An automatic image analysis system for RLGS films

The RLGS (Restriction Landmark Genome Scanning) method was originally developed as a powerful method for enabling viewing of thousands of restriction landmarks. It offers a tool for obtaining information about genetic loci, with a single RLGS profile displaying approximately 2000 restriction landmarks as spots. One of the most useful applications is RLGS spot mapping, which allows the efficient, low-cost construction of the genetic map of any organism. However, analyses of the profiles depend mainly on human visual observation and are tedious and laborious. Although several commercially available image analyzing systems for profile comparison have been examined, they cannot be used for the RLGS spot mapping system owing to the background characteristics of the RLGS profiles, unsatisfactory rates of correspondence, and inefficient correction of informative genetic data. We therefore developed a novel automatic image analysis system for RLGS spot mapping, using an original algorithm based on the binary image transferred from the original RLGS profile. This system was employed for identifying non-polymorphic and parental strain-specific polymorphic spots of the F₁ mouse profile and yielded efficient initial screening of RLGS profiles. Received: 5 December 1997 / Accepted: 6 April 1998     

High-Speed Positional Cloning Based on Restriction Landmark Genome Scanning

Restriction landmark genome scanning (RLGS) was developed as a method of genome analysis that is based on the concept that restriction enzyme sites can be used as landmarks. In this article, we demonstrate how this method can be used for the systematic, successful positional cloning of mouse mutantreelergene. The major advantage of the RLGS method is that it allows the scanning of several thousand spots/loci throughout the genome with one RLGS profile. High-speed positional cloning based on the RLGS method includes (1) high-speed construction of a linkage map (RLGS spot mapping), (2) high-speed detection of RLGS spot markers tightly linked to the mutant phenotype (RLGS spot bombing method), and (3) construction of YAC contigs covering the region where tightly linked spot markers are located (RLGS-based YAC contig mapper). We introduced a series of these procedures by using them to positionally clone thereelergene. High-speed construction of the whole genetic map and spots/loci (less than 1 cM) within the closest flanking markers is demonstrated. The RLGS-based YAC contig mapper also efficiently yielded the YAC physical contig map of the target region. Finally, we cloned thereelergene, which is the causal gene for the perturbation of the three-dimensional brain architecture due to the abnormal migration of neuroblasts inreelermouse. Since the RLGS method itself can be used for any organism, we conclude that the total RLGS-based positional cloning system can be used to identify any mutant gene of any organism.     

Linkage map of Syrian hamster with restriction landmark genomic scanning

We have constructed the linkage map with precise genetic analysis of the Syrian hamster, Mesocricetus auratus, according to the restriction landmark genomic scanning (RLGS) spot mapping method. Although only 3.2–6.6% of the total RLGS spots between the two strains, ACN and BIO 14.6, showed genetic variance, 572 loci were found to be polymorphic. Out of 569 RLGS loci and 3 other loci, 531 were mapped with the backcross (ACN × BIO 14.6) F₁× BIO 14.6. The cumulative map was 1111.6 cM, indicating that the spots/loci are located throughout the genome at 1.94 cM intervals on average. Thus, RLGS provides us with a rapid tool to construct the genetic map of any species, even if it has less genetic variation. Received: 15 July 1996 / Accepted: 25 September 1996     

A genetic linkage map of the MSM Japanese wild mouse strain with restriction landmark genomic scanning (RLGS)

A high-resolution genetic map of the Mus musculus molossinus (MSM) Japanese wild mouse strain was constructed with restriction landmark genomic scanning (RLGS) and compared with that of the laboratory strain C3H. MSM is phylogenetically 1 million years apart from common laboratory mouse strains and is distinctly resistant to chemical carcinogenesis. Since it exhibits frequent genetic polymorphisms with laboratory mice but can still be easily crossed with laboratory strains, hybrids between MSM and carcinogen-sensitive laboratory mouse strains provide excellent materials for analysis of modifier genes and genetic changes during carcinogenesis. We have generated MSM backcross progeny with the C3H strain, which is extremely sensitive to hepatocarcinogenesis, to construct the present map. RLGS profiles with two combinations of restriction enzymes (NotI–PvuII–PstI, NotI–PstI–PvuII) yielded more than 2000 spots each. The polymorphism rate was about 39.2%, and of a total of 1732 polymorphic spot loci identified, 1371 could be assigned to specific chromosomes by comparison with 79 microsatellite marker loci. Thus, 1450 loci, on all chromosomes except for Y, effectively mapped 90% of the genome (1431.7 cM length). Although some spots might be derived from the same NotI site, each NotI site potentially generating two fragments, the presence of at least 515 loci groups with different progeny distribution patterns dispersed through the genome with an average spacing of 3 cM, means that this genetic map should be useful for analysis of various biological phenomena, including carcinogenesis and ontogenesis, at the gene level. Received: 25 August 1999 / Accepted: 20 December 1999     

