Linkage: from particulate to interactive genetics

Genetics was established on a strictparticulate conception of heredity. Geneticlinkage, the deviation from independentsegregation of Mendelian factors, was conceivedas a function of the material allocation of thefactors to the chromosomes, rather than to themultiple effects (pleiotropy) of discretefactors. Although linkage maps wereabstractions they provided strong support forthe chromosomal theory of inheritance. DirectCytogenetic evidence was scarce until X-rayinduced major chromosomal rearrangementsallowed direct correlation of genetic andcytological rearrangements. Only with thediscovery of the polytenic giant chromosomes inDrosophila larvae in the 1930s were thevirtual maps backed up by physical maps of thegenetic loci. Genetic linkage became a pivotalexperimental tool for the examination of theintegration of genetic functions in developmentand in evolution. Genetic mapping has remaineda hallmark of genetic analysis. The location ofgenes in DNA is a modern extension of thenotion of genetic linkage.     

Mouse linkage cytogenetics (L-C) probes

We have identified 149 hybridization probes at 10-cM intervals in the mouse and have confirmed their order and linkage by fluorescence in situ hybridization. These probes represent a new resource for mapping in the mouse and can be used to correlate linkage and cytogenetic maps, to map novel sequences to within a few centimorgans, to relate cytogenetic abnormalities to the genetic map, and to make cross-species comparisons.     

Estimating the power of a proposed linkage study for a complex genetic trait.

Many genetic traits have complex modes of inheritance; they may exhibit incomplete or age-dependent penetrance or fail to show any clear Mendelian inheritance pattern. As primary linkage maps for the human genome near completion, it is becoming increasingly possible to map these traits. Prior to undertaking a linkage study, it is important to consider whether the pedigrees available for the proposed study are likely to provide sufficient information to demonstrate linkage, assuming a linked marker is tested. In the current paper, we describe a computer simulation method to estimate the power of a proposed study to detect linkage for a complex genetic trait, given a hypothesized genetic model for the trait. Our method simulates trait locus genotypes consistent with observed trait phenotypes, in such a way that the probability to detect linkage can be estimated by sample statistics of the maximum lod score distribution. The method uses terms available when calculating the likelihood of the trait phenotypes for the pedigree and is applicable to any trait determined by one or a few genetic loci; individual-specific environmental effects can also be dealt with. Our method provides an objective answer to the question, Will these pedigrees provide sufficient information to map this complex genetic trait?     

Improvements to the GDB Human Genome Data Base.

Version 6.0 of the Human Genome Data Base introduces a number of significant improvements over previous releases of GDB. The most important of these are revised data representations for genes and genomic maps and a new curatorial model for the database. GDB 6.0 is the first major genomic database to provide read/write access directly to the scientific community, including capabilities for third-party annotation. The revised database can represent all major categories of genetic and physical maps, along with the underlying order and distance information used to construct them. The improved representation permits more sophisticated map queries to be posed and supports the graphical display of maps. In addition the new GDB has a richer model for gene information, better suited for supporting cross-references to databases describing gene function, structure, products, expression and associated phenotypes.     

Segregation distortion in Lolium: evidence for genetic effects

Segregation distortion (SD) is the deviation of genetic segregation ratios from their expected Mendelian fraction and is a common phenomenon found in most genetic mapping studies. In this study two segregating Lolium perenne populations were used to construct two genetic maps: an 'F(2) biomass' consisting of 360 genotypes and an 'F(1) late flowering' sibling based population consisting of 182 genotypes. Additionally two parental maps were generated for the 'F(1) late flowering' population. SD was detected and p-values for SD were calculated for each marker locus. The 'F(1) late flowering' map had only half of the extent of SD (32%) compared to the map based on the 'F(2) biomass' population (63%). Molecular marker data have been supplemented with genomic in situ hybridization (GISH) data to show non major non-recombined segments of Fescue chromosomes within the parental inbred ryegrass lines with a Festuca x Lolium pedigree. We conclude that SD in our study is more likely caused by genetic effects rather than by population structure and marker types. Two new L. perenne mapping populations including their genetic maps are introduced; one of them is the largest reported Lolium mapping population consisting of 360 individuals.     

