A BAC-based physical map of the channel catfish genome

Catfish is the major aquaculture species in the United States. To enhance its genome studies involving genetic linkage and comparative mapping, a bacterial artificial chromosome (BAC) contig-based physical map of the channel catfish (Ictalurus punctatus) genome was generated using four-color fluorescence-based fingerprints. Fingerprints of 34,580 BAC clones (5.6x genome coverage) were generated for the FPC assembly of the BAC contigs. A total of 3307 contigs were assembled using a cutoff value of 1x10(-20). Each contig contains an average of 9.25 clones with an average size of 292 kb. The combined contig size for all contigs was 0.965 Gb, approximately the genome size of the channel catfish. The reliability of the contig assembly was assessed by both hybridization of gene probes to BAC clones contained in the fingerprinted assembly and validation of randomly selected contigs using overgo probes designed from BAC end sequences. The presented physical map should greatly enhance genome research in the catfish, particularly aiding in the identification of genomic regions containing genes underlying important performance traits.     

A physical map of the bovine genome

Background

Cattle are important agriculturally and relevant as a model organism. Previously described genetic and radiation hybrid (RH) maps of the bovine genome have been used to identify genomic regions and genes affecting specific traits. Application of these maps to identify influential genetic polymorphisms will be enhanced by integration with each other and with bacterial artificial chromosome (BAC) libraries. The BAC libraries and clone maps are essential for the hybrid clone-by-clone/whole-genome shotgun sequencing approach taken by the bovine genome sequencing project.

Results

A bovine BAC map was constructed with HindIII restriction digest fragments of 290,797 BAC clones from animals of three different breeds. Comparative mapping of 422,522 BAC end sequences assisted with BAC map ordering and assembly. Genotypes and pedigree from two genetic maps and marker scores from three whole-genome RH panels were consolidated on a 17,254-marker composite map. Sequence similarity allowed integrating the BAC and composite maps with the bovine draft assembly (Btau3.1), establishing a comprehensive resource describing the bovine genome. Agreement between the marker and BAC maps and the draft assembly is high, although discrepancies exist. The composite and BAC maps are more similar than either is to the draft assembly.

Conclusion

Further refinement of the maps and greater integration into the genome assembly process may contribute to a high quality assembly. The maps provide resources to associate phenotypic variation with underlying genomic variation, and are crucial resources for understanding the biology underpinning this important ruminant species so closely associated with humans.     

A physical map of the Mycoplasma genitalium genome

We report the construction of a physical map of the genome of the human pathogen Mycoplasma genitalium through the use of pulse-field gel electrophoresis. The small size and relative simplicity of this genome permit the arrangement of restriction fragments without having to construct linking clones. The size of the genome has been calculated to be approximately 600 kb and several important genetic determinants have been assigned specific loci on the map.     

A physical map of the sorghum chloroplast genome

Summary The chloroplast genome of the IS1112C cytoplasm of sorghum was mapped by the construction of a Bam-HI library in pUC8, and hybridization with BamHI, SalI, and PstI digests of chloroplast DNA (ctDNA) of sorghum and maize. The molecules are extensively colinear, with only one of 13 SalI fragments differing slightly from maize. Seven of 70 restriction sites differed in the two species. A total molecular size of ca. 138 kb was estimated for sorghum. The inverted repeat was not conserved between sorghum and maize, as revealed by a slightly larger BamHI 16S rDNA fragment in sorghum. Homology of a sequence adjacent to the bcl gene and one end of the inverted repeat was detected. These homologies were also observed in maize, and suggest that the ctDNA genomes of sorghum and maize share small reiterations of sequences of the inverted repeat.USDA-ARS     

A BAC-based physical map of Zhikong scallop (Chlamys farreri Jones et Preston)

