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Physical and genetic map of the Finegoldia magna (formerly Peptostreptococcus magnus) ATCC 29328 genome.

A physical and genetic map of Finegoldia magna (formerly Peptostreptococcus magnus) ATCC 29328 chromosome was constructed. The order of rare cleavage restriction fragments was determined by double digestion with the restriction enzymes I-CeuI, SgrAI, ApaI and PmeI, cross-hybridization and ApaI-linking clones. The size of the circular chromosome of F. magna was estimated to be 1.9 Mb. This strain also had a 200-kb megaplasmid. The chromosome contained four rrn operons, and the orientation of two rrn operons was opposite to the others. Fragment analysis of Peptostreptococcus anaerobius ATCC 27337(T) chromosome suggested that its size was much smaller than that of F. magna ATCC 29328.

Chromosome Mapping↗

Genetic map construction with constraints.

A pilot program, CME, is described for generating a physical genetic map from hybridization fingerprinting data. CME is implemented in the parallel constraint logic programming language ElipSys. The features of constraint logic programming are used to enable the integration of pre-existing mapping information (partial probe orders from cytogenetic maps and local physical maps) into the global map generation process, while parallelism enables the search space to be traversed more efficiently. CME was tested using data from chromosome 2 of Schizosaccharomyces pombe and was found able to generate maps as well as (and sometimes better than) a more traditional method. This paper illustrates the practical benefits of using a symbolic logic programming language and shows that the features of constraint handling and parallel execution bring the development of practical systems based on AI programming technologies nearer to being a reality.

Algorithms↗

Genetic mapping of 20 novel expressed sequence tags from midgestation mouse embryos suggests chromosomal clustering.

Current knowledge of genes that regulate pattern formation and differentiation processes during mammalian embryonic development is limited. In an effort to isolate developmentally relevant genes, 20 novel, end-sequenced cDNAs selected from a Day 10.5 postcoitum mouse embryo library were genetically mapped in intersubspecific backcross mice. Eleven of 20 cDNA clones mapped to three mouse autosomes (Chr 5, 11, and 14), a result that was unlikely (P < 0.03) if the distribution of genes expressed in embryos is random within the mouse genome. Several clones were candidates for mouse developmental mutations by virtue of genetic colocalization and concordance of embryonic expression patterns with the distribution of defects in mutant mice: Estm11 was a candidate for the mouse mutation wabbler-lethal (wl), since Estm11 mapped in the vicinity of wl on mouse Chr 14 and was expressed in those regions of embryonic brain that exhibit axonal degeneration in wl. End-sequence analysis, genetic mapping, and in situ hybridization appeared to be an effective combination of methods for identification and characterization of genes with potential regulatory functions during mammalian embryogenesis.

Animals↗

Heteroduplexes of phiX174 and G4 DNAs: orientation to genetic map and comparison with predictions from nucleotide sequences.

Heteroduplexes between the viral DNA of phiX174 and DNA from the replicative form (RF) of phage G4 were examined by electron microscopy. The single Eco RI site of G4-RF was utilized as a physical marker by preparing the heteroduplexes from the denatured, linear DNA obtained by restricting G4-RF with Eco RI endonuclease. Restriction fragments of phiX were used in a separate series of heteroduplexes to align the heteroduplex map and the G4 Eco RI site with the similar genetic maps of the two phages. The positions of the branch migrating junctions of recombinant phiX-G4 figure-8s, previously located only with respect to the G4-Eco RI site, have now been located with high proability within the gene A region of the two genomes. The degree of mismatch between the known nucleotide sequences of phi X and G4 accounts for positions of all of the regions of single-strandedness in the observed heteroduplexes, but unexplained discrepancies were also found.

Base Sequence↗

Development of a microsatellite genetic map spanning 5q31-q33 and subsequent placement of the LGMD1A locus between D5S178 and IL9.

Limb-girdle muscular dystrophy (LGMD) is a genetically and clinically heterogeneous group of disorders. We previously localized an autosomal dominant form of the disorder (LGMD1A) to chromosome 5q22-31 by linkage analysis in a single large pedigree. After developing a microsatellite genetic map incorporating six loci in q31-33 of chromosome 5 and spanning 35 cM, we have refined the original localization. Using multipoint analysis, LGMD1A is localised to a 7 cM region between the markers IL9 and D5S178 with odds > 1000:1.

