The physical map of the chromosome of a serogroup A strain of Neisseria meningitidis shows complex rearrangements relative to the chromosomes of the two mapped strains of the closely related species N. gonorrhoeae.

A physical map of the chromosome of N. meningitidis Z2491 (serogroup A, subgroup IV-1) has been constructed. Z2491 DNA was digested with NheI, SpeI, SgfI, PacI, BglII, or PmeI, resulting in a limited number of fragments that were resolved by contour-clamped homogeneous electric field (CHEF) electrophoresis. The estimated genome size for this strain was 2,226 kb. To construct the map, probes corresponding to single-copy genes or sequences were used on Southern blots of chromosomal DNA digested with the different mapping enzymes and subjected to CHEF electrophoresis. By determining which fragments from different digests hybridized to each specific probe, it was possible to walk back and forth between digests to form a circular macrorestriction map. The intervals between mapped restriction sites range from 10 to 143 kb in size. A total of 117 markers have been placed on the map; 75 represent identified genes, with the remaining markers defined by anonymous cloned fragments of neisserial DNA. Comparison of the arrangement of genetic loci in Z2491 with that in gonococcal strain FA1090, for which a physical map was previously constructed, revealed complex genomic rearrangements between the two strains. Although gene order is generally conserved over much of the chromosome, a region of approximately 500 kb shows translocation and/or inversion of multiple blocks of markers between the two strains. Even within the relatively conserved portions of the maps, several genetic markers are in different positions in Z2491 and FA1090.     

An alpha satellite DNA polymorphism specific for the centromeric region of chromosome 13

Alpha satellite DNA is composed of variants of a short consensus sequence that are repeated in tandem arrays in the centromeric heterochromatin of each human chromosome. To define centromeric markers for linkage studies, we screened human genomic DNA for restriction fragment length polymorphisms using a probe detecting alphoid sequences on chromosomes 13 and 21. We describe one such DNA polymorphism. Analysis of linkage of this DNA marker to other polymorphic markers in the CEPH pedigrees demonstrates linkage to markers on the proximal long arm of chromosome 13 and defines the centromeric end of the linkage map of this chromosome.     

The CEPH consortium primary linkage map of human chromosome 10

The first CEPH consortium map, that of chromosome 10, is presented. This primary linkage map contains 28 continuously linked loci defined by genotypes generated from CEPH family DNAs with 37 probe and enzyme combinations. Cytogenetic localization of some of the genetic markers indicates that the consortium map extends, at least, from 10p13 to 10q26. The order of loci on the consortium map agrees with the physical localization data. The female map spans 309 cM (206 cM if an approximation of interference is included in the mapping function used to construct the map), and the mean genetic distance of intervals is 11 cM (7 cM). Also presented are maps of chromosome 10 from each of five CEPH collaborating laboratories, based on genotypes for all relevant markers in the CEPH database. The CEPH consortium map of chromosome 10 should be useful for localization of any gene of interest falling within the span covered. The genotypes in the chromosome 10 consortium map database are now available to the scientific community.     

A sex-averaged genetic linkage map in coastal Douglas-fir (Pseudotsuga menziesii[Mirb.] Franco var ‘menziesii’) based on RFLP and RAPD markers

 We have constructed a sex-averaged genetic linkage map in coastal Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco var ‘menziesii’) using a three-generation outcrossed pedigree and molecular markers. Our research objectives are to learn about genome organization and to identify markers associated with adaptive traits. The map reported here is comprised of 141 markers organized into 17 linkage groups and covers 1,062 centiMorgans (cM). Of the markers positioned on the map, 94 were derived from a Douglas-fir complimentary-DNA (cDNA) library that was constructed from new-growth needle tissue. Other markers include 11 Douglas-fir genomic-DNAs, 20 loblolly pine (Pinus taeda L.) cDNAs, 15 random amplified polymorphic DNAs (RAPDs) and a PCR-amplified phytochrome probe. A high degree of variation was detected in each of the two parents of our mapping population, and many of the restriction fragment length polymorphism (RFLP) and RAPD phenotypes were complex. Marker data were analyzed for linkage using mapping software JOINMAP version 2.0. Received: 16 March 1998 / Accepted: 22 April 1998     