Genetic profile of the SMXA recombinant inbred mouse strains revealed with restriction landmark genomic scanning

We have applied the restriction landmark genomic scanning (RLGS) method to the SMXA recombinant inbred (RI) mouse strain set to reveal its detailed genetic profile. A total of 663 polymorphic RLGS spot loci were identified, 576 of which were assigned to chromosomes. Strain distribution patterns (SDPs) at 55 microsatellite marker loci were also obtained. As a result, the total number of loci with distinct SDPs on chromosomes increased to 400. These loci were dispersed on all chromosomes, except for the Chromosome (Chr) Y, and effectively covered the genome with an average spacing of 4 cM. The SMXA RI strain set, hereby, would be of value for genetic study. Received: 20 February 1998 / Accepted: 19 May 1998     

Comparison of DNA methylation patterns among mouse cell lines by restriction landmark genomic scanning.

Restriction landmark genomic scanning (RLGS) is a novel method which enables us to simultaneously visualize a large number of loci as two-dimensional gel spots. By this method, the status of DNA methylation can efficiently be determined by monitoring the appearance or disappearance of spots by using a methylation-sensitive restriction enzyme. In the present study, using RLGS with NotI, we examined, in comparison with a brain RLGS profile, the status of DNA methylation of more than 900 loci among three types of mouse cell lines: the embryonal carcinoma cell line P19, the stable mesenchymal cell line 10T1/2, and our established neuroepithelial (EM) cell lines. We found that the relative numbers of RLGS spots which appeared were less than 3.3% of those surveyed in all cell lines examined. However, 5 to 14% of spots disappeared, the numbers increasing with an increase in the length of the culture period, and many spots were commonly lost in 10T1/2 and in three EM cell lines. Thus, for these cell lines, many more spots disappeared than appeared. However, the numbers of spots disappearing and appearing were well balanced, and the ratio in P19 cells was almost equal to that in liver cells in vivo. These RLGS experimental observations suggested that permanent cell lines such as 10T1/2 are hypermethylated and that our newly established EM cell lines are also becoming heavily methylated at common loci. On the other hand, methylation and demethylation seem to be balanced in P19 cells in a manner similar to that in in vivo liver tissue.     

Spot Mapping on the Standard Profile of Restriction Landmark Genomic Scanning (RLGS) of Sorted Chromosome 20 Using Methylation-Insensitive Enzyme

We established the spot mapping system on a restriction landmark genomic scanning (RLGS) profile using sorted chromosome as RLGS material. In this mapping system, we can mapped RLGS spots physically, regardless of their polymorphism, using methylation-insensitive enzymes in all RLGS steps. Here, we report that we identified 28 spots derived from human chromosome 20 on an RLGS profile, and that number was in good agreement with the number predicted from the length of the chromosome 20.     

A New Tool for the Rapid Cloning of Amplified and Hypermethylated Human DNA Sequences from Restriction Landmark Genome Scanning Gels

Restriction landmark genome scanning (RLGS) is an effective genome-scanning technique capable of identifying DNA amplification and aberrant DNA methylation. Previously published methods for the cloning of human DNA fragments from RLGS gels have been successful only for high-copy-number fragments (repetitive elements or DNA amplifications). We present here the first technique capable of efficiently cloning single-copy human DNA fragments ("spots") identified in RLGS profiles. This technique takes advantage of a plasmid-based, human genomic DNA, NotI/EcoRV boundary library. The library is arrayed in microtiter plates. When clones from a single plate are pooled and mixed with genomic DNA, the resultant RLGS gel is a normal profile with a defined set of spots showing enhanced intensity for that particular plate. This was performed for a set of 32 plates as well as their pooled rows and columns. Thus, we have mapped individual RLGS spots to exact plate, row, and column addresses in the library and have thereby obtained immediate access to these clones. The feasibility of the technique is demonstrated in examples of cloning methylated DNA fragments identified in human breast tumor and testicular tumor RLGS profiles and in the cloning of an amplified DNA fragment identified in a human medulloblastoma RLGS profile.     