Chromosome Mapping↗

Transformation of the cyanobacterium Synechocystis sp. PCC 6803 as a tool for genetic mapping: optimization of efficiency.

The cyanobacterium Synechocystis sp. PCC 6803 is transformable at high efficiency and integrates DNA by homologous double recombination. However, several genetic mapping procedures depend on the ability to generate transformants even with very small amounts of added DNA. This study is aimed at optimizing the transformation efficiency at limiting concentrations of exogenous DNA. The transformation efficiency showed little sensitivity to experimental conditions. Transformation with circular plasmid DNA was found to be no more than 30% more efficient than with linearized plasmid DNA. The efficiency of transformation remained essentially the same in the presence of competing DNA, indicating that the capacity of DNA uptake by the cells is not limiting. The incubation time of cells with DNA before plating (0-8 h) affected the transformation efficiency by up to 3-fold. Only minor changes in the efficiency were observed as a function of the presence of a membrane filter on the plate or the presence of TAE or TBE gel buffer residues in the transformation mixture. However, transformability of the host strain of Synechocystis sp. PCC 6803 was increased by two orders of magnitude if the sll1354 gene encoding the exonuclease RecJ was deleted. Therefore, the transformation efficiency of Synechocystis sp. PCC 6803 with exogenous DNA appears to be determined primarily by intracellular processes such as the efficiency of DNA processing and homologous recombination.

Bacterial Proteins↗

A genetic map of Cottus gobio (Pisces, Teleostei) based on microsatellites can be linked to the physical map of Tetraodon nigroviridis.

To initiate QTL studies in the nonmodel fish Cottus gobio we constructed a genetic map based on 171 microsatellite markers. The mapping panel consisted of F1 intercrosses between two divergent Cottus lineages from the River Rhine System. Basic local alignment search tool (BLAST) searches with the flanking sequences of the microsatellite markers yielded a significant (e < 10(-5)) hit with the Tetraodon nigroviridis genomic sequence for 45% of the Cottus loci. Remarkably, most of these hits were due to short highly conserved noncoding stretches. These have an average length of 40 bp and are on average 92% conserved. Comparison of the map locations between the two genomes revealed extensive conserved synteny, suggesting that the Tetraodon genomic sequence will serve as an excellent genomic reference for at least the Acanthopterygii, which include evolutionarily interesting fish groups such as guppies (Poecilia), cichlids (Tilapia) or Xiphophorus (Platy). The apparent high density of short conserved noncoding stretches in these fish genomes will highly facilitate the identification of genes that have been identified in QTL mapping strategies of evolutionary relevant traits.

Animals↗

Escherichia coli molecular genetic map (1500 kbp): update II.

The DNA sequence data for Escherichia coli deposited in the EMBL library (release 27), together with miscellaneous data obtained from several laboratories, have been localized on an updated and corrected version of the restriction map of the chromosome generated by Kohara et al. (1987) and modified by others. This second update adds a further 500 kbp, increasing the amount of the E. coli chromosome sequenced to about one third of the total: 1510 kbp of sequenced DNA is included in the present data base. The accuracy of the map is assessed, and allows us to propose a precise genetic map position for every sequenced gene. The location of rare-cutting sites such as AvrII, NotI and SfiI have also been included in the update in order to combine the data obtained from different sources into one single file. The distribution of palindromic sequences (to which most restriction sites belong) has been studied in coding sequences. There appears to be a significant counter-selection against several such sequences in E. coli coding sequences (but not in other organisms such as Saccharomyces cerevisiae), suggesting the existence of constraints on DNA structure in E. coli, perhaps indicative of a functional role for horizontal gene transfer, preserving coding sequences, in this type of bacteria.

Base Sequence↗

High-resolution genetic map of bovine chromosome 29 through focused marker development.