A molecular genetic map and electrophoretic karyotype of the plant pathogenic fungus Cochliobolus sativus

A molecular genetic map was constructed and an electrophoretic karyotype was resolved for Cochliobolus sativus, the causal agent of spot blotch of barley and wheat. The genetic map consists of 27 linkage groups with 97 amplified fragment length polymorphism (AFLP) markers, 31 restriction fragment length polymorphism (RFLP) markers, two polymerase chain reaction amplified markers, the mating type locus (CsMAT), and a gene (VHv1) conditioning high virulence on barley cv. Bowman. These linkage groups covered a map distance of 849 cM. The virulence gene VHv1 cosegregated with six AFLP markers and was mapped on one of the major linkage groups. Fifteen chromosome-sized DNAs were resolved in C. sativus isolates ND93-1 and ND9OPr with contour-clamped homogeneous electric field (CHEF) electrophoresis combined with telomere probe analysis of comigrating chromosome-sized DNAs. The chromosome sizes ranged from 1.25 to 3.80 Mbp, and the genome size of the fungus was estimated to be approximately 33 Mbp. By hybridizing genetically mapped RFLP and AFLP markers to CHEF blots, 25 of the 27 linkage groups were assigned to specific chromosomes. The barley-specific virulence locus VHv1 was localized on a chromosome of 2.80 Mbp from isolate ND9OPr in the CHEF gel. The total map length of the fungus was estimated to be at least 1,329 cM based on the map distance covered by the linked markers and the estimated gaps. Therefore, the physical to genetic distance ratio is approximately 25 kb/cM. Construction of a high-resolution map around target loci will facilitate the cloning of the genes conferring virulence and other characters in C. sativus by a map-based cloning strategy.     

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.     

梨遗传连锁图谱的构建及其与苹果图谱的比较

以‘丰水’为母本、‘砀山酥梨’为父本杂交所得的F1代104株单体为作图群体,利用SSR分子标记进行遗传连锁分析,应用Jionmap 3.0作图软件,构建了一张包含104个SSR分子标记,分属于18个连锁群的梨遗传连锁图谱,覆盖梨基因组总长831.8cM,平均图距为8.0cM。根据定位到该图谱上的SSR标记与苹果‘Fiesta’图谱进行比较,25个共有的SSR标记将该图谱和苹果图谱各连锁群连接起来,这些标记不仅呈现良好的共线性而且它们之间的相对遗传距离也很相近。研究认为,SSR标记作为锚定引物,可以与不同物种的遗传图谱相比较整合,为不同物种之间遗传信息的转移提供参考依据;同时该研究为梨树相关性状的基因定位、分离以及克隆奠定了基础。     

An integrated and comparative genetic map of the turkey genome

An integrated genetic linkage map was developed for the turkey (Meleagris gallopavo) that combines the genetic markers from the three previous mapping efforts. The UMN integrated map includes 613 loci arranged into 41 linkage groups. An additional 105 markers are tentatively placed within linkage groups based on two-point LOD scores and 19 markers remain unlinked. A total of 210 previously unmapped markers has been added to the UMN turkey genetic map. Markers from each of the 20 linkage groups identified in the Roslin map and the 22 linkage groups of the Nte map are incorporated into the new integrated map. Overall map distance contained within the 41 linkage groups is 3,365 cM (sex-averaged) with the largest linkage group (94 loci) measuring 533.1 cM. Average marker interval for the map was 7.86 cM. Sequences of markers included in the new map were compared to the chicken genome sequence by 'BLASTN'. Significant similarity scores were obtained for 95.6% of the turkey sequences encompassing an estimated 91% of the chicken genome. A physical map of the chicken genome based on positions of the turkey sequences was built and 36 of the 41 turkey linkage groups were aligned with the physical map, five linkage groups remain unassigned. Given the close similarities between the turkey and chicken genomes, the chicken genome sequence could serve as a scaffold for a genome sequencing effort in the turkey.     