High-resolution single-copy gene fluorescence in situ hybridization and its use in the construction of a cytogenetic map of maize chromosome 9

High-resolution cytogenetic maps provide important biological information on genome organization and function, as they correlate genetic distance with cytological structures, and are an invaluable complement to physical sequence data. The most direct way to generate a cytogenetic map is to localize genetically mapped genes onto chromosomes by fluorescence in situ hybridization (FISH). Detection of single-copy genes on plant chromosomes has been difficult. In this study, we developed a squash FISH procedure allowing successful detection of single-copy genes on maize (Zea mays) pachytene chromosomes. Using this method, the shortest probe that can be detected is 3.1 kb, and two sequences separated by approximately 100 kb can be resolved. To show the robust nature of this protocol, we localized nine genetically mapped single-copy genes on chromosome 9 in one FISH experiment. Integration of existing information from genetic maps and the BAC contig-based physical map with the cytological structure of chromosome 9 provides a comprehensive cross-referenced cytogenetic map and shows the dramatic reduction of recombination in the pericentromeric heterochromatic region. To establish a feasible mapping system for maize, we also developed a probe cocktail for unambiguous identification of the 10 maize pachytene chromosomes. These results provide a starting point toward constructing a high-resolution integrated cytogenetic map of maize.     

Centromere mapping in the Pacific abalone (Haliotis discus hannai) through half-tetrad analysis in gynogenetic diploid families

Centromere mapping is an essential prerequisite for our understanding of the composition and structure of genomes. For centromere mapping, in two meiogynogenetic families of the Pacific abalone (Haliotis discus hannai), we screened 97 microsatellite markers that cover all linkage groups from a currently available abalone linkage map. Microsatellite analysis showed that no unique paternal allele was found in all gynogenetic progeny, which confirmed 100% success of induction of gynogenesis. In the control crosses, all 97 microsatellite loci were compatible with Mendelian inheritance, while in meiogynogenetic progeny, 5.2% of the microsatellite loci showed segregation distortions from an expected 1:1 ratio of two homozygote classes. The second division segregation frequency of the microsatellites ranged from 0.037 to 0.950 with a mean of 0.399, indicating the existence of interference. Heterogeneity among linkage groups in the crossover distribution was observed. Centromere location was mostly in accordance with the abalone karyotype, but differences in marker order between linkage and centromere maps occurred. Information on the positions of centromeres in relation to the microsatellite loci will represent a contribution towards assembly of genetic maps in the commercially important abalone species.     

Whole-genome linkage disequilibrium screening for complex traits in horses

The identification of candidate genes for significant traits is crucial. In this study, we developed and tested effective and systematic methods based on linkage disequilibrium (LD) for the identification of candidate regions for genes with Mendelian inheritance and those associated with complex traits. Our approach entailed the combination of primary screening using pooled DNA samples based on ΔTAC, secondary screening using an individual typing method and tertiary screening using a permutation test based on the differences in the haplotype frequency between two neighbouring microsatellites. This series of methods was evaluated using horse coat colour traits (chestnut/non-chestnut) as a simple Mendelian inheritance model. In addition, the methods were evaluated using a complex trait model constructed by mixing samples from chestnut and non-chestnut horses. Using both models, the methods could detect the expected regions for the horse coat colour trait. The results revealed that LD extends up to several centimorgans in horses, indicating that whole-genome LD screening in horses could be performed systematically and efficiently by combining the above-mentioned methods. Since genetic maps based on microsatellites have been constructed for many other species, the approaches present here could have wide applicability.     

Viability effects and not meoitic drive cause dramatic departures from Mendelian inheritance for malic enzyme in hybrids of Tigriopus californicus populations

The genetic basis of post-zygotic reproductive isolation is beginning to be untangled in closely related species, but less is known about the genetics of reproductive isolation between divergent populations. Here, two genes encoding malic enzyme (ME) are isolated from the copepod Tigriopus californicus and their influence upon lowered viability in F(2) hybrids of genetically divergent populations is determined. Each ME gene has diverged extensively between T. californicus populations and one gene shows evidence for a recent selective sweep. Segregation patterns of genotypes for both ME genes in adult F(2) hybrids reveal dramatic departures from Mendelian inheritance, deviations that are not seen in F(2) nauplii implying that selection is acting during development based upon the genotype at these ME genes. These results imply that selection against deleterious gene combinations and not aberrant segregation (i.e. meiotic drive) is likely to lead to dramatic departures from Mendelian inheritance observed in these crosses.     

ODS2: a multiplatform software application for creating integrated physical and genetic maps

A contig map is a physical map that shows the native order of a library of overlapping genomic clones. One common method for creating such maps involves using hybridization to detect clone overlaps. False- positive and false-negative hybridization errors, the presence of chimeric clones, and gaps in library coverage lead to ambiguity and error in the clone order. Genomes with good genetic maps, such as Neurospora crassa, provide a means for reducing ambiguities and errors when constructing contig maps if clones can be anchored with genetic markers to the genetic map. A software application called ODS2 for creating contig maps based on clone-clone hybridization data is presented. This application is also designed to exploit partial ordering information provided by anchorage of clones to a genetic map. This information, along with clone-clone hybridization data, is used by a clone ordering algorithm and is represented graphically, allowing users to interactively align physical and genetic maps. ODS2 has a graphical user interface and is implemented entirely in Java, so it runs on multiple platforms. Other features include the flexibility of storing data in a local file or relational database and the ability to create full or minimum tiling contig maps.     

