was mapped successfully to the distal region of the long arm of rice chromosome 4 (4q2.1). For precise localisation of the nucleotide sequences within the chromosome region, image analyses were effective. The BAC clone was localised to the specific region, 2q2.1:96.16, by image analysis. The result was compared with the known location of the BAC clone on the genetic map and the consistency was confirmed. The effectiveness and reliability in physically mapping nucleotide sequences on small plant chromosomes achieved by the FISH method using a variety of probes was unequivocally demonstrated.     

人Xp21．1－p21．3上3．5MbYAC重叠群构建及物理图谱分析

用Ａｌｕ－ＰＣＲ指纹图谱法分析了人Ｘｐ２１．１－ｐ２１．３上一系列的酵母人工染色体（ｙｅａｓｔａｒｔｉｆｉｃｉａｌｃｈｒｏｍｏｓｏｍｅ,ＹＡＣ）克隆,发现其中的两个ＹＡＣ克隆构成包含ＤＸＳ１６６位点的重叠群,而且这一重叠群与以前构建的包含ＤＭＤ基因全序列的ＹＡＣ重叠群相连接,ＹＡＣ克隆末端探针交叉杂交证实了这一重叠,使这一ＹＡＣ重叠群至少延伸至ＤＸＳ１６６位点,形成一个跨度为３．５Ｍｂ的ＹＡＣ重叠群。基于这些重叠的ＹＡＣ克隆绘制了这一区域的大尺度限制酶切图谱,并在这一图谱上定位了ＤＸＳ１６６位点,从而确定了ＤＸＳ１６６位点与ＤＭＤ基因的物理关系。这一工作为ＤＭＤ基因的５＇远端调控作用研究及该区域未知基因的克隆奠定了基础。     

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。     

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/.     

Physical mapping of the Mycoplasma capricolum genome

A physical map of Mycoplasma capricolum ATCC 27343 genome was constructed, based on estimation of the restriction fragment sizes by pulse-field electrophoresis. The linkage order of restriction fragments was determined by two-dimensional electrophoresis of partial and complete single digests and complete double digests and by Southern hybridization analysis. The genome size was established at 1155.5 kb, and 26 cleavage sites for 7 endonucleases were assigned to the map.     

Bacterial genome mapper: A comparative bacterial genome mapping tool

Recently, next generation sequencing (NGS) technologies have led to a revolutionary increase in sequencing speed and costefficacy. Consequently, a vast number of contigs from many recently sequenced bacterial genomes remain to be accurately mapped and annotated, requiring the development of more convenient bioinformatics programs. In this paper, we present a newly developed web-based bioinformatics program, Bacterial Genome Mapper, which is suitable for mapping and annotating contigs that have been assembled from bacterial genome sequence raw data. By constructing a multiple alignment map between target contig sequences and two reference bacterial genome sequences, this program also provides very useful comparative genomics analysis of draft bacterial genomes. AVAILABILITY: The database is available for free at http://mbgm.kribb.re.kr.