Molecular map of the Chlamydomonas reinhardtii nuclear genome

We have prepared a molecular map of the Chlamydomonas reinhardtii genome anchored to the genetic map. The map consists of 264 markers, including sequence-tagged sites (STS), scored by use of PCR and agarose gel electrophoresis, and restriction fragment length polymorphism markers, scored by use of Southern blot hybridization. All molecular markers tested map to one of the 17 known linkage groups of C. reinhardtii. The map covers approximately 1,000 centimorgans (cM). Any position on the C. reinhardtii genetic map is, on average, within 2 cM of a mapped molecular marker. This molecular map, in combination with the ongoing mapping of bacterial artificial chromosome (BAC) clones and the forthcoming sequence of the C. reinhardtii nuclear genome, should greatly facilitate isolation of genes of interest by using positional cloning methods. In addition, the presence of easily assayed STS markers on each arm of each linkage group should be very useful in mapping new mutations in preparation for positional cloning.     

Linkage Map of the Honey Bee, Apis Mellifera, Based on Rapd Markers

A linkage map was constructed for the honey bee based on the segregation of 365 random amplified polymorphic DNA (RAPD) markers in haploid male progeny of a single female bee. The X locus for sex determination and genes for black body color and malate dehydrogenase were mapped to separate linkage groups. RAPD markers were very efficient for mapping, with an average of about 2.8 loci mapped for each 10-nucleotide primer that was used in polymerase chain reactions. The mean interval size between markers on the map was 9.1 cM. The map covered 3110 cM of linked markers on 26 linkage groups. We estimate the total genome size to be ~3450 cM. The size of the map indicated a very high recombination rate for the honey bee. The relationship of physical to genetic distance was estimated at 52 kb/cM, suggesting that map-based cloning of genes will be feasible for this species.     

Dinucleotide Repeat Loci Contribute Highly Informative Genetic Markers to the Human Chromosome 2 Linkage Map

Microsatellite repeat loci can provide informative markers for genetic linkage. Currently, the human chromosome 2 genetic linkage map has very few highly polymorphic markers. Being such a large chromosome, it will require a large number of informative markers for the dense coverage desired to allow disease genes to be mapped quickly and accurately. Dinucleotide repeat loci from two anonymous chromosome 2 genomic DNA clones were sequenced so that oligonucleotide primers could be designed for amplifying each locus using the polymerase chain reaction (PCR). Five sets of PCR primers were also generated from nucleotide sequences in the GenBank Database of chromosome 2 genes containing dinucleotide repeats. In addition, one PCR primer pair was made that amplifies a restriction fragment length polymorphism on the TNP1 gene (Hoth and Engel, 1991). These markers were placed on the CEPH genetic linkage map by screening the CEPH reference DNA panel with each primer set, combining these data with those of other markers previously placed on the map, and analyzing the combined data set using CRI-MAP and LINKAGE. The microsatellite loci are highly informative markers and the TNP1 locus, as expected, is only moderately informative. A map was constructed with 38 ordered loci (odds 1000:1) spanning 296 cM (male) and 476 cM (female) of chromosome 2 compared with 306 cM (male) and 529 cM (female) for a previous map of 20 markers.     

A radiation hybrid map of the region on human chromosome 22 containing the neurofibromatosis type 2 locus.

We describe a high-resolution radiation hybrid map of the region on human chromosome 22 containing the neurofibromatosis type 2 (NF2) gene. Eighty-five hamster-human somatic cell hybrids generated by X-irradiation and cell fusion were used to generate the radiation hybrid map. The presence or absence of 18 human chromosome 22-specific markers was determined in each hybrid by using Southern blot hybridization. Sixteen of the 18 markers were distinguishable by X-ray breakage in the radiation hybrids. Analysis of these data using two different mathematical models and two different statistical methods resulted in a single framework map consisting of 8 markers ordered with odds greater than 1000:1. The remaining nonframework markers were all localized to regions consisting of two adjoining intervals on the framework map with odds greater than 1000:1. Based on the RH map, the NF2 region of chromosome 22, defined by the flanking markers D22S1 and D22S28, is estimated to span a physical distance of approximately 6 Mb and is the most likely location for 9 of the 18 markers studied: D22S33, D22S41, D22S42, D22S46, D22S56, LIF, D22S37, D22S44, and D22S15.     