Epigenetics: application of virtual image restriction landmark genomic scanning (Vi-RLGS)

Restriction landmark genomic scanning (RLGS) is a powerful method for the systematic detection of genetic mutations in DNA length and epigenetic alteration due to DNA methylation. However, the identification of polymorphic spots is difficult because the resulting RLGS spots contain very little target DNA and many non-labeled DNA fragments. To overcome this, we developed a virtual image restriction landmark genomic scanning (Vi-RLGS) system to compare actual RLGS patterns with computer-simulated RLGS patterns (virtual RLGS patterns). Here, we demonstrate in detail the contents of the simulation program (rlgssim), based on the linear relationship between the reciprocal of mobility plotted against DNA fragment length and Vi-RLGS profiling of Arabidopsis thaliana.     

A Genetic Linkage Map of the Mouse Using Restriction Landmark Genomic Scanning (Rlgs)

We have developed a multiplex method of genome analysis, restriction landmark genomic scanning (RLGS) that has been used to construct genetic maps in mice. Restriction landmarks are end-labeled restriction fragments of genomic DNA that are separated by using high resolution, two-dimensional gel electrophoresis identifying as many as two thousand landmark loci in a single gel. Variation for several hundred of these loci has been identified between laboratory strains and between these strains and Mus spretus. The segregation of more than 1100 RLGS loci has been analyxed in recombinant inbred (RI) strains and in two separate interspecific genetic crosses. Genetic maps have been derived that link 1045 RLGS loci to reference loci on all of the autosomes and the X chromosome of the mouse genome. The RLGS method can be applied to genome analysis in many different organisms to identify genomic loci because it used end-labeling of restriction landmarks rather than probe hybridization. Different combinations of restriction enzymes yield different sets of RLGS loci providing expanded power for genetic mapping.     

Punctuated Distribution of Recombination Hotspots and Demarcation of Pericentromeric Regions in Phaseolus vulgaris L.

High density genetic maps are a reliable tool for genetic dissection of complex plant traits. Mapping resolution is often hampered by the variable crossover and non-crossover events occurring across the genome, with pericentromeric regions (pCENR) showing highly suppressed recombination rates. The efficiency of linkage mapping can further be improved by characterizing and understanding the distribution of recombinational activity along individual chromosomes. In order to evaluate the genome wide recombination rate in common beans (Phaseolus vulgaris L.) we developed a SNP-based linkage map using the genotype-by-sequencing approach with a 188 recombinant inbred line family generated from an inter gene pool cross (Andean x Mesoamerican). We identified 1,112 SNPs that were subsequently used to construct a robust linkage map with 11 groups, comprising 513 recombinationally unique marker loci spanning 943 cM (LOD 3.0). Comparative analysis showed that the linkage map spanned >95% of the physical map, indicating that the map is almost saturated. Evaluation of genome-wide recombination rate indicated that at least 45% of the genome is highly recombinationally suppressed, and allowed us to estimate locations of pCENRs. We observed an average recombination rate of 0.25 cM/Mb in pCENRs as compared to the rest of genome that showed 3.72 cM/Mb. However, several hot spots of recombination were also detected with recombination rates reaching as high as 34 cM/Mb. Hotspots were mostly found towards the end of chromosomes, which also happened to be gene-rich regions. Analyzing relationships between linkage and physical map indicated a punctuated distribution of recombinational hot spots across the genome.     

