Identification of the sex-determining locus of Atlantic salmon (Salmo salar) on chromosome 2

We have integrated data from linkage mapping, physical mapping and karyotyping to gain a better understanding of the sex-determining locus, SEX, in Atlantic salmon (Salmo salar). SEX has been mapped to Atlantic salmon linkage group 1 (ASL1) and is associated with several microsatellite markers. We have used probes designed from the flanking regions of these sex-linked microsatellite markers to screen a bacterial artificial chromosome (BAC) library, representing an 11.7x coverage of the Atlantic salmon genome, which has been HindIII fingerprinted and assembled into contigs. BACs containing sex-linked microsatellites and their related contigs have been identified and representative BACs have been placed on the Atlantic salmon chromosomes by fluorescent in situ hybridization (FISH). This identified chromosome 2, a large metacentric, as the sex chromosome. By positioning several BACs on this chromosome by FISH, it was possible to orient ASL1 with respect to chromosome 2. The region containing SEX appears to lie on the long arm between marker Ssa202DU and a region of heterochromatin identified by DAPI staining. BAC end-sequencing of clones within sex-linked contigs revealed five hitherto unmapped genes along the sex chromosome. We are using an in silico approach coupled with physical probing of the BAC library to extend the BAC contigs to provide a physical map of ASL1, with a view to sequencing chromosome 2 and, in the process, identifying the sex-determining gene.     

TOPAAS, a tomato and potato assembly assistance system for selection and finishing of bacterial artificial chromosomes

We have developed the software package Tomato and Potato Assembly Assistance System (TOPAAS), which automates the assembly and scaffolding of contig sequences for low-coverage sequencing projects. The order of contigs predicted by TOPAAS is based on read pair information; alignments between genomic, expressed sequence tags, and bacterial artificial chromosome (BAC) end sequences; and annotated genes. The contig scaffold is used by TOPAAS for automated design of nonredundant sequence gap-flanking PCR primers. We show that TOPAAS builds reliable scaffolds for tomato (Solanum lycopersicum) and potato (Solanum tuberosum) BAC contigs that were assembled from shotgun sequences covering the target at 6- to 8-fold coverage. More than 90% of the gaps are closed by sequence PCR, based on the predicted ordering information. TOPAAS also assists the selection of large genomic insert clones from BAC libraries for walking. For this, tomato BACs are screened by automated BLAST analysis and in parallel, high-density nonselective amplified fragment length polymorphism fingerprinting is used for constructing a high-resolution BAC physical map. BLAST and amplified fragment length polymorphism analysis are then used together to determine the precise overlap. Assembly onto the seed BAC consensus confirms the BACs are properly selected for having an extremely short overlap and largest extending insert. This method will be particularly applicable where related or syntenic genomes are sequenced, as shown here for the Solanaceae, and potentially useful for the monocots Brassicaceae and Leguminosea.     

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.     

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.     

A physical map of the genome of Atlantic salmon, Salmo salar

A physical map of the Atlantic salmon (Salmo salar) genome was generated based on HindIII fingerprints of a publicly available BAC (bacterial artificial chromosome) library constructed from DNA isolated from a Norwegian male. Approximately 11.5 haploid genome equivalents (185,938 clones) were successfully fingerprinted. Contigs were first assembled via FPC using high-stringency (1e-16), and then end-to-end joins yielded 4354 contigs and 37,285 singletons. The accuracy of the contig assembly was verified by hybridization and PCR analysis using genetic markers. A subset of the BACs in the library contained few or no HindIII recognition sites in their insert DNA. BglI digestion fragment patterns of these BACs allowed us to identify three classes: (1) BACs containing histone genes, (2) BACs containing rDNA-repeating units, and (3) those that do not have BglI recognition sites. End-sequence analysis of selected BACs representing these three classes confirmed the identification of the first two classes and suggested that the third class contained highly repetitive DNA corresponding to tRNAs and related sequences.     

Five-color-based high-information-content fingerprinting of bacterial artificial chromosome clones using type IIS restriction endonucleases

We have developed a high-information-content fingerprinting (HICF) system for bacterial artificial chromosome (BAC) clones using a Type IIS restriction endonuclease, HgaI, paired with a Type II restriction endonuclease, RsaI. In the method described, unknown five-base overhangs generated with HgaI are partially or fully sequenced by modified fluorescent dideoxy terminators. Using an in-lane size standard labeled with a fifth dye, fragments are characterized by both the size and the sequence of its terminal one to five bases. The enhanced information content associated with this approach significantly increases the accuracy and efficiency of detecting shared fragments among BAC clones. We have compared data obtained from this method to predicted HICF patterns of 10 fully sequenced BACs. We have further applied HICF to 555 BAC clones to assemble contigs spanning 16p11.2 to 16p13.1 of human chromosome 16.     