A mapped set of DNA markers for human chromosome 17

We have developed and mapped by genetic linkage a primary set of markers for chromosome 17. The map consists of 21 loci derived from 27 probe/enzyme systems, including eight highly informative markers at loci containing a variable number of tandemly repeated DNA sequences (VNTRs). The map is continuous from the telomeric region of the short arm to the telomeric region of the long arm, covering estimated genetic distances of 218 cM in males and 279 cM in females. The average heterozygosity among all 21 loci in the population sample analyzed is 58%; 77% heterozygosity was observed among the eight VNTR markers that were highly informative. This map will make it possible to detect by linkage the location of genetic defects associated with chromosome 17 and will also provide anchor points for a high-resolution map of this chromosome.     

Comparison between genetic and physical maps in Zea mays L. of molecular markers linked to resistance against Diatraea spp

In the pachytene stage, chromosomes are maximally extended and can easily be distinguished. Therefore, by applying fluorescence in situ hybridization (FISH) to pachytene chromosomes, it is possible to generate a high-resolution physical map of chromosome 9 in maize. Molecular markers ( umc105a on the short arm of chromosome 9, csu145a on the long arm) were used that flank quantitative trait loci (QTL) for sugarcane borer (SCB) and southwestern corn borer (SWCB) resistance. As reference markers, a centromere-specific probe (CentC) and a knob-specific probe (pZm4-21) were utilized. Two fluorescent dyes with four probes were used to physically position these markers. Signals of repetitive DNA sequences in cosmid probes were suppressed by chromosome in situ suppression (CISS) hybridization. FISH signals were strong and reproducible for all probes. We measured the distances in micrometers for four subchromosomal regions and estimated the corresponding number of base pairs. The physical locations of the markers were compared on mitotic metaphase and pachytene chromosomes to the genetic map of chromosome 9. Genetic analysis positioned the two markers for SCB resistance in a central interval representing approximately 33.7% of the genetic length. However, the physical distance between these probes was determined to encompass about 70% of the physical length of chromosome 9. The two markers were located at distal positions on opposite arms of chromosome 9. Physical maps provide valuable information for gene isolation and understanding recombination.     

A Genetic Map of Lettuce (Lactuca sativa L.) with Restriction Fragment Length Polymorphism, Isozyme, Disease Resistance and Morphological Markers

A detailed linkage map of lettuce was constructed using 53 genetic markers including 41 restriction fragment length polymorphism (RFLP) loci, five downy mildew resistance genes, four isozyme loci and three morphological markers. The genetic markers were distributed into nine linkage groups and cover 404 cM which may be 25-30% of the lettuce genome. The majority (31 of 34) of the RFLP probes detected single segregating loci, although seven of these may have been homologous to further monomorphic loci. When several loci were detected by a single probe, the loci were generally linked, suggesting tandem duplications. One probe, however, detected loci in three linkage groups suggesting translocations. The five downy mildew resistance genes (Dm1, Dm3, Dm4, Dm5/8 and Dm13), segregating in the Calmar x Kordaat cross, represented each of the four resistance gene linkage groups. Dm5/8 is flanked by two cDNA loci, each located 10 cM away. These flanking markers will be used to study the source of variation in downy mildew genes and are also part our strategy to clone resistance genes.     

An integrated comparative map of the porcine X chromosome

The objectives of this study were to assign both microsatellite and gene-based markers on porcine chromosome X to two radiation hybrid (RH) panels and to develop a more extensive integrated map of SSC-X. Thirty-five microsatellite and 20 gene-based markers were assigned to T43RH, and 16 previously unreported microsatellite and 15 gene-based markers were added to IMpRH map. Of these, 30 microsatellite and 12 gene-based markers were common to both RH maps. Twenty-two gene-based markers were submitted to BLASTN analysis for identification of orthologues of genes on HSA-X. Single nucleotide polymorphisms (SNPs) were detected for 12 gene-based markers, and nine of these were placed on the genetic map. A total of 92 known loci are present on at least one porcine chromosome X map. Thirty-seven loci are present on all three maps; 31 loci are found on only one map. Location of 33 gene-based markers on the comprehensive map translates into an integrated comparative map that supports conservation of gene order between SSC-X and HSA-X. This integrated map will be valuable for selection of candidate genes for porcine quantitative trait loci (QTLs) that map to SSC-X.     

