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.     

Characterization of three maize bacterial artificial chromosome libraries toward anchoring of the physical map to the genetic map using high-density bacterial artificial chromosome filter hybridization

Three maize (Zea mays) bacterial artificial chromosome (BAC) libraries were constructed from inbred line B73. High-density filter sets from all three libraries, made using different restriction enzymes (HindIII, EcoRI, and MboI, respectively), were evaluated with a set of complex probes including the 185-bp knob repeat, ribosomal DNA, two telomere-associated repeat sequences, four centromere repeats, the mitochondrial genome, a multifragment chloroplast DNA probe, and bacteriophage lambda. The results indicate that the libraries are of high quality with low contamination by organellar and lambda-sequences. The use of libraries from multiple enzymes increased the chance of recovering each region of the genome. Ninety maize restriction fragment-length polymorphism core markers were hybridized to filters of the HindIII library, representing 6x coverage of the genome, to initiate development of a framework for anchoring BAC contigs to the intermated B73 x Mo17 genetic map and to mark the bin boundaries on the physical map. All of the clones used as hybridization probes detected at least three BACs. Twenty-two single-copy number core markers identified an average of 7.4 +/- 3.3 positive clones, consistent with the expectation of six clones. This information is integrated into fingerprinting data generated by the Arizona Genomics Institute to assemble the BAC contigs using fingerprint contig and contributed to the process of physical map construction.     

Construction of a binary BAC library for an apomictic monosomic addition line of<Emphasis Type="Italic"> Beta corolliflora</Emphasis> in sugar beet and identification of the clones derived from the alien chromosome

A plant-transformation-competent binary BAC library was constructed from the genomic DNA of the chromosome 9 monosomic addition line of Beta corolliflora Zoss. in sugar beet (B. vulgaris. L). This monosomic addition line (designated M14) is characterized by diplosporic reproduction caused by the alien chromosome carrying the gene(s) responsible for diplospory. The library consists of 49,920 clones with an average insert size of 127 kb, representing approximately 7.5 haploid genome equivalents and providing a greater than 99% probability of isolating a single-copy DNA sequence from the library. To develop the scaffold of a physical map for the alien chromosome, B. corolliflora genome-specific dispersed repetitive DNA sequences were used as probes to isolate BAC clones derived from the alien chromosome in the library. A total of 2,365 positive clones were obtained and arrayed into a sublibrary specific for B. corolliflora chromosome 9 (designated bcBAC-IX). The bcBAC-IX sublibrary was further screened with a subtractive cDNA pool generated from the ovules of M14 and the floral buds of B. vulgaris by the suppression subtractive hybridization method. One hundred and three positive binary BACs were obtained, which potentially contain the genes of the alien chromosome specifically expressed during the ovule and embryo development of M14, and may be associated with apomictic reproduction. Thus, these binary BAC clones will be useful for identification of the genes for apomixis by genetic transformation.Communicated by H. C. Becker     

BAC-derived diagnostic markers for sex determination in asparagus

A HindIII BAC (bacterial artificial chromosome) library of asparagus (Asparagus officinalis L.) was established from a single male plant homozygous for the male flowering gene (MM). The library represents approximately 5.5 haploid genome equivalents with an average insert size of 82 kb. A subset of the library (2.6 haploid genome equivalents) was arranged into DNA pools. Using nine sex-linked amplified fragment length polymorphism (AFLP) and two sequence-tagged site (STS) markers, 13 different BAC clones were identified from this part of the library. The BACs were arranged into a first-generation physical map around the sex locus. Four PCR-derived markers were developed from the BAC ends, one of which could be scored in a co-dominant way. Using a mapping population of 802 plants we mapped the BAC-derived markers to the same position close to the M gene as the corresponding AFLP and STS markers. The markers are useful for further chromosome walking studies and as diagnostic markers for selecting male plants homozygous for the M gene.Communicated by G. Wenzel     

Use of the IGF BAC library for physical mapping of the Arabidopsis thaliana genome

In order to generate a physical map of the Arabidopsis thaliana genome based on bacterial artificial chromosome clones (BACs), an iterative high throughput hybridisation strategy was applied and its efficiency was evaluated. Thus, probes generated from both ends of 500 BAC clones selected from the Arabidopsis –IGF–BAC library were hybridised to the entire library gridded on high density filters. The 1000 hybridisation reactions identified 4496 clones (41.8% of the complete library, or 50.3% if organellar, centromeric, and ribosomal DNA carrying clones are excluded) which were assembled into a minimum of 220 contigs. These results demonstrate the viability of the applied ‘double-end clone-limited/sampling without replacement’ hybridisation strategy for the generation of a high resolution physical map, and provide a highly useful resource for map-based gene cloning approaches and further genome analysis.     

