Prime plasmids derived from the IncP-10 plasmid R91-5 in Pseudomonas putida

Abstract Plasmid primes carrying various fragments of Pseudomonas putida chromosome have been derived from pMO22, a derivative of R91-5 loaded with Tn 501 . These prime plasmids transfer efficiently to P. aeruginosa where they effectively complement various auxotrophic markers. Proof of prime plasmid formation has been provided by the high-frequency transfer of plasmid and chromosomal markers, the unselected cotransfer of either plasmid or chromosomal markers into P. aeruginosa and by transformation of both plasmid and chromosomal markers using prime plasmid DNA. Such prime plasmids have been used to map the location of new markers on the P. putida chromosome.     

Integration of R91-5::Tn501 into the Pseudomonas putida PPN chromosome and genetic circularity of the chromosomal map

Derivatives of the Pseudomonas aeruginosa plasmid R91-5, loaded with the transposon Tn501, were transferred to P. putida PPN. Over 90% of exconjugants, which arose at a frequency of ca. 10(-6) per donor cell, exhibited high-frequency (greater than 10(-2) per donor cell) polarized transfer of chromosomal markers. In one instance it was demonstrated by transduction that the plasmid had been inserted into a gene required for serine biosynthesis. The integrated nature of the plasmid in this and other P. putida (R91-5::Tn501) derivatives was supported by the failure to detect covalently closed circular DNA in these strains. The transfer origins of six different Hfr donors have been characterized genetically, and time-of-entry kinetics obtained from interrupted matings have enabled the construction of a circular genetic map 103 min in length and containing 35 markers. The genetic map of P. putida PPN shows significant differences in marker order to that of P. aeruginosa PAO.     

Isolation of High Frequency of Recombination Donors from Tn5 Chromosomal Mutants of Pseudomonas Putida Ppn and Recalibration of the Genetic Map

A Tn5 loaded derivative of the IncP-10 plasmid R91-5 (pMO75) was used as a suicide vector to generate random chromosomal insertion mutations in Pseudomonas putida PPN. Reintroduction of pMO75 into such mutants resulted in integration of the plasmid at the site of Tn5 insertion, giving rise to two classes of high frequency of donors recombination (Hfr) donors, transferring chromosome at high frequency (greater than 10(-1) per donor cell) in opposite directions. Consequently, Tn5 induced auxotrophic mutations could be equated with or distinguished from previously mapped mutations, and closely linked markers ordered, on the basis of marker recovery using the two classes of Hfr donor. The isolation of many new transfer origins allowed more accurate time-of-entry analysis than previously possible and resulted in the reduction of the genetic map from 103 min to 88 min.     

Linkage map of Escherichia coli K-12, edition 8.

The linkage map of Escherichia coli K-12 depicts the arrangement of genes on the circular chromosome of this organism. The basic units of the map are minutes, determined by the time-of-entry of markers from Hfr into F- strains in interrupted-conjugation experiments. The time-of-entry distances have been refined over the years by determination of the frequency of cotransduction of loci in transduction experiments utilizing bacteriophage P1, which transduces segments of DNA approximately 2 min in length. In recent years, the relative positions of many genes have been determined even more precisely by physical techniques, including the mapping of restriction fragments and the sequencing of many small regions of the chromosome. On the whole, the agreement between results obtained by genetic and physical methods has been remarkably good considering the different levels of accuracy to be expected of the methods used. There are now few regions of the map whose length is still in some doubt. In some regions, genetic experiments utilizing different mutant strains give different map distances. In other regions, the genetic markers available have not been close enough to give accurate cotransduction data. The chromosome is now known to contain several inserted elements apparently derived from lambdoid phages and other sources. The nature of the region in which the termination of replication of the chromosome occurs is now known to be much more complex than the picture given in the previous map. The present map is based upon the published literature through June of 1988. There are now 1,403 loci placed on the linkage group, which may represent between one-third and one-half of the genes in this organism.     

A genetic map constructed using a doubled haploid population derived from two elite Chinese common wheat varieties

Genetic mapping provides a powerful tool for the analysis of quantitative trait loci (QTLs) at the genomic level.Herein,we report a new genetic linkage map developed from an F1-derived doubled haploid (DH) population of 168 lines,which was generated from the cross between two elite Chinese common wheat (Triticum aestivum L.) varieties,Huapei 3 and Yumai 57.The map contained 305 loci,represented by 283 simple sequence repeat (SSR) and 22 expressed sequence tag (EST)-SSR markers,which covered a total length of 2141.7 cM with an average distance of 7.02 cM between adjacent markers on the map.The chromosomal locations and map positions of 22 new SSR markers were determined,and were found to distribute on 14 linkage groups.Twenty SSR loci showed different chromosomal locations from those reported in other maps.Therefore,this map offers new information on the SSR markers of wheat.This genetic map provides new opportunities to detect and map QTLs controlling agronomically important traits.The unique features of this map are discussed.     