Chromosome-specific libraries aid in the development of genetic maps and focus marker development in areas of the genome with identified quantitative trait loci (QTL). A small-insert BTA29 library constructed by microdissection of a 1:29 Rb-fusion cell line, was screened for dinucleotide repeats (CA)(15) and/or (GA)(15) with the goal of generating new genetic markers for this, the smallest bovine autosome. A total of 90 primer pairs were designed and 82 of these successfully amplified bovine genomic DNA by PCR. In addition to these 82 loci, primer pairs were developed for nine putative genes identified from the sequenced clones by BLAST searches of GenBank. A somatic cell panel was used to test for synteny of the new loci with two previously mapped BTA29 markers located on the MARC bovine linkage map. A total of 75 of the 82 microsatellite (ms) loci were integrated into the MARC bovine linkage map. Linkage analysis placed 69 ms markers on BTA29, five on BTAX and one on BTA1. Combined results of the somatic cell and linkage analyses place 79 new markers (ms and gene-related) on BTA29, six loci on BTAX and two loci on BTA1. The results of this effort significantly increase the marker density on BTA29, expanding the ability to fine map QTL associated with this chromosome.

Animals↗

An intraspecific genetic map of velvetbean (Mucuna sp.) based on AFLP markers.

Velvetbean ( Mucuna sp., n=11), a self-pollinated species, is an important legume used in tropical agricultural systems in rotation with other crops for nematode management and/or soil improvement. A genetic map of velvetbean was constructed in order to identify potential molecular markers linked to important morphological and agronomic traits that would be particularly useful for developing and improving the species. Traits such as seed coat color, pod color, and pod pubescence were among the main parameters observed in a process of genetic diversity estimation. Two slightly divergent velvetbean accessions, PI364362 and 'Edgar Farm White', a land race from Alabama, were used to make an intraspecific F(1) hybrid. Amplified fragment length polymorphism analysis (AFLP) detected an average of six polymorphic fragments per primer pair between the two parents. As expected for dominant markers, the sum of all AFLP bands from both parents was generally observed to be present in the AFLP profiles of the F(1) progeny, indicating full penetrance and the dominant nature of AFLP markers. An F(2) population was generated by self-pollinating a single F(1) plant. Using 37 AFLP primer pairs, we detected 233 polymorphic markers of which 164 (70.4%) segregated in 3:1 Mendelian ratios, while the remaining 69 (29.6%) both segregated and were scorable. The genetic linkage map constructed from this population comprised 166 markers, including two morphological traits (pod color and pod pubescence). Twenty linkage groups were found with an average distance between markers of 34.4 cM, covering a total of 687.9 cM. The linkage groups contained from 2 to 12 loci each and the distance between two consecutive loci ranged from 0 to 21.8 cM. The newly designated morphological traits pod color ( pdc) and pod pubescence ( pdp) co-segregated with each other at a distance of 4.2 cM. Two DNA markers designated ACGCAG2 and ACTCTG1 were located in the same group as pdc and pdp. The AFLP linkage map provides opportunities for use in marker-assisted selection and in the detection of loci controlling morphologically important traits.

Genetic Linkage↗

Genetic map of the Staphylococcal bacteriophage phi11.

Ten sus mutants of the staphylococcal bacteriophage phi 11, each a representative from a different complementation group, have been used in three-factor cross experiments. The results of these crosses indicate a circular genetic map for phi 11. Functional studies of the mutants have been limited to electron microscopic examinations of lysates after prophage induction (or infection). One gene is an early gene, five genes are concerned with tail formation, and three are concerned with head formation. The tenth gene is possibly a head gene. The contribution by phi 11 to the genomic content of the plasmid-phage hybrid phi 11 de has been investigated. Phi 11 de contains most of the late genes and appears to be missing a continuous phi 11 segment that includes the early gene flanked by two late genes.

Bacteriolysis↗

An integrated physical and genetic map of the nematode Pristionchus pacificus.

The free-living nematode Pristionchus pacificus is one of several species that have recently been developed as a satellite system for comparative functional studies in evolutionary developmental biology. Comparisons of developmental processes between P. pacificus and the well established model organism Caenorhabditis elegans at the cellular and genetic levels provide detailed insight into the molecular changes that shape evolutionary transitions. To facilitate genetic analysis and cloning of mutations in P. pacificus, we previously generated a BAC-based genetic linkage map for this organism. Here, we describe the construction of a physical map of the P. pacificus genome based on AFLP fingerprint analysis of 7747 BAC clones. Most of the SSCP markers used to generate the genetic linkage map were derived from BAC ends, so that the physical genome map and the genetic map can be integrated. The contigs that make up the physical map are evenly distributed over the genetic linkage map and no clustering is observed, indicating that the physical map provides a valid representation of the P. pacificus genome. The integrated genome map thus provides a framework for positional cloning and the study of genome evolution in nematodes.