Construction and characterization of a bacterial artificial chromosome library of thermo-sensitive genie male-sterile rice 5460S

In order to develop a detailed physical map of the thermo-sensitive genie male-sterile (TGMS) gene-encompassing region and finally clone the TGMS gene, a high-quality rice bacterial artificial chromosome (BAC) library from TGMS rice 5460S was constructed. The method of constructing BAC library was examined and optimized. The 5460S library consists of 19 584 BAC clones with an average insert size of 110 kb, which represents about 5 times rice haploid genome equivalents. Rice inserts of up to 140 kb and 250 kb were isolated and appeared stable after 100 generations of serial growth. Hybridization of BAC clones with mitochondrial and chloroplastic genes as probes demonstrated that this library has no organellar contamination. The 5460S library was screened with 3 molecular markers linked to tmsl gene as probes and at least 1 BAC clone was identified with each probe. The insert ends of positive clones were successfully isolated using thermal asymmetric interlaced PCR (TAIL-PCR) technique.     

Construction and characterization of a papaya BAC library as a foundation for molecular dissection of a tree-fruit genome

A bacterial artificial chromosome (BAC) library was constructed from high-molecular-weight DNA isolated from young leaves of papaya (Carica papaya L.). This BAC library consists of 39168 clones from two separate ligation reactions. The average insert size of the library is 132 kb; 96.5% of the 18700 clones from the first ligation contained inserts that averaged 86 kb in size, 95.7% of the 20468 clones from the second ligation contained inserts that averaged 174 kb in size. Two sorghum chloroplast probes hybridized separately to the library and revealed a total of 504 chloroplast clones or 1.4% of the library. The entire BAC library was estimated to provide 13.7× papaya-genome equivalents, excluding the false-positive and chloroplast clones. High-density filters were made containing 94% or 36864 clones of the library with 12.7× papaya-genome equivalents. Eleven papaya-cDNA and ten Arabidopsis-cDNA probes detected an average of 22.8 BACs per probe in the library. Because of its relatively small genome (372 Mbp/1 C) and its ability to produce ripe fruit 9 to 15 months after planting, papaya shows promise as a model plant for studying genes that affect fruiting characters. A rapid approach to locating fruit-controlling genes will be to assemble a physical map based on BAC contigs to which ESTs have hybridized. A physical map of the papaya genome will significantly enhance our capacity to clone and manipulate genes of economic importance. Received: 11 April 2000 / Accepted: 28 July 2000     

Construction of a 1.5-Mb Bacterial Artificial Chromosome Contig in Xp11.23, a Region of High Gene Content

To generate sequence-ready templates for the gene-rich Xp11.23 region, we have constructed a 1.5-Mb bacterial artificial chromosome (BAC) contig spanning the interval between the DNA markers OATL1 and DXS255. The contig includes 28 BACs, ranging in size from 58 to 285 kb with an average size of 135 kb, which provide 2.5-fold coverage of the region. The BAC contig was constructed based entirely on the content of 40 DNA markers from a previously established YAC contig and 11 new markers developed from BAC-end DNA sequences, 4 of which were required to close gaps in the map. There was no evidence of rearrangement, instability, or chimerism in any of the BAC clones. The BAC cloning system appears to provide robust and total physical coverage of this gene-rich region with clones that are suitable for DNA sequencing.     

Construction and characterization of a half million clone BAC library of durum wheat (<Emphasis Type="Italic">Triticum turgidum</Emphasis> ssp. <Emphasis Type="Italic">durum</Emphasis>)

Durum wheat (Triticum turgidum ssp. durum, 2n = 4x = 28, genomes AB) is an economically important cereal used as the raw material to make pasta and semolina. In this paper we present the construction and characterization of a bacterial artificial chromosome (BAC) library of tetraploid durum wheat cv. Langdon. This variety was selected because of the availability of substitution lines that facilitate the assignment of BACs to the A and B genome. The selected Langdon line has a 30-cM segment of chromosome 6BS from T. turgidum ssp. dicoccoides carrying a gene for high grain protein content, the target of a positional cloning effort in our laboratory. A total of 516,096 clones were organized in 1,344 384-well plates and blotted on 28 high-density filters. Ninety-eight percent of these clones had wheat DNA inserts (0.3% chloroplast DNA, 1.4% empty clones and 0.3% empty wells). The average insert size of 500 randomly selected BAC clones was 131 kb, resulting in a coverage of 5.1-fold genome equivalents for each of the two genomes, and a 99.4% probability of recovering any gene from each of the two genomes of durum wheat. Six known copy-number probes were used to validate this theoretical coverage and gave an estimated coverage of 5.8-fold genome equivalents. Screening of the library with 11 probes related to grain storage proteins and starch biosynthesis showed that the library contains several clones for each of these genes, confirming the value of the library in characterizing the organization of these important gene families. In addition, characterization of fingerprints from colinear BACs from the A and B genomes showed a large differentiation between the A and B genomes. This library will be a useful tool for evolutionary studies in one of the best characterized polyploid systems and a source of valuable genes for wheat. Clones and high-density filters can be requested at Communicated by P. LangridgeThe first two authors contributed equally to the investigation     