Development of forward genetics in Toxoplasma gondii

The development of forward genetics as a functional system in Toxoplasma gondii spanned more than three decades from the mid-1970s until now. The initial demonstration of experimental genetics relied on chemically induced drug-resistant mutants that were crossed by co-infecting cats, collecting oocysts, sporulating and hatching progeny in vitro. To capitalise on this, genetic markers were employed to develop linkage maps by tracking inheritance through experimental crosses. In all, three generations of genetic maps were developed to define the chromosomes, estimate recombination rates and provide a system for linkage analysis. Ultimately this genetic map would become the foundation for the assembly of the T. gondii genome, which was derived from whole genome shotgun sequencing, into a chromosome-centric view. Finally, application of forward genetics to multigenic biological traits showed the potential to map and identify specific genes that control complex phenotypes including virulence.     

Bovine genome mapping: evolutionary inference and the power of comparative genomics

All bovine chromosomes are now represented by a syntenic group, a linkage map and at least one in situ hybridization. Almost 1000 loci are mapped, about 300 of which are coding sequences useful for comparative mapping and evolutionary inference. Economically important loci are beginning to appear on bovine linkage maps and enhanced comparative maps are likely to be necessary to identify many of these genes by a comparative positional candidate gene approach.     

A 2-cM genetic linkage map of human chromosome 7p that includes 47 loci.

A new high-resolution genetic linkage map for human chromosome 7p has been constructed. The map is composed of 47 loci (54 polymorphic systems), 19 of which are uniquely placed with odds of at least 1000:1. Four genes are represented, including glucokinase (GCK, ATP:D-hexose-6-phosphotransferase, EC 2.7.1.2) which was mapped via a (CA)n dinucleotide repeat polymorphism. The sex-average map measures 94.4 cM and the male and female maps measure 73.2 and 116.1 cM, respectively. We believe that the genetic map extends nearly the full length of the short arm of chromosome 7 since a centromere marker has been incorporated, and the most distal marker, D7S21, has been cytogenetically localized by in situ hybridization to 7p22-pter. The average marker spacing is 2 cM, and the largest interval between uniquely placed markers is 13 cM (sex-average map). Overall, female recombination was observed to be about 1.5 times that of males, and a statistically significant sex-specific recombination frequency was found for a single interval. The map is based on genotypic data gathered from 40 CEPH reference pedigrees and was constructed using the CRI-MAP program package. The map presented here represents a combined and substantially expanded dataset compared to previously published chromosome 7 maps, and it will serve as a "baseline" genetic map that should prove useful for future efforts to develop a 1-cM map and for construction of a contiguous clone-based physical map for this chromosome.     

A Physical Map of the X Chromosome of Drosophila Melanogaster: Cosmid Contigs and Sequence Tagged Sites

A physical map of the euchromatic X chromosome of Drosophila melanogaster has been constructed by assembling contiguous arrays of cosmids that were selected by screening a library with DNA isolated from microamplified chromosomal divisions. This map, consisting of 893 cosmids, covers ~64% of the euchromatic part of the chromosome. In addition, 568 sequence tagged sites (STS), in aggregate representing 120 kb of sequenced DNA, were derived from selected cosmids. Most of these STSs, spaced at an average distance of ~35 kb along the euchromatic region of the chromosome, represent DNA tags that can be used as entry points to the fruitfly genome. Furthermore, 42 genes have been placed on the physical map, either through the hybridization of specific probes to the cosmids or through the fact that they were represented among the STSs. These provide a link between the physical and the genetic maps of D. melanogaster. Nine novel genes have been tentatively identified in Drosophila on the basis of matches between STS sequences and sequences from other species.     

Linkage mapping of the Mediterranean cypress, Cupressus sempervirens, based on molecular and morphological markers

Gene mapping for a Cupressus species is presented for the first time. Two linkage maps for the Mediterranean cypress (Cupressus sempervirens) varieties, C. sempervirens var. horizontalis and C. sempervirens var. pyramidalis, were constructed following the pseudo-testcross mapping strategy and employing RAPD, SCAR and morphological markers. A total of 427 loci (425 RAPDs, two SCARs) representing parents and F(1) progeny were screened for polymorphism with 32 random decamer and two SCAR primers. A morphological marker defined as "crown form" was also included. Of 274 polymorphic loci, the 188 that presented Mendelian inheritance formed the mapping dataset. Of these loci, 30% were mapped into seven linkage groups for the horizontalis (maternal) and four linkage groups for the pyramidalis (paternal) map. The putative "crown form" locus was included in a linkage group of both maps. The horizontalis and the pyramidalis maps covered 160.1 and 144.5 cM, respectively, while genome length was estimated to be 1696 cM for the former variety and 1373 cM for the latter. The four RAPD markers most tightly linked to crown form were cloned and converted to SCARs. Each of the cloned RAPD markers yielded two to three different sequences behaving as co-migrating fragments. Two SCAR markers, SC-D05(432) and SC-D09(667), produced amplified bands of the expected sizes and maintained linkage with the appropriate phenotype, but to a lesser extent compared to their original RAPD counterparts. These linkage maps represent a first step towards the localization of QTLs and genes controlling crown form and other polygenic traits in cypress.     