Isolation and characterization of Pseudomonas aeruginosa R'' plasmids constructed by interspecific mating

Plasmid R68.45 was used to construct R' plasmids carrying a maximum of 4 to 5 map minutes of the Pseudomonas aeruginosa PAO chromosome by interspecific mating, using P. putida PPN as the recipient. These R' plasmids were used to determine the map location of the amiE locus and to identify tentatively a number of P. putida auxotrophic mutations. Some of these R' plasmids could not be maintained in recombination-deficient P. aeruginosa strains.     

A high-resolution radiation hybrid map of the proximal region of rat Chromosome 4

Radiation hybrid (RH) mapping has been used to produce genome maps in the human and mouse, but as yet the technique has been applied little to other species. We describe the use of RH mapping in the rat, using a newly available rat/hamster RH panel, to construct an RH map of the proximal part of rat Chromosome (Chr) 4. This region is of interest because quantitative trait loci (QTLs) for defective insulin and catecholamine action, hypertension, and dyslipidemia map to this region. The RH map includes 23 rat genes or microsatellites previously mapped to this part of Chr 4, one rat gene not previously mapped in the rat, and markers for four new genes, homologs of which map to the syntenic region of the mouse genome. The RH map integrates genetic markers previously mapped on several rat crosses, increases the resolution of existing maps, and may provide a suitable basis for physical map construction and gene identification in this chromosomal region. Our results demonstrate the utility of RH mapping in the rat genome and show that RH mapping can be used to localize, in the rat genome, the homologs of genes from other species such as the mouse. This will facilitate identification of candidate genes underlying QTLs on this chromosomal segment. Received: 4 December 1998 / Accepted: 19 January 1999     

A second-generation genetic linkage map of the baboon (Papio hamadryas) genome

Construction of genetic linkage maps for nonhuman primate species provides information and tools that are useful for comparative analysis of chromosome structure and evolution and facilitates comparative analysis of meiotic recombination mechanisms. Most importantly, nonhuman primate genome linkage maps provide the means to conduct whole genome linkage screens for localization and identification of quantitative trait loci that influence phenotypic variation in primate models of common complex human diseases such as atherosclerosis, hypertension, and diabetes. In this study we improved a previously published baboon whole genome linkage map by adding more loci. New loci were added in chromosomal regions that did not have sufficient marker density in the initial map. Relatively low heterozygosity loci from the original map were replaced with higher heterozygosity loci. We report in detail on baboon chromosomes 5, 12, and 18 for which the linkage maps are now substantially improved due to addition of new informative markers.     

Towards an integrated linkage map of common bean. 4. Development of a core linkage map and alignment of RFLP maps

 Three RFLP maps, as well as several RAPD maps have been developed in common bean (Phaseolus vulgaris L.). In order to align these maps, a core linkage map was established in the recombinant inbred population BAT93×Jalo EEP558 (BJ). This map has a total length of 1226 cM and comprises 563 markers, including some 120 RFLP and 430 RAPD markers, in addition to a few isozyme and phenotypic marker loci. Among the RFLPs mapped were markers from the University of California, Davis (established in the F₂ of the BJ cross), University of Paris-Orsay, and University of Florida maps. These shared markers allowed us to establish a correspondence between the linkage groups of these three RFLP linkage maps. In total, the general map location (i.e., the linkage group membership and approximate location within linkage groups) has been determined for some 1070 markers. Approaches to align this core map with other current or future maps are discussed. Received: 10 March 1998 / Accepted: 22 April 1998     

A physical and genetic map of Neisseria meningitidis B1940

A physical map of the chromosome of Neisseria meningitidis B1940 has been constructed by one- and two-dimensional pulsed-field gel electrophoresis techniques. Complete macrorestriction maps for the enzymes Nhe I (16 sites), Sgf I (13 sites), Sfi I (11 sites) and I-Ceu I (4 sites), as well as a partial restriction map for the restriction enzyme Spe I (15 of c. 28 sites) could be established. Altogether, 59 restriction sites were mapped on a single circular chromosome of 2.3 Mbp. By restriction endonuclease digestion and Southern hybridization of cloned genetic markers, 39 genetic loci were assigned to this map. Comparison with the metabolic maps of Neisseria gonorrhoeae MS11-N198 and FA1090 revealed a high degree of conservation in the arrangement of gene loci among these two species, although four out of 24 genetic loci are located at different chromosomal positions, indicating several genomic rearrangements.     