Fine physical mapping of the rice stripe resistance gene locus, Stvb-i

The Stvb-i gene confers stripe disease resistance to rice. For positional cloning, we constructed a physical map spanning 1.8-cM distance between flanking markers, consisting of 18 bacterial artificial chromosome (BAC) clones, around the Stvb-i locus on rice chromosome 11. The 18 clones were isolated by screening a BAC library derived from a japonica cultivar, Shimokita, with three Stvb-i-linked RFLP markers and DraI-digested DNAs of a yeast artificial chromosome (YAC) clone. The results of Southern hybridization and restriction enzyme analyses indicated that these BAC clones are contiguous and cover about a 700-kb region containing the Stvb-i allele. Utilizing end and internal fragments of the BAC insert DNAs, 33 molecular markers were generated within a small chromosomal region including the Stvb-i locus. Genotyping analysis with these markers for a resistant cultivar and four nearby recombinants selected from 120 F₂ individuals indicated that Stvb-i is contained within an approximately 286-kb region covered with two overlapping BAC clones. Received: 25 August 1999 / Accepted: 16 November 1999     

A bacterial artificial chromosome library for the Chinese alligator (Alligator sinensis)

Chinese alligator (Alligator sinensis) is a rare and endangered species endemic to China. To better understand genetic details of the Chinese alligator genomic structure, a highly redundant bacterial artificial chromosome (BAC) library was constructed. This library consists of 216,238 clones with an average insert size of about 90kb, indicating that the library contains 6.8-fold genome equivalents. Subsequently, we constructed a 516kb contig map for the Chinese alligator olfactory receptor (OR) genes, which spans nine BAC clones, and subjected the BACs to full sequencing. The sequence analysis revealed that this contig contained 16 OR functional genes and meanwhile demonstrated that the nine BACs, which constituted the contig, overlapped correctly, proving the usability of this genome library. As a result, this BAC library could provide a useful platform for physical mapping, genome sequencing or complex analysis of targeted genomic regions for this rare species.     

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 Sorghum propinquum BAC library,suitable for cloning genes associated with loss-of-function mutations during crop domestication

A large insert Sorghum propinquum BAC library has been constructed to analyze the physical organization of the sorghum genome and to facilitate positional cloning of sorghum genes and QTLs associated with the early stages of grain crop domestication. This library was established from 12 different ligations using high-molecular-weight DNA generated from either one cycle or two cycles of size selection. This library consists of 38 016 BAC clones with an estimated average insert size of 126 kb and coverage of 6.6 genome equivalents. The 6.6 genome-equivalent BAC library of S. propinquum provides a 99.7% probability of finding one or more BACs that contain genes of interest. Twenty mapped DNA probes, ten homologous and ten heterologous, were used to screen the library, and 121 positive clones were identified, 6.05 per locus or 6.37 per probe.     

A physical map of the 20q12-q13.1 region associated with type 2 diabetes

Several recent genetic studies have suggested linkage of Type 2 diabetes (non-insulin-dependent diabetes mellitus) susceptibility to a region of chromosome 20q12-q13.1. To facilitate the identification and cloning of a diabetes susceptibility gene(s) in this region, we have constructed correlated radiation hybrid and YAC/BAC contig physical maps of the region. A high-resolution radiation hybrid map encompassing 9.5 Mb between the PLC and the CEBPB genes was constructed using 68 markers: 25 polymorphic markers, 15 known genes, 21 ESTs, and 7 random genomic sequences. The physical order of the polymorphic markers within this radiation hybrid map is consistent with published genetic maps. A YAC/BAC contig that gives continuous coverage between PLC and CEBPB was also constructed. This contig was constructed from 24 YACs, 34 BACs, and 1 P1 phage clone onto which 71 markers were mapped: 23 polymorphic markers, 12 genes, 24 ESTs, and 12 random genomic sequences. The radiation hybrid map and YAC/BAC physical map enable precise mapping of newly identified transcribed sequences and polymorphic markers that will aid in linkage and linkage disequilibrium studies and facilitate identification and cloning of candidate Type 2 diabetes susceptibility genes residing in 20q12-q13.1.     