The genetic and molecular basis of epilepsy

In the past decade, studies of large families in which epilepsy has been inherited in an autosomal dominant fashion have revealed several mutated genes, most of which encode ion channel subunits. Despite these exciting findings, only a few families with similar phenotypes have mutations in these known genes. More frustrating has been the genetic research into idiopathic epilepsies with complex inheritance. Although these forms are more common than those with Mendelian inheritance, their unknown mode of inheritance, phenotypic heterogeneity and the uncertainty of the genetic overlap among syndrome subtypes have hampered gene mapping. New techniques of molecular analysis could help the dissection of genes for epilepsies with complex inheritance. Hopefully, in the near future, successful genetic studies will make possible the discovery of new and more-targeted anti-epileptic drugs.     

Physical map of the genome of Rhodobacter capsulatus SB 1003.

A map of the chromosome of Rhodobacter capsulatus was constructed by overlapping the large restriction fragments generated by endonucleases AseI and XbaI. The analyses were done by hybridization of single fragments with the restriction fragments blotted from pulsed-field gels and by grouping cosmids of a genomic library of R. capsulatus into contigs, corresponding to the restriction fragments, and further overlapping of the contigs. A technical difficulty due to a repeated sequence made it necessary to use hybridization with cloned genes and prior knowledge of the genetic map in order to close the physical circle in a unique way. In all, 41 restriction sites were mapped on the 3.6-Mb circular genome and 22 genes were positioned at 26 loci of the map. Cosmid clones were grouped in about 80 subcontigs, forming two groups, one corresponding to the chromosome of R. capsulatus and the other corresponding to a 134-kb plasmid. cos site end labeling and partial digestion of cosmids were used to construct a high-resolution EcoRV map of the 134-kb plasmid. The same method can be extended to the entire chromosome. The cosmid clones derived in this work can be used as a hybridization panel for the physical mapping of new genes as soon as they are cloned.     

A unified approach to the evolutionary consequences of genetic and nongenetic inheritance

Inheritance-the influence of ancestors on the phenotypes of their descendants-translates natural selection into evolutionary change. For the past century, inheritance has been conceptualized almost exclusively as the transmission of DNA sequence variation from parents to offspring in accordance with Mendelian rules, but advances in cell and developmental biology have now revealed a rich array of inheritance mechanisms. This empirical evidence calls for a unified conception of inheritance that combines genetic and nongenetic mechanisms and encompasses the known range of transgenerational effects, including the transmission of genetic and epigenetic variation, the transmission of plastic phenotypes (acquired traits), and the effects of parental environment and genotype on offspring phenotype. We propose a unified theoretical framework based on the Price equation that can be used to model evolution under an expanded inheritance concept that combines the effects of genetic and nongenetic inheritance. To illustrate the utility and generality of this framework, we show how it can be applied to a variety of scenarios, including nontransmissible environmental noise, maternal effects, indirect genetic effects, transgenerational epigenetic inheritance, RNA-mediated inheritance, and cultural inheritance.     

Conservation of gene order between horse and human X chromosomes as evidenced through radiation hybrid mapping

A radiation hybrid (RH) map of the equine X chromosome (ECAX) was obtained using the recently produced 5000(rad) horse x hamster hybrid panel. The map comprises 34 markers (16 genes and 18 microsatellites) and spans a total of 676 cR(5000), covering almost the entire length of ECAX. Cytogenetic alignment of the RH map was improved by fluorescent in situ hybridization mapping of six of the markers. The map integrates and refines the currently available genetic linkage, syntenic, and cytogenetic maps, and adds new loci. Comparison of the physical location of the 16 genes mapped in this study with the human genome reveals similarity in the order of the genes along the entire length of the two X chromosomes. This degree of gene order conservation across evolutionarily distantly related species has up to now been reported only between human and cat. The ECAX RH map provides a framework for the generation of a high-density map for this chromosome. The map will serve as an important tool for positional cloning of X-linked diseases/conditions in the horse.     

A physical and genetic linkage map of the distal long arm of human chromosome 5.

By in situ hybridization of probes for three cloned genes and eight genetically-linked polymorphic DNA markers, we have prepared a physical map of the distal long arm of chromosome 5. These results, together with the localizations of 11 genes and the genetic linkage map reported previously by us and by other investigators, represent a map that spans 55 cM.