A genetic linkage map for the ectomycorrhizal fungus Laccaria bicolor and its alignment to the whole-genome sequence assemblies

A genetic linkage map for the ectomycorrhizal basidiomycete Laccaria bicolor was constructed from 45 sib-homokaryotic haploid mycelial lines derived from the parental S238N strain progeny. For map construction, 294 simple sequence repeats (SSRs), single-nucleotide polymorphisms (SNPs), amplified fragment length polymorphisms (AFLPs) and random amplified polymorphic DNA (RAPD) markers were employed to identify and assay loci that segregated in backcross configuration. Using SNP, RAPD and SSR sequences, the L. bicolor whole-genome sequence (WGS) assemblies were aligned onto the linkage groups. A total of 37.36 Mbp of the assembled sequences was aligned to 13 linkage groups. Most mapped genetic markers used in alignment were colinear with the sequence assemblies, indicating that both the genetic map and sequence assemblies achieved high fidelity. The resulting matrix of recombination rates between all pairs of loci was used to construct an integrated linkage map using JoinMap. The final map consisted of 13 linkage groups spanning 812 centiMorgans (cM) at an average distance of 2.76 cM between markers (range 1.9-17 cM). The WGS and the present linkage map represent an initial step towards the identification and cloning of quantitative trait loci associated with development and functioning of the ectomycorrhizal symbiosis.     

Direct Determination of NotI Cleavage Sites in the Genomic DNA of Adult Mouse Kidney and Human Trophoblast Using Whole-Range Restriction Landmark Genomic Scanning

Restriction landmark genomic scanning (RLGS) is a method forvisualizing restriction landmarks, employing direct labelingof restriction sites of genomic DNA and high-resolution two-dimensionalelectrophoresis. We determined the conditions for both the firstand second dimensions of RLGS that define all of the restrictionfragments which carry the NotI landmark. Using this system,we determined the number of cleavable NotI sites of genomicDNA from the mouse kidney (C57BL/6) and from the human placenta.The mouse and human genomes were cleaved at 2,380±80sites (4,760±160 spots) and 3,240±110 sites (6,480±220spots), respectively with NotI.     

Construction of a genetic linkage map in man using restriction fragment length polymorphisms.

We describe a new basis for the construction of a genetic linkage map of the human genome. The basic principle of the mapping scheme is to develop, by recombinant DNA techniques, random single-copy DNA probes capable of detecting DNA sequence polymorphisms, when hybridized to restriction digests of an individual's DNA. Each of these probes will define a locus. Loci can be expanded or contracted to include more or less polymorphism by further application of recombinant DNA technology. Suitably polymorphic loci can be tested for linkage relationships in human pedigrees by established methods; and loci can be arranged into linkage groups to form a true genetic map of "DNA marker loci." Pedigrees in which inherited traits are known to be segregating can then be analyzed, making possible the mapping of the gene(s) responsible for the trait with respect to the DNA marker loci, without requiring direct access to a specified gene's DNA. For inherited diseases mapped in this way, linked DNA marker loci can be used predictively for genetic counseling.     

Mapping genes for common diseases: the case for genetic (LD) maps

We examine the current effort to develop a haplotype map of the human genome and suggest an alternative approach which represents linkage disequilibrium patterns in the form of a metric LD map. LD maps have some of the useful properties of genetic linkage maps but have a much higher resolution which is optimal for SNP-based association mapping of common diseases. The studies that have been undertaken to date suggest that LD and recombination maps show some close similarities because of abundant, narrow, recombination hot spots. These hot spots are co-localised in all populations but, unlike linkage maps, LD maps differ in scale for different populations because of differences in population history. The prospects for developing optimized panels of SNPs and the use of linkage disequilibrium maps in disease gene localisation are assessed in the light of recent evidence.     

Second-generation integrated genetic linkage/radiation hybrid maps of the domestic cat (Felis catus)

We report construction of second-generation integrated genetic linkage and radiation hybrid (RH) maps in the domestic cat (Felis catus) that exhibit a high level of marker concordance and provide near-full genome coverage. A total of 864 markers, including 585 coding loci (type I markers) and 279 polymorphic microsatellite loci (type II markers), are now mapped in the cat genome. We generated the genetic linkage map utilizing a multigeneration interspecies backcross pedigree between the domestic cat and the Asian leopard cat (Prionailurus bengalensis). Eighty-one type I markers were integrated with 247 type II markers from a first-generation map to generate a map of 328 loci (320 autosomal and 8 X-linked) distributed in 47 linkage groups, with an average intermarker spacing of 8 cM. Genome coverage spans approximately 2,650 cM, allowing an estimate for the genetic length of the sex-averaged map as 3,300 cM. The 834-locus second-generation domestic cat RH map was generated from the incorporation of 579 type I and 255 type II loci. Type I markers were added using targeted selection to cover either genomic regions underrepresented in the first-generation map or to refine breakpoints in human/feline synteny. The integrated linkage and RH maps reveal approximately 110 conserved segments ordered between the human and feline genomes, and provide extensive anchored reference marker homologues that connect to the more gene dense human and mouse sequence maps, suitable for positional cloning applications.     