Physical mapping of resistant and susceptible soybean genomes near the soybean cyst nematode resistance gene Rhg4.

The soybean cyst nematode (SCN), Heterodera glycines Ichinohe, is the foremost pest of soybean (Glycine max L. Merr.). The rhg1 allele on linkage group (LG) G and the Rhg4 allele on LG A2 are important in conditioning resistance. Markers closely linked to the Rhg4 locus were used previously to screen a library of bacterial artificial chromosome (BAC) clones from susceptible 'Williams 82' and identified a single 150-kb BAC, Gm_ISb001_056_G02 (56G2). End-sequenced subclones positioned onto a restriction map provided landmarks for identifying the corresponding region from a BAC library from accession PI 437654 with broad resistance to SCN. Seventy-three PI 437654 BACs were assigned to contigs based upon HindIII restriction fragment profiles. Four contigs represented the PI 437654 counterpart of the 'Williams 82' BAC, with PCR assays connecting these contigs. Some of the markers on the PI 437654 contigs are separated by a greater physical distance than in the 'Williams 82' BAC and some primers amplify bands from BACs in the mid-portion of the connected PI 437654 BAC contigs that are not amplified from the 'Williams 82' BAC. These observations suggest that there is an insertion in the PI 437654 genome relative to the 'Williams 82' genome in the Rhg4 region.     

PCR筛选BAC文库和直接BAC末端测序方法的建立

建立了一种用PCR方法筛选富含高度重复序列的大麦BAC DNA 文库和直接对 BAC DNA进行末端测序的方法.用PCR技术进行大麦BAC DNA 文库(含816个平板,每个平板含384个克隆)的筛选分4步进行.在实验中,建立了两个水平的BAC DNA池(一级池和二级池).一个二级池由一个平板(含有384个克隆)的DNA 组成,一个一级池由连续10个稀释100倍的二级池的DNA混合而成(如1～10,11～20等),共82个一级池.BAC DNA 文库筛选的第一步是对82个一级池的筛选.得到阳性一级池后(如2号一级池),对其所含的10个二级池(从11～20)进行第二步筛选.得到阳性二级池后,培养相应的阳性平板的所有克隆(384个),从头开始(左上侧),每相邻的4个克隆为一组,在96孔板上(4 X 96=384) 进行第三轮PCR反应;之后对筛选结果为阳性的4个克隆分别进行菌落 PCR(第四轮)得到单一阳性克隆.根据BAC DNA Hind III 酶切指纹图谱,对同一引物筛选的BACs进行重叠群作图(Contig).对代表contig 的两端的BAC DNA直接进行末端测序并对测序结果Blast,以检测其在大麦中是否属于单拷贝序列.根据测序和Blast结果设计引物,用中国春附加系(附加大麦染色体)对来自BAC克隆的引物进行染色体定位并用分离群体进行遗传学作图,以确定是否可以用作下一步的染色体步行.     

A physical map of large segments of pig Chromosome 7q11–q14: comparative analysis with human Chromosome 6p21

The aim of this study was to establish a porcine physical map along the chromosome SSC7q by construction of BAC contigs between microsatellites Sw1409 and S0102. The SLA class II contig, located on SSC7q, was lengthened. Four major BAC contigs and 10 short contigs span a region equivalent to 800 cR measured by IMpRH7000 mapping. The BAC contigs were initiated by PCR screening with primers derived from human orthologous segments, extended by chromosome walking, and controlled and oriented by RH mapping with the two available panels, IMpRH7000Rad and IMNpRH12000Rad. The location of 43 genes was revealed by sequenced segments, either from BAC ends or PCR products from BAC clones. The 220 BAC end sequences (BES) were also used to analyze the different marks of evolution. Comparative mapping analysis between pigs and humans demonstrated that the gene organization on HSA6p21 and on SSC7p11 and q11-q14 segments was conserved during evolution, with the exception of long fragments of HSA6p12 which shuffled and spliced the SLA extended class II region. Additional punctual variations (unique gene insertion/deletion) were observed, even within conserved segments, revealing the evolutionary complexity of this region. In addition, 18 new polymorphic microsatellites have been selected in order to cover the entire SSC7p11-q14 region.     