Animals↗

Rapid genetic mapping of ESTs using SNP pyrosequencing and indel analysis.

We describe an effective systematic approach to genetic mapping of cDNA clones, including those obtained from EST sequencing. The EST of interest is first partially sequenced from the 3'-end. PCR primers which bracket the 3'-UTR segment of the cDNA are designed. The corresponding gene segment is amplified from the parents of the mapping population, using primers equipped with 3'- and 5'-extensions to facilitate direct sequencing of PCR products. Comparison of the sequences obtained from the mapping parents frequently reveals single nucleotide polymorphisms or insertion / deletion polymorphisms, which can then be genotyped in a mapping population. The genotyping of SNPs is performed by pyrosequencing, a sequencing-by-synthesis method that has been used successfully in SNP diagnostics. SNP analysis of up to 96 samples, a number required to produce meaningful genetic segregation data, can be rapidly accomplished in parallel. The parental genotype of three loci, stearoyl-ACP desaturase, nucleoside-diphosphate kinase and sucrose synthetase-1 were determined by conventional sequencing, and the polymorphism so identified were scored by the pyrosequencing of 94 individuals of a maize recombinant-inbred population. These loci were successfully placed onto chromosomes 3, 7 and 9 respectively. This method is generally applicable to most plant species, which show sufficient sequence diversity in the 3'-UTR region of genes.

3' Untranslated Regions↗

The Oenococcus oeni genome: physical and genetic mapping of strain GM and comparison with the genome of a 'divergent' strain, PSU-1.

The physical and genetic maps of the Oenococcus oeni strains GM and PSU-1, which represent two genomic divergent groups on the basis of macrorestriction and ribotyping analysis, were compared. To achieve this comparison, the GM maps were constructed and the PSU-1 maps, already established, were improved. All the recognition sites of the restriction enzymes ASC:I, I-CEU:I, FSE:I, NOT:I and SFI:I were located in both chromosomes and the position of 26 genetic markers, including two rrn operons and 14 new putative oenococcal genes, were allocated to the restriction fragments generated by the five enzymes. The comparative analysis of O. oeni GM and PSU-1 genomes revealed extensive conservation of loci order. As for the differences encountered in the locations of restriction sites, they seem to be a reflection of the differences in restriction fragment sizes, explainable by insertion/deletion events and point mutations. No evidence for major genomic rearrangements was found. The genomic conservation between the two strains is in agreement and suggests homogeneity within the species, which was not unexpected in view of the restricted ecological niche of O. oeni. Further comparisons of physical maps, both of O. oeni strains and related species, will certainly help to assess whether O. oeni is really an homogeneous species and physical mapping is suitable for taxonomic purposes, both at the supra- and intraspecific levels.

Chromosome Mapping↗

An analytical formula to estimate confidence interval of QTL location with a saturated genetic map as a function of experimental design.

Analytical formulae are derived for the confidence interval for location of a quantitative trait locus (QTL) using a saturated genetic map, as a function of the experimental design, the QTL allele substitution effect, and the number of individuals genotyped and phenotyped. The formulae are derived assuming evenly spaced recombination events, rather than the actual unevenly spaced distribution. The formulae are useful for determining desired sample size when designing a wide variety of QTL mapping experiments, and for evaluating a priori the potential of a given mapping population for defining the location of a QTL. The formulae do not take into account the finite number of recombination events in a given sample.

Animals↗

Fine genetic mapping and BAC contig development for the citrus tristeza virus resistance gene locus in Poncirus trifoliata (Raf.).