A chromosomal genetic map of Rhizobium sp. NGR234 generated with Tn5-Mob

Summary Rhizobium sp. NGR234 in a fast-growing Rhizobium strain with a broad host range. The location and role of chromosomal genes involved in cellular metabolism or in the legume symbioses is unknown. We isolated a series of auxotrophic and antibiotic resistant mutants of NGR234 and utilized a chromosome mobilization system based on Tn5-Mob and pJB3JI; Tn5-Mob donor strains behaved like Hfr strains, transferring the chromosome polarly at high frequency from a fixed point of insertion. The use of four different strains with Tn5-Mob located at different nutritional loci in crosses with double auxotrophic recipients, allowed us to build up a circular linkage map of NGR234 based on relative recombination frequencies. Also, symbiotically important genes identified by site-directed mutagenesis, such as hemA and ntrA, could be located and mapped on the chromosome.Abbreviations Tc tetracycline - Sp spectinomycin - Rif rifampicin - Km kanamycin     

A Primary Genetic Linkage Map of 14 Polymorphic Loci for the Short Arm of Human Chromosome 8

A genetic linkage map of markers for the short arm of human chromosome 8 has been constructed with 14 polymorphic DNA markers on the basis of genotypes obtained in 40 CEPH reference families. This unbroken map spans 45 cM in males and 79 cM in females. The 14 markers include three genes, MSR, LPL, and NEFL, and one anonymous DNA segment that were previously assigned to chromosome 8. The other 10 marker had been isolated from a chromosome 8-specific cosmid library and physically localized to chromosomal bands by fluorescence in situ hybridization. The order of loci determined by genetic linkage was consistent with their physical locations. This map will facilitate efficient linkage studies of human genetic diseases that may be segregating on chromosome 8p and will provide anchor points for development of high-resolution maps for this chromosomal region.     

A genetic linkage map for coho salmon (Oncorhynchus kisutch)

Construction of genetic linkage maps is an important first step for a variety of genomic applications, such as selective breeding in aquaculture, comparative studies of chromosomal evolution and identification of loci that have played key roles in the evolution of a species. Here we present a sex-specific linkage map for coho salmon. The map was constructed using 148 AFLP markers, 133 microsatellite loci and the phenotypic locus SEX . Twenty-four linkage groups spanning 287.4 cM were mapped in males, and 33 linkage groups spanning 429.7 cM were mapped in females. Several male linkage groups corresponded to two female linkage groups. The combination of linkage groups across both sexes appeared to characterize regions of 26 chromosomes. Two homeologous chromosomes were identified based on information from duplicated loci. Homologies between the coho and rainbow trout maps were examined. Eighty-six loci were found to form common linkage relationships between the two maps; these relationships provided evidence for whole-arm fissions, fusions and conservation of chromosomal regions in the evolution of these two species.     

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.     

Towards an expanded and integrated linkage map of cucumber (Cucumis sativus L.).

Linkage maps in cucumber (Cucumis sativus var. sativus L.) have been constructed using morphological traits, isozymes, restriction fragment length polymorphisms (RFLPs), and random amplified polymorphic DNAs (RAPDs). The lack of polymorphism in cucumber has led to the construction of relatively unsaturated maps (13- to 80-point). We have added amplified fragment length polymorphism (AFLP) markers to existing narrow-based (within C. sativus) and wide-based (C. sativus x C. sativus var. hardwickii) maps. JOINMAP v. 2.0 was used to construct maps and to join these with historical maps from several previous studies. Our narrow- and wide-based merged maps contain 255 and 197 markers, respectively, including morphological traits, disease resistance loci, isozymes, RFLPs, RAPDs, and AFLPs. Condensation of total map distance occurred in merged maps compared to historic maps using many of the same markers. This phenomenon is most likely due to differences in map construction algorithms. The merged maps represent the best fit of the data used and are an important first step towards the construction of a comprehensive linkage map for cucumber. Identification of additional anchor markers between the narrow- and wide-based maps presented here may allow their future integration into a unified model.     

Physical map of the DNA of bacteriophage tf of Pseudomonas putida

A physical map has been constructed for P. putida bacteriophage tf DNA containing single-strand breaks (nicks). Localization of cleavage sites for EcoRI, HindIII, HpaI ClaI, BamHI, SalI, XbaI and XhoI restriction endonucleases was determined. Position of single-strand breaks was mapped by electrophoretic analysis of denatured tf DNA and electron microscopy of partially denatured DNA samples. The tf genome is characterized by the presence of two classes of nicks differing in the frequency of their presence in population of bacteriophage DNA molecules.     