Development of simple sequence repeat markers from bacterial artificial chromosomes without subcloning

Simple sequence repeats (SSRs) were isolated from pearl millet bacterial artificial clones (BACs) without any subcloning steps. SSR sequences were targeted using 3' end-anchored SSR primers. Flanking sequences were isolated by suppression PCR. In this pilot study, 25 SSR markers have been developed from 40 BAC pools, comprising a total of 384 clones. This novel way to develop new markers has the added advantage that mapping the SSR markers will anchor individual BACs to the genetic maps and, thus, facilitate the construction of BAC contigs.     

An integrated gene and SSLP BAC map framework of mouse chromosome 11

Physical maps are important resources both in sequencing and in functional analyses of large genomes. Global contig-building approaches are regarded to be more efficient relative to the cumulative outcome of scattered and more localized physical mapping studies accompanying positional cloning. This work is part of an effort to assemble a complete physical map of mouse chromosome 11 in which selection of clones containing specific genetic markers from genomic libraries is the first step in the process. Using a previously developed strategy, we identified 361 bacterial artificial chromosomes (BACs) containing 88 gene markers. Since the linkage positions of markers chosen for these studies are known, the BAC framework obtained is anchored to the genetic map and represents about 13% of the length of the entire chromosome. Together with similar assignments of BACs generated previously using D11Mit markers (Cai et al., 1988, Genomics, 54: 387-397), 36-40% of the chromosome 11 is now assembled into contigs, and these contigs correlate through 51 clones carrying both gene and simple sequence length polymorphism markers.     

Insertion of modifications in the beta-globin locus using GET recombination with single-stranded oligonucleotides and denatured PCR fragments

We describe the use of the GET recombination system with oligonucleotides or single-stranded polymerase chain reaction (PCR) fragments to insert modifications in the human beta-globin locus without counterselection. The method involves recombination between oligonucleotides or denatured PCR fragments and homologous sequences in the beta-globin gene in a clone of 205-kb bacterial artificial chromosome (BAC), based on the inducible expression of the recE, recT, and gam genes. In this method, oligonucleotides or denatured PCR fragments are electroporated directly into cells carrying both the globin BAC and the pGETrec plasmid, after induction of the GET recombination system. Recombinant BAC clones are identified by PCR, using allele-specific amplification for the mutated sequences. We have used this approach to insert a unique restriction site as well as a common thalassemia mutation (stop codon 39, C-->T) into the human beta-globin locus. We have observed the frequency of recombinant clones to be as high as 1 in 100-200 clones. Therefore, this approach provides a simple and efficient method for introducing point mutations and other fine modifications into BACs, and should greatly facilitate the use of BACs for functional studies and therapeutic applications.     

A BAC contig map over the proximal approximately 3.3 Mb region of horse chromosome 21

A total of 207 BAC clones containing 155 loci were isolated and arranged into a map of linearly ordered overlapping clones over the proximal part of horse chromosome 21 (ECA21), which corresponds to the proximal half of the short arm of human chromosome 19 (HSA19p) and part of HSA5. The clones form two contigs - each corresponding to the respective human chromosomes - that are estimated to be separated by a gap of approximately 200 kb. Of the 155 markers present in the two contigs, 141 (33 genes and 108 STS) were generated and mapped in this study. The BACs provide a 4-5x coverage of the region and span an estimated length of approximately 3.3 Mb. The region presently contains one mapped marker per 22 kb on average, which represents a major improvement over the previous resolution of one marker per 380 kb obtained through the generation of a dense RH map for this segment. Dual color fluorescence in situ hybridization on metaphase and interphase chromosomes verified the relative order of some of the BACs and helped to orient them accurately in the contigs. Despite having similar gene order and content, the equine region covered by the contigs appears to be distinctly smaller than the corresponding region in human (3.3 Mb vs. 5.5-6 Mb) because the latter harbors a host of repetitive elements and gene families unique to humans/primates. Considering limited representation of the region in the latest version of the horse whole genome sequence EquCab2, the dense map developed in this study will prove useful for the assembly and annotation of the sequence data on ECA21 and will be instrumental in rapid search and isolation of candidate genes for traits mapped to this region.     