A map of the distal region of the long arm of human chromosome 21 constructed by radiation hybrid mapping and pulsed-field gel electrophoresis

We have used radiation hybrid (RH) mapping and pulsed-field gel electrophoresis (PFGE) to determine the order and positions of 28 DNA markers from the distal region of the long arm of human chromosome 21. The maps generated by these two methods are in good agreement. This study, combined with that of D. R. Cox et al. (1990, Science 250:245-250), results in an RH map that covers the long arm of chromosome 21 (21q). We have used a subtelomeric probe to show that our map includes the telomere and have identified single-copy genes and markers within 200 kbp of the telomere. Comparison of the physical and RH maps with genetic linkage maps shows "hot spots" of meiotic recombination in the distal region, one of which is close to the telomere, in agreement with previous cytogenetic observations of increased recombination frequency near telomeres.     

A genetic linkage map of the rat derived from recombinant inbred strains

We have constructed a genetic linkage map in the rat by analyzing the strain distribution patterns of 500 genetic markers in a large set of recombinant inbred strains derived from the spontaneously hypertensive rat and the Brown-Norway rat (HXB and BXH recombinant inbred strains). 454 of the markers could be assigned to specific chromosomes, and the amount of genome covered by the mapped markers was estimated to be 1151 centimorgans. By including a variety of morphologic, biochemical, immunogenetic, and molecular markers, the current map integrates and extends existing linkage data and should facilitate rat gene mapping and genetic studies of hypertension and other complex phenotypes of interest in the HXB and BXH recombinant inbred strains. Received: 21 June 1995 / Accepted: 11 September 1995     

Construction of a 10,000-marker ultradense genetic recombination map of potato: providing a framework for accelerated gene isolation and a genomewide physical map

An ultradense genetic linkage map with >10,000 AFLP loci was constructed from a heterozygous diploid potato population. To our knowledge, this is the densest meiotic recombination map ever constructed. A fast marker-ordering algorithm was used, based on the minimization of the total number of recombination events within a given marker order in combination with genotyping error-detection software. This resulted in "skeleton bin maps," which can be viewed as the most parsimonious marker order. The unit of distance is not expressed in centimorgans but in "bins." A bin is a position on the genetic map with a unique segregation pattern that is separated from adjacent bins by a single recombination event. Putative centromeres were identified by a strong clustering of markers, probably due to cold spots for recombination. Conversely, recombination hot spots resulted in large intervals of up to 15 cM without markers. The current level of marker saturation suggests that marker density is proportional to physical distance and independent of recombination frequency. Most chromatids (92%) recombined once or never, suggesting strong chiasma interference. Absolute chiasma interference within a chromosome arm could not be demonstrated. Two examples of contig construction and map-based cloning have demonstrated that the marker spacing was in accordance with the expected physical distance: approximately one marker per BAC length. Currently, the markers are used for genetic anchoring of a physical map of potato to deliver a sequence-ready minimal tiling path of BAC contigs of specific chromosomal regions for the potato genome sequencing consortium (http://www.potatogenome.net).     

Genetic linkage map of the guppy,Poecilia reticulata,and quantitative trait loci analysis of male size and colour variation

We report construction of a genetic linkage map of the guppy genome using 790 single nucleotide polymorphism markers, integrated from six mapping crosses. The markers define 23 linkage groups (LGs), corresponding to the known haploid number of guppy chromosomes. The map, which spans a genetic length of 899 cM, includes 276 markers linked to expressed genes (expressed sequence tag), which have been used to derive broad syntenic relationships of guppy LGs with medaka chromosomes. This combined linkage map should facilitate the advancement of genetic studies for a wide variety of complex adaptive phenotypes relevant to natural and sexual selection in this species. We have used the linkage data to predict quantitative trait loci for a set of variable male traits including size and colour pattern. Contributing loci map to the sex LG for many of these traits.