Zhikong scallop (Chlamys farreri) is one of the most economically important aquaculture species in China. Physical maps are crucial tools for genome sequencing, gene mapping and cloning, genetic improvement and selective breeding. In this study, we have developed a genome-wide, BAC-based physical map for the species. A total of 81,408 clones from two BAC libraries of the scallop were fingerprinted using an ABI 3130xl Genetic Analyzer and a fingerprinting kit developed in our laboratory. After data processing, 63,641 (～5.8× genome coverage) fingerprints were validated and used in the physical map assembly. A total of 3,696 contigs were assembled for the physical map. Each contig contained an average of 10.0 clones, with an average physical size of 490 kb. The combined total physical size of all contigs was 1.81 Gb, equivalent to approximately 1.5 fold of the scallop haploid genome. A total of 10,587 BAC end sequences (BESs) and 167 markers were integrated into the physical map. We evaluated the physical map by overgo hybridization, BAC-FISH (fluorescence in situ hybridization), contig BAC pool screening and source BAC library screening. The results have provided evidence of the high reliability of the contig physical map. This is the first physical map in mollusc; therefore, it provides an important platform for advanced research of genomics and genetics, and mapping of genes and QTL of economical importance, thus facilitating the genetic improvement and selective breeding of the scallop and other marine molluscs.     

A physical genome map of the Burkholderia cepacia type strain

Burkholderia cepacia (basonym Pseudomonas cepaci a), the type speciesof the new genus Burkholderia , is of interest, not only because of its broad catabolic capacity and its ability to antagonize soil-borne plant pathogens, but also because of its causative role in infections in man, which are particularly evident in patients with cystic fibrosis. A physical map of the 8.1 Mb genome of the B. cepacia type-strain ATCC 25416 was constructed by applying two-dimensional pulsed-field gel electrophoresis techniques. Placed onto the macrorestriction map were 38 Spel , 11 Swal , 11 Pacl , 11 Pmel and six l-Ceul sites, resulting in an average resolution of 1O5 kbp. Random single-hit linearization by irradiation and restriction mapping uncovered the presence of four circular replicons of 3.65 Mb, 3.17 Mb, 1.07 Mb and 200 kbp in size. The largest replicon harbours four rrn operons while the other two Megabase-size replicons each contain a single rrn operon, suggesting that the genome has three chromosomes and a large plasmid. Within the beta subdivision of proteobacteria, the existence of multiple replicons is not confined to B. cepacia . The phylogenetically related species Burkholderia glumae , Burkholderia pickettii , Burkholderia solanacearum , Alcaligenes eutrophus and the so far unassigned Pseudomonas glathei were also found to harbour more than one Megabase-size replicon.     

A BAC-based integrated linkage map of the silkworm Bombyx mori

Background

In 2004, draft sequences of the model lepidopteran Bombyx mori were reported using whole-genome shotgun sequencing. Because of relatively shallow genome coverage, the silkworm genome remains fragmented, hampering annotation and comparative genome studies. For a more complete genome analysis, we developed extended scaffolds combining physical maps with improved genetic maps.

Results

We mapped 1,755 single nucleotide polymorphism (SNP) markers from bacterial artificial chromosome (BAC) end sequences onto 28 linkage groups using a recombining male backcross population, yielding an average inter-SNP distance of 0.81 cM (about 270 kilobases). We constructed 6,221 contigs by fingerprinting clones from three BAC libraries digested with different restriction enzymes, and assigned a total of 724 single copy genes to them by BLAST (basic local alignment search tool) search of the BAC end sequences and high-density BAC filter hybridization using expressed sequence tags as probes. We assigned 964 additional expressed sequence tags to linkage groups by restriction fragment length polymorphism analysis of a nonrecombining female backcross population. Altogether, 361.1 megabases of BAC contigs and singletons were integrated with a map containing 1,688 independent genes. A test of synteny using Oxford grid analysis with more than 500 silkworm genes revealed six versus 20 silkworm linkage groups containing eight or more orthologs of Apis versus Tribolium, respectively.

Conclusion

The integrated map contains approximately 10% of predicted silkworm genes and has an estimated 76% genome coverage by BACs. This provides a new resource for improved assembly of whole-genome shotgun data, gene annotation and positional cloning, and will serve as a platform for comparative genomics and gene discovery in Lepidoptera and other insects.     

A physical genome map of Pseudomonas aeruginosa PAO.

A complete macrorestriction map of the 5.9 Mb genome of Pseudomonas aeruginosa PAO (DSM 1707) was constructed by the combination of various one- and two-dimensional pulsed field gel electrophoresis techniques. A total of 51 restriction sites (36 SpeI sites, 15 DpnI sites) were placed on the physical map yielding an average resolution of 110 kb. Several genes encoding virulence factors and enzymes of metabolic pathways were located on the anonymous map by Southern hybridization. Distances between the gene loci were similar on the genetic and physical maps, suggesting an even distribution of genome mobility throughout the bacterial chromosome. The four rRNA operons were organized in pairs of inverted repeats. The two-dimensional macro-restriction techniques described herein are generally applicable for the genome mapping of any prokaryote and lower eukaryote which yields resolvable fragment patterns on two-dimensional pulsed field gels.     