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.     

Construction, analysis, and application of a radiation hybrid mapping panel surrounding the mouse agouti locus.

The region surrounding the agouti coat color locus on mouse Chromosome 2 contains several genes required for peri-implantation development, limb morphogenesis, and segmentation of the nervous system. We have applied radiation hybrid mapping, a somatic cell genetic technique for constructing long-range maps of mammalian chromosomes, to eight molecular markers in this region. Using a mathematical model to estimate the frequency of radiation-induced breakage, we have constructed a map that spans approximately 20 recombination units and 475 centirays8000. The predicted order of markers, Prn-p-Pygb-Emv-13-Psp-Xmv-10-Emv-15-Src-Ada, is consistent with a previously derived multipoint meiotic map for six of the eight markers and suggests that Xmv-10 may lie relatively close to one or more of the agouti recessive lethal mutations. The resolution of our map is approximately 40-fold higher than the meiotic map, but the median retention frequency of mouse DNA in hybrid cells, 0.12, is 4-fold lower than similar experiments with human chromosomes. From one of the radiation hybrid lines that contained a minimum amount of mouse DNA, 25 independent cosmids were isolated with a mouse-specific hybridization probe. Single-copy fragments from two of these cosmids were shown to originate from mouse Chromosome 2, and the meiotic map position of one was found to be within 10 recombination units of the region of interest. Our results indicate more precise map positions for Pygb and Xmv-10, demonstrate that radiation hybrid mapping can provide high-resolution map information for the mouse genome, and establish a new method for isolating large fragments of DNA from a specific subchromosomal region.     

The second generation of the International Equine Gene Mapping Workshop half-sibling linkage map

A low-density, male-based linkage map was constructed as one of the objectives of the International Equine Gene Mapping Workshop. Here we report the second generation map based on testing 503 half-sibling offspring from 13 sire families for 344 informative markers using the CRIMAP program. The multipoint linkage analysis localized 310 markers (90%) with 257 markers being linearly ordered. The map included 34 linkage groups representing all 31 autosomes and spanning 2262 cM with an average interval between loci of 10.1 cM. This map is a milestone in that it is the first map with linkage groups assigned to each of the 31 automosomes and a single linkage group to all but three chromosomes.     

A gene controlling sex in grapevines placed on a molecular marker-based genetic map.

Genetic maps of Vitis (2n = 38) have been constructed from an interspecific hybrid population of 58 seedlings of the cross 'Horizon' ('Seyval' x 'Schuyler') x Illinois 547-1 (V. cinerea B9 x V. rupestris B38). The maps were initially constructed based on 277 RAPD (random amplified polymorphic DNA) markers using a double-pseudotestcross strategy. Subsequently, 25 microsatellites, 4 CAPS (cleaved amplified polymorphic sequence), and 12 AFLP (amplified fragment length polymorphism) markers were added to the maps. Another 120 markers, mostly those segregating 3:1, were also assigned but not positioned on the linkage groups in the two maps. The 'Horizon' map consisted of 153 markers covering 1199 cM, with an average map distance of 7.6 cM between markers. The Illinois 547-1 map had 179 markers covering 1470 cM, with an average map distance of 8.1 cM. There were 20 linkage groups in each map, one more than the basic number of chromosomes in grapes. Ten linkage groups in each map were identified as homologous using 16 microsatellite and 2 CAPS markers polymorphic in both parents. A single locus controlling sex in grapes mapped close to a microsatellite marker. These maps provide enough coverage of the genome for QTL (quantitative trait loci) analysis and as a starting point for positional gene cloning in grapes.