A bacterial artificial chromosome library for soybean PI 437654 and identification of clones associated with cyst nematode resistance

We have constructed a soybean bacterial artificial chromosome (BAC) library using the plant introduction (PI) 437654. The library contains 73728 clones stored in 192384-well microtiter plates. A random sampling of 230 BACs indicated an average insert size of 136 kb with a range of 20 to 325 kb, and less than 4% of the clones do not contain inserts. Ninety percent of BAC clones in the library have an average insert size greater than 100 kb. Based on a genome size of 1115 Mb, library coverage is 9 haploid genome equivalents. Screening the BAC library colony filters with cpDNA sequences showed that contamination of the genomic library with chloroplast clones was low (1.85%). Library screening with three genomic RFLP probes linked to soybean cyst nematode (SCN) resistance genes resulted in an average of 18 hits per probe (range 7 to 30). Two separate pools of forward and reverse suppression subtractive cDNAs obtained from SCN-infected and uninfected roots of PI 437654 were hybridized to the BAC library filters. The 488 BACs identified from positive signals were fingerprinted and analyzed using FPC software (version 4.0) resulting in 85 different contigs. Contigs were grouped and analyzed in three categories: (1) contigs of BAC clones which hybridized to forward subtracted cDNAs, (2) contigs of BAC clones which hybridized to reverse subtracted cDNAs, and (3) contigs of BAC clones which hybridized to both forward and reverse subtracted cDNAs. This protocol provides an estimate of the number of genomic regions involved in early resistance response to a pathogenic attack.     

High-throughput fingerprinting of bacterial artificial chromosomes using the snapshot labeling kit and sizing of restriction fragments by capillary electrophoresis

We have developed an automated, high-throughput fingerprinting technique for large genomic DNA fragments suitable for the construction of physical maps of large genomes. In the technique described here, BAC DNA is isolated in a 96-well plate format and simultaneously digested with four 6-bp-recognizing restriction endonucleases that generate 3' recessed ends and one 4-bp-recognizing restriction endonuclease that generates a blunt end. Each of the four recessed 3' ends is labeled with a different fluorescent dye, and restriction fragments are sized on a capillary DNA analyzer. The resulting fingerprints are edited with a fingerprint-editing computer program and contigs are assembled with the FPC computer program. The technique was evaluated by repeated fingerprinting of several BACs included as controls in plates during routine fingerprinting of a BAC library and by reconstruction of contigs of rice BAC clones with known positions on rice chromosome 10.     

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.     

BAC representation of two low‐copy regions of the genome of Arabidopsis thaliana

Two regions of Arabidopsis chromosome 4, totalling 4.7 Mb, were assayed for representation in the TAMU and IGF BAC libraries. A directed approach to BAC identification was developed. Gel-purified DNA samples of YACs selected from the YAC-based physical map of chromosome 4 were used to probe high-density colony arrays of the BAC libraries. Strategies were developed that allowed the efficient construction of restriction maps and BAC contigs. Four hundred and sixty-four BACs were mapped, assembled into two complete contigs and used to analyse genomic representation. These BACs provided a mean of 9.4-fold redundant coverage, with a range of 2- to 22-fold. The representation provided by the two libraries showed almost coincident peaks and troughs, with a periodicity of approximately 200 kb. These results demonstrate that, provided both TAMU and IGF libraries are used in their entirety, BACs should provide an excellent resource for both physical mapping and sequencing of the Arabidopsis genome.     

Homology-driven assembly of a sequence-ready mouse BAC contig map spanning regions related to the 46-Mb gene-rich euchromatic segments of human chromosome 19

Draft sequence derived from the 46-Mb gene-rich euchromatic portion of human chromosome 19 (HSA19) was utilized to generate a sequence-ready physical map spanning homologous regions of mouse chromosomes. Sequence similarity searches with the human sequence identified more than 1000 individual orthologous mouse genes from which 382 overgo probes were developed for hybridization. Using human gene order and spacing as a model, these probes were used to isolate and assemble bacterial artificial chromosome (BAC) clone contigs spanning homologous mouse regions. Each contig was verified, extended, and joined to neighboring contigs by restriction enzyme fingerprinting analysis. Approximately 3000 mouse BACs were analyzed and assembled into 44 contigs with a combined length of 41.4 Mb. These BAC contigs, covering 90% of HSA19-related mouse DNA, are distributed throughout 15 homology segments derived from different regions of mouse chromosomes 7, 8, 9, 10, and 17. The alignment of the HSA19 map with the ordered mouse BAC contigs revealed a number of structural differences in several overtly conserved homologous regions and more precisely defined the borders of the known regions of HSA19-syntenic homology. Our results demonstrate that given a human draft sequence, BAC contig maps can be constructed quickly for comparative sequencing without the need for preestablished mouse-specific genetic or physical markers and indicate that similar strategies can be applied with equal success to genomes of other vertebrate species.