BAC-based upgrading and physical integration of a genetic SNP map in Atlantic salmon

A better understanding of the genotype–phenotype correlation of Atlantic salmon is of key importance for a whole range of production, life history and conservation biology issues attached to this species. High-density linkage maps integrated with physical maps and covering the complete genome are needed to identify economically important genes and to study the genome architecture. Linkage maps of moderate density and a physical bacterial artificial chromosome (BAC) fingerprint map for the Atlantic salmon have already been generated. Here, we describe a strategy to combine the linkage mapping with the physical integration of newly identified single nucleotide polymorphisms (SNPs). We resequenced 284 BAC-ends by PCR in 14 individuals and detected 180 putative SNPs. After successful validation of 152 sequence variations, genotyping and genetic mapping were performed in eight salmon families comprising 376 individuals. Among these, 110 SNPs were positioned on a previously constructed linkage map containing SNPs derived from expressed sequence tag (EST) sequences. Tracing the SNP markers back to the BACs enabled the integration of the genetic and physical maps by assigning 73 BAC contigs to Atlantic salmon linkage groups.     

A physical map of the permuted genome of bacteriophage T1

Summary A restriction map has been constructed for the DNA of coliphage T1 which locates the cleavage sites of the restriction endonucleases, BglI (6 cuts), BglII (16 cuts), EcoRI (2 cuts), HindIII (2 cuts) and PstI (2 cuts). Digestions with BglI and BglII reveal fragments which are present in sub-molar quantities. Two methods, one using the selective removal of molecular ends with exonuclease III and the other involving the comparison of digestion patterns of concatemeric and virion DNA, have shown that the submolar fragments are at or close to the ends of the molecules. Digestions with BglI show that one terminal fragment has a very precise molecular weight whereas all the others are of heterogenous molecular weight. These results are consistent with the model for DNA packaging in which maturation is initiated at a precise site on a concatemeric precursor and proceeds by the encapsidation of up to four successive headfuls of 1.065 genome equivalents (MacHattie and Gill 1977).     

A physical and genetic map of the Spiroplasma citri genome.

A physical and genetic map of the Spiroplasma citri genome has been constructed using several restriction enzymes and pulsed field gel electrophoresis. A number of genes were subsequently localized on the map by the use of appropriate probes. The genome size of the spiroplasma estimated from restriction fragments is close to 1780 kbp, the largest of all Mollicutes studied so far. It contains multisite insertions of Spiroplasma virus 1 (SpV1) sequences. The physical and genetic map of the S. citri genome shares several features with that of other Mollicutes, especially those in the Mycoplasma mycoides cluster. This supports the finding that S. citri and these Mycoplasma spp. are phylogenetically related.     

Construction of a high-density,high-resolution genetic map and its integration with BAC-based physical map in channel catfish

Construction of genetic linkage map is essential for genetic and genomic studies. Recent advances in sequencing and genotyping technologies made it possible to generate high-density and high-resolution genetic linkage maps, especially for the organisms lacking extensive genomic resources. In the present work, we constructed a high-density and high-resolution genetic map for channel catfish with three large resource families genotyped using the catfish 250K single-nucleotide polymorphism (SNP) array. A total of 54,342 SNPs were placed on the linkage map, which to our knowledge had the highest marker density among aquaculture species. The estimated genetic size was 3,505.4 cM with a resolution of 0.22 cM for sex-averaged genetic map. The sex-specific linkage maps spanned a total of 4,495.1 cM in females and 2,593.7 cM in males, presenting a ratio of 1.7 : 1 between female and male in recombination fraction. After integration with the previously established physical map, over 87% of physical map contigs were anchored to the linkage groups that covered a physical length of 867 Mb, accounting for ∼90% of the catfish genome. The integrated map provides a valuable tool for validating and improving the catfish whole-genome assembly and facilitates fine-scale QTL mapping and positional cloning of genes responsible for economically important traits.     

A physical map of the genome of Haemophilus influenzae type b.