A high density barley microsatellite consensus map with 775 SSR loci

A microsatellite or simple sequence repeat (SSR) consensus map of barley was constructed by joining six independent genetic maps based on the mapping populations 'Igri x Franka', 'Steptoe x Morex', 'OWB(Rec) x OWB(Dom)', 'Lina x Canada Park', 'L94 x Vada' and 'SusPtrit x Vada'. Segregation data for microsatellite markers from different research groups including SCRI (Bmac, Bmag, EBmac, EBmag, HVGeneName, scsssr), IPK (GBM, GBMS), WUR (GBM), Virginia Polytechnic Institute (HVM), and MPI for Plant Breeding (HVGeneName), generated in above mapping populations, were used in the computer program RECORD to order the markers of the individual linkage data sets. Subsequently, a framework map was constructed for each chromosome by integrating the 496 "bridge markers" common to two or more individual maps with the help of the computer programme JoinMap 3.0. The final map was calculated by following a "neighbours" map approach. The integrated map contained 775 unique microsatellite loci, from 688 primer pairs, ranging from 93 (6H) to 132 (2H) and with an average of 111 markers per linkage group. The genomic DNA-derived SSR marker loci had a higher polymorphism information content value (average 0.61) as compared to the EST/gene-derived SSR loci (average 0.48). The consensus map spans 1,068 cM providing an average density of one SSR marker every 1.38 cM. Such a high-density consensus SSR map provides barley molecular breeding programmes with a better choice regarding the quality of markers and a higher probability of polymorphic markers in an important chromosomal interval. This map also offers the possibilities of thorough alignment for the (future) physical map and implementation in haplotype diversity studies of barley.     

Genome structure in soybean revealed by a genomewide genetic map constructed from a single population

A complete genetic linkage map of the soybean, in which sequence-based (SB) genetic markers are evenly distributed genomewide, was constructed from an F(12) population composed of 113 recombinant inbred lines derived from an interspecific cross involving Korean genotypes Hwangkeum and IT182932. Several approaches were employed for the development of 112 novel SB markers targeting both the gaps and the ends of the linkage groups (LGs). The resultant map harbored 20 well-resolved LGs presumed to correspond to the 20 pairs of soybean chromosomes. The map allowed us to identify the important chromosomal structures that were not observed in the integrated genetic maps, to identify the new potentially gene-rich regions, to detect segregation distortion regions within the whole genome, and to extend the ends of the LGs. The results will facilitate the further discovery of agronomically relevant genetic loci in the heretofore neglected chromosomal regions and should also provide some important links between the soybean genetic, physical, and genome sequence maps in the regions.     

Combined RAPD and RFLP molecular linkage map of asparagus.

Two linkage maps of asparagus (Asparagus officinalis L.) were constructed using a double pseudotestcross mapping strategy with restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNAs (RAPDs), and allozymes as markers in a population generated from crossing MW25 x A19, two heterozygous parents. All data were inverted and combined with the natural data to detect linkages in repulsion phase. Two sets of data, one for each parent, were formed according to the inheritance patterns of the markers. The maternal MW25 map has a total of 163 marker loci placed in 13 linkage groups covering 1281 cM, with an average and a maximum distance between adjacent loci of 7.9 and 29 cM, respectively. The paternal A19 map has 183 marker loci covering 1324 cM in 9 linkage groups, with an average and a maximum distance between two adjacent loci of 7.7 and 29 cM, respectively. Six multiallelic RFLPs segregating in the pattern a/c x b/c and eight heterozygous loci (four RAPDs, and four RFLPs segregating in the pattern a/b x a/b (HZ loci)) were common to both maps. These 14 loci were used as bridges to align homologous groups between the two maps. In this case, RFLPs were more frequent and informative than RAPDs. Nine linkage groups in the MW25 map were homologous to six groups in the A19 map. In two cases, two or more bridge loci were common to a group; thus, the orientation of homologous linkage groups was also determined. In four other cases, only one locus was common to the two homologous groups and the orientation was unknown. Mdh, four RFLPs, and 14 RAPDs were assigned to chromosome L5, which also has the sex locus M.     

A human genome map of comparative anchor tagged sequences.

Effective comparative mapping inference utilizing developing gene maps of animal species requires the inclusion of anchored reference loci that are homologous to genes mapped in the more "gene-dense" mouse and human maps. Nominated anchor loci, termed comparative anchor tagged sequences (CATS), have been ordered in the mouse linkage map, but due to the dearth of common polymorphisms among human coding genes have not been well represented in human linkage maps. We present here an ordered framework map of 314 comparative anchor markers in humans based on mapping analysis in the Genebridge 4 panel of radiation hybrid cell lines, plus empirically optimized CATS PCR primers which detect these markers. The ordering of these homologous gene markers in human and mouse maps provides a framework for comparative gene mapping of representative mammalian species.