A map-based cloning strategy has been employed to isolate Ctv, a single dominant gene from Poncirus trifoliata that confers resistance to citrus tristeza virus (CTV), the most important viral pathogen of citrus. Cloning of this gene will allow development of commercially acceptable, virus-resistant cultivars. A high-resolution genetic linkage map of the Ctv locus region was developed using a backcross population of 678 individuals. Three DNA markers that were closely linked or co-segregated with Ctv were identified and used to screen BAC libraries derived from an intergeneric hybrid of Poncirus and Citrus. Through chromosome walking and landing, two BAC contigs were developed: one encompassing the Ctv region, and the other spanning the allelic susceptibility gene region. The resistance gene contig consists of 20 BAC clones and is approximately 550 kb in length; the susceptibility gene contig consists of 16 BAC clones and extends about 450 kb. The Ctv locus was localized within a genomic region of approximately 180 kb by genetic mapping of BAC insert ends. The BAC contigs were integrated with the genetic map; variation in the ratio of genetic to physical distance was observed in the vicinity of Ctv. Southern hybridization data indicated that a few copies of NBS-LRR class sequences are distributed at or around the Ctv locus. Efforts are being made to assign the Ctv locus to a smaller genomic fragment whose function can be confirmed through genetic complementation of a CTV susceptible phenotype. These results indicate that map-based gene cloning is feasible in a woody perennial.

Blotting, Southern↗

High-resolution genetic mapping with ordered arrays of Saccharomyces cerevisiae deletion mutants.

We present a method for high-resolution genetic mapping that takes advantage of the ordered set of viable gene deletion mutants, which form a set of colinear markers covering almost every centimorgan of the Saccharomyces cerevisiae genome, and of the synthetic genetic array (SGA) system, which automates the construction of double mutants formed by mating and meiotic recombination. The Cbk1 kinase signaling pathway, which consists minimally of CBK1, MOB2, KIC1, HYM1, and TAO3 (PAG1), controls polarized morphogenesis and activation of the Ace2 transcription factor. Deletion mutations in the Cbk1 pathway genes are tolerated differently by common laboratory strains of S. cerevisiae, being viable in the W303 background but dead in the S288C background. Genetic analysis indicated that the lethality of Cbk1 pathway deletions in the S288C background was suppressed by a single allele specific to the W303 background. SGA mapping (SGAM) was used to locate this W303-specific suppressor to the SSD1 locus, which contains a known polymorphism that appears to compromise SSD1 function. This procedure should map any mutation, dominant or recessive, whose phenotype is epistatic to wild type, that is, a phenotype that can be scored from a mixed population of cells obtained by germination of both mutant and wild-type spores. In principle, SGAM should be applicable to the analysis of multigenic traits. Large-scale construction of ordered mutations in other model organisms would broaden the application of this approach.

Adaptor Proteins, Signal Transducing↗

QTL mapping of ten agronomic traits on the soybean ( Glycine max L. Merr.) genetic map and their association with EST markers.

A set of 184 recombinant inbred lines (RILs) derived from soybean vars. Kefeng No.1 x Nannong 1138-2 was used to construct a genetic linkage map. The two parents exhibit contrasting characteristics for most of the traits that were mapped. Using restricted fragment length polymorphisms (RFLPs), simple sequence repeats (SSRs) and expressed sequence tags (ESTs), we mapped 452 markers onto 21 linkage groups and covered 3595.9 cM of the soybean genome. All of the linkage groups except linkage group F were consistent with those of the consensus map of Cregan et al. (1999). Linkage group F was divided into two linkage groups, F1 and F2. The map consisted of 189 RFLPs, 219 SSRs, 40 ESTs, three R gene loci and one phenotype marker. Ten agronomic traits-days to flowering, days to maturity, plant height, number of nodes on main stem, lodging, number of pods per node, protein content, oil content, 100-seed weight, and plot yield-were studied. Using winqtlcart, we detected 63 quantitative trait loci (QTLs) that had LOD>3 for nine of the agronomic traits (only exception being seed oil content) and mapped these on 12 linkage groups. Most of the QTLs were clustered, especially on groups B1 and C2. Some QTLs were mapped to the same loci. This pleiotropism was common for most of the QTLs, and one QTL could influence at most five traits. Seven EST markers were found to be linked closely with or located at the same loci as the QTLs. EST marker GmKF059a, encoding a repressor protein and mapped on group C2, accounted for about 20% of the total variation of days to flowering, plant height, lodging and nodes on the main stem, respectively.

Agriculture↗