Contour-clamped homogeneous electric field pulsed-field gel electrophoresis (PFGE) was used in combination with Southern hybridization to construct a genomic restriction map for the pathogen Haemophilus influenzae serotype b, strain Eagan. Sites for four restriction endonucleases, EagI, NaeI, RsrII and SmaI, were located on the 2100 kbp circular chromosome. Twelve potential virulence loci have been placed on the map together with certain loci essential for growth of the bacteria (e.g. ribosomal RNA operons). PFGE also provided a valuable tool for characterizing ten capsulated, type b isolates (other than Eagan) known to be genetically heterogeneous and two laboratory-derived variants (transformants) derived through complex recombinational events involving random uptake of high-molecular-mass donor genomic DNA.     

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.     

A physical and genetic map of the Mycoplasma hyopneumoniae strain J genome

A macrorestriction map of the genome of Mycoplasma hyopneumoniae strain J, the type strain of the causative agent of enzootic pneumonia in pigs, was constructed using pulsed-field gel electrophoresis (PFGE) and DNA hybridization. The size of the genome as determined by PFGE was approximately 1070 kb. Assembly of the M. hyopneumoniae genomic map was facilitated and complimented by the simultaneous construction of an ordered cosmid library. Five contigs of overlapping cosmids were assembled, which together represent coverage of approximately 728 kb. Forty-two genetic markers (including three types of repeated elements) were placed on the M. hyopneumoniae map. Closer examination of an ApaI restriction fragment contained entirely within a single cosmid insert suggests that the genome size may be overestimated by PFGE.     

A 4,103 marker integrated physical and comparative map of the horse genome

A comprehensive second-generation whole genome radiation hybrid (RH II), cytogenetic and comparative map of the horse genome (2n = 64) has been developed using the 5000rad horse x hamster radiation hybrid panel and fluorescence in situ hybridization (FISH). The map contains 4,103 markers (3,816 RH; 1,144 FISH) assigned to all 31 pairs of autosomes and the X chromosome. The RH maps of individual chromosomes are anchored and oriented using 857 cytogenetic markers. The overall resolution of the map is one marker per 775 kilobase pairs (kb), which represents a more than five-fold improvement over the first-generation map. The RH II incorporates 920 markers shared jointly with the two recently reported meiotic maps. Consequently the two maps were aligned with the RH II maps of individual autosomes and the X chromosome. Additionally, a comparative map of the horse genome was generated by connecting 1,904 loci on the horse map with genome sequences available for eight diverse vertebrates to highlight regions of evolutionarily conserved syntenies, linkages, and chromosomal breakpoints. The integrated map thus obtained presents the most comprehensive information on the physical and comparative organization of the equine genome and will assist future assemblies of whole genome BAC fingerprint maps and the genome sequence. It will also serve as a tool to identify genes governing health, disease and performance traits in horses and assist us in understanding the evolution of the equine genome in relation to other species.     

A genetic linkage map of an apple rootstock progeny anchored to the Malus genome sequence

An apple rootstock progeny raised from the cross between the very dwarfing ??M.27?? and the more vigorous ??M.116?? (??M.M.106???×???M.27??) was used for the construction of a linkage map comprising a total of 324 loci: 252 previously mapped SSRs, 71 newly characterised or previously unmapped SSR loci (including 36 amplified by 33 out of the 35 novel markers reported here), and the self-incompatibility locus. The map spanned the 17 linkage groups (LG) expected for apple covering a genetic distance of 1,229.5?cM, an estimated 91% of the Malus genome. Linkage groups were well populated and, although marker density ranged from 2.3 to 6.2?cM/SSR, just 15 gaps of more than 15?cM were observed. Moreover, only 17.5% of markers displayed segregation distortion and, unsurprisingly in a semi-compatible backcross, distortion was particularly pronounced surrounding the self-incompatibility locus (S) at the bottom of LG17. DNA sequences of 273 SSR markers and the S locus, representing a total of 314 loci in this investigation, were used to anchor to the ??Golden Delicious?? genome sequence. More than 260 of these loci were located on the expected pseudo-chromosome on the ??Golden Delicious?? genome or on its homeologous pseudo-chromosome. In total, 282.4?Mbp of sequence from 142 genome sequence scaffolds of the Malus genome were anchored to the ??M.27???×???M.116?? map, providing an interface between the marker data and the underlying genome sequence. This will be exploited for the identification of genes responsible for traits of agronomic importance such as dwarfing and water use efficiency.