Strategy to sequence the genome of Corynebacterium glutamicum ATCC 13032: use of a cosmid and a bacterial artificial chromosome library

The initial strategy of the Corynebacterium glutamicum genome project was to sequence overlapping inserts of an ordered cosmid library. High-density colony grids of approximately 28 genome equivalents were used for the identification of overlapping clones by Southern hybridization. Altogether 18 contiguous genomic segments comprising 95 overlapping cosmids were assembled. Systematic shotgun sequencing of the assembled cosmid set revealed that only 2.84 Mb (86.6%) of the C. glutamicum genome were represented by the cosmid library. To obtain a complete genome coverage, a bacterial artificial chromosome (BAC) library of the C. glutamicum chromosome was constructed in pBeloBAC11 and used for genome mapping. The BAC library consists of 3168 BACs and represents a theoretical 63-fold coverage of the C. glutamicum genome (3.28 Mb). Southern screening of 2304 BAC clones with PCR-amplified chromosomal markers and subsequent insert terminal sequencing allowed the identification of 119 BACs covering the entire chromosome of C. glutamicum. The minimal set representing a 100% genome coverage contains 44 unique BAC clones with an average overlap of 22 kb. A total of 21 BACs represented linking clones between previously sequenced cosmid contigs and provided a valuable tool for completing the genome sequence of C. glutamicum.     

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 clone contig of 12q24.3 encompassing the distal hereditary motor neuropathy type II gene

We previously assigned the disease locus for autosomal dominant hereditary motor neuropathy type II (distal HMN II) within a 13-cM interval at chromosome 12q24.3. We constructed a physical map of the distal HMN II region based on yeast artificial chromosomes (YACs), P1 artificial chromosomes (PACs), and bacterial artificial chromosomes (BACs) using an STS content mapping approach. The contig contains 26 YAC, 15 PAC, and 60 BAC clones and covers a physical distance of approximately 5 Mb. A total of 99 STS markers, including 25 known STSs and STRs, 49 new STSs generated from clone end-fragments, 20 ESTs, and 5 known genes, were located on the contig. This physical map provides a valuable resource for mapping genes and markers located within the distal HMN II region and facilitates the positional cloning of the distal HMN II gene.     

Characterization of a new BAC library for rainbow trout: evidence for multi-locus duplication

A 10X rainbow trout bacterial artificial chromosome (BAC) library was constructed to aid in the physical and genetic mapping efforts of the rainbow trout genome. The library was derived from the Swanson clonal line (YY male) and consists of 184,704 clones with an average insert size of 137,500 bp (PFGE) or 118,700 bp (DNA fingerprinting). The clones were gridded onto 10 large nylon membranes to produce high-density arrays for screening the library by hybridization. The library was probed with 11 cDNAs from the NCCCWA EST project chosen because of interest in their homology to known gene sequences, seven known genes, and a Y-specific sex marker. Putative positive clones identified by hybridization were re-arrayed and gridded for secondary confirmation. FPC analysis of HindIII and EcoRV DNA fingerprinting was used to estimate the level of redundancy in the library, to construct BAC contigs and to detect duplicated loci in the semi-duplicated rainbow trout genome. A good correlation (R2 = 0.7) was found between the number of hits per probe and the number of contigs that were assembled from the positive BACs. The average number of BACs per contig was 9.6, which is in good agreement with 10X genome coverage of the library. Two-thirds of the loci screened were predicted to be duplicated as the positive BACs for those genes were assembled into two or three different contigs, which suggests that most of the rainbow trout genome is duplicated.     

Physical mapping across an avirulence locus of Phytophthora infestans using a highly representative, large-insert bacterial artificial chromosome library

The oomycete plant pathogen Phytophthora infestans is the causal agent of late blight, one of the most devastating diseases of potato worldwide. As part of efforts to clone avirulence (Avr) genes and pathogenicity factors from P. infestans, we have constructed a bacterial artificial chromosome (BAC) library from an isolate containing six Avr genes. The BAC library comprises clones with an average insert size of 98 kb and represents an estimated 10 genome equivalents. A three-dimensional pooling strategy was developed to screen the BAC library for amplified fragment length polymorphism (AFLP) markers, as this type of marker has been extensively used in construction of a P. infestans genetic map. Multiple positive clones were identified for each AFLP marker tested. The pools were used to construct a contig of 11 BAC clones in a region of the P. infestans genome containing a cluster of three avirulence genes. The BAC contig is predicted to encompass the Avr11 locus but mapping of the BAC ends will be required to determine if the Avr3 and Avr10 loci are also present in the BAC contig. These results are an important step towards the positional cloning of avirulence genes from P. infestans, and the BAC library represents a valuable resource for largescale studies of oomycete genome organisation and gene content.     

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/].