Use of DNA fingerprints for the detection of major genes for quantitative traits in domestic species

The detection of marker loci linked to major genes or quantitative trait loci (QTL) of large effect in farm animal populations is of great potential value, both because it allows the easy manipulation of the major genes and because it provides a possible route to their ultimate isolation. At present the number of markers available is limited in farm animals. DNA fingerprints provide a promising source of informative marker loci and have the advantage that several loci can be detected on a single Southern hybridization. The disadvantage of DNA fingerprints is the difficulty in determining allelism of DNA fingerprint bands in different pedigrees and the fact that not all potentially resolvable loci can be resolved in a single pedigree. With probes capable of detecting 50 randomly distributed loci, about 50% of the genome of a typical domestic mammal might be expected to be closely linked to a marker (at a distance of 0.2 Morgans or less). If a proportion of DNA fingerprint loci prove to be clustered near chromosomal telomeres or elsewhere in the genome, coverage will be less. In order to detect linkage to a major gene, sires known or suspected to be heterozygous are used to produce large half-sibships, all animals in the pedigree are DNA fingerprinted and the phenotypes of the offspring are recorded. Where several heterozygous sires are available, sires can be selected in an attempt to maximize the number of marker loci resolved. The optimum number of sires needed to produce pedigrees will depend upon the size of the major gene, the number of DNA fingerprint probes available and the characteristics of the DNA fingerprints produced, but often one or two pedigrees will be optimum. Monte Carlo simulation was used to explore the power of detection of linkage between a major gene and a marker locus in a backcross. Maximum likelihood and analysis of variance of mean differences between marker genotypes were of similar power, but maximum likelihood provided reasonable estimates of the major gene effect and its linkage to the marker under some circumstances. One hundred offspring informative for the segregation of a marker would provide reasonable power for the detection of a gene causing a difference between the heterozygote and the homozygote of at least one within-sire, within-genotype standard deviation when linkage was very close (0.05 or less).(ABSTRACT TRUNCATED AT 400 WORDS)     

The efficiency of multilocus DNA fingerprint probes for individualization and establishment of family relationships, determined from extensive casework.

The properties of human DNA fingerprints detected by multilocus minisatellite probes 33.6 and 33.15 have been investigated in 36 large sibships and in 1,702 Caucasian paternity cases involving the analysis of over 180,000 DNA fingerprint bands. The degree of overlap of minisatellite loci detected by these two probes is shown to be negligible (approximately 1%), and the resulting DNA fingerprints are therefore derived from independent sets of hypervariable loci. The level of allelism and linkage between different hypervariable DNA fragments scored with these probes is also low, implying substantial statistical independence of DNA fragments. Variation between the DNA fingerprints of different individuals indicates that the probability of chance identity is very low (much less than 10(-7) per probe). Empirical observations and theoretical considerations both indicate that genetic heterogeneity between subpopulations is unlikely to affect substantially the statistical evaluation of DNA fingerprints, at least among Caucasians. In paternity analysis, the proportion of nonmaternal DNA fragments in a child which cannot be attributed to the alleged father is shown to be an efficient statistic for distinguishing fathers from nonfathers, even in the presence of minisatellite mutation. Band-sharing estimates between a claimed parent and a child can also distinguish paternity from nonpaternity, though with less efficiency than comparison of a trio of mother, child, and alleged father.     

DNA fingerprints of farm animals generated by microsatellite and minisatellite DNA probes

A multi-locus DNA probe, R18.1, derived from a bovine genomic library, detected DNA fingerprints of highly polymorphic loci in hybridization to genomic DNA from poultry and sheep, and of moderate polymorphic loci in cattle and human DNA. The average numbers of detected bands in chickens and sheep were 27.8 and 21.4, and the average band sharing levels were 0.25 and 0.33, respectively. In hybridization to cattle and human DNA, the results were less polymorphic; nevertheless, individual identification is feasible using probe R18.1. The results obtained by R18.1 were compared to results obtained by Jeffreys minisatellite probe 33.6 and two microsatellite oligonucleotides, (GT)12 and (GTG)5. The total number of detected loci using probes R18.1 and 33.6 were estimated in chickens through family analysis of broilers and the maximal number of detectable loci was calculated.     

DNA fingerprinting analysis of Booroola pedigrees: a search for linkage to the Booroola gene

Seven minisatellite probes from a variety of sources were used to analyse 11 paternal half-sib families in which the Booroola gene was segregating. A total of 402 bands that showed segregation in the pedigrees were examined for linkage to the Booroola gene. None of the bands showed segregation with the Booroola gene. The most likely evidence for a linked band was produced by the HaRas HVR probe in Family 902 (=0.0; LOD 2.3). The conclusion, however, is that the minisatellite probes used in this study could not be used as markers for the Booroola gene. The study highlighted problems associated with the use of minisatellite probes in linkage studies in half-sib families. The complex banding patterns found on fingerprinting gels was a major source of scoring error. In a few cases both of the sire's alleles could be identified at a particular locus, but in most cases only one of the alleles could be identified. For the most part, the bands had to be treated as dominant alleles. The contribution of dam alleles to the banding pattern could only be estimated. There was an indication that minisatellite loci in sheep are clustered in particular regions of the sheep genome as the rate at which bands segregated with each other was higher than one would expect from loci randomly distributed throughout the genome.     

DNA "fingerprints" and segregation analysis of multiple markers in human pedigrees

Tandem-repetitive DNA hybridization probes based on a putative human recombination signal detect multiple polymorphic minisatellite fragments in human DNA. The genetic complexity of the resulting individual-specific DNA "fingerprints" was investigated by studying a large sibship affected by neurofibromatosis and a more extensive pedigree segregating for two different hemoglobinopathies. The segregation of up to 41 different heterozygous DNA fragments from each parent could be analyzed in a single sibship, using two different repeat probes. Most of these variable DNA fragments could not be paired as alleles, to an extent which suggests that the DNA fingerprints are together derived from approximately 60 heterozygous loci (approximately 120 variable fragments), only a proportion of which can be scored in a given individual. Two or three of the DNA fragments detected by one probe showed tight linkage and may be derived from long minisatellite(s) that are cleaved to produce more than one polymorphic DNA fragment. Excluding allelic and linked DNA fragments, almost all remaining scorable fragments segregated independently, allowing up to 34 unlinked loci to be examined simultaneously. These loci are scattered over most or all of the human autosomes. Minisatellite probes are therefore suitable for rapid marker generation and can be applied to linkage analysis in human pedigrees.     

DNA fingerprints of poultry

Summary. Human minisatellite probes cross-hybridize to DNA of several species of poultry (chicken, duck, turkey and goose), and detect high levels of polymorphism. The resulting DNA fingerprints are individual specific, and allow the discrimination even between closely related birds. The pattern of poultry DNA fingerprints is different from that of humans and other animals, having a higher average proportion of large DNA fragments. Pedigree analysis revealed a low number of allelic pairs of variable DNA fragments, indicating that most of the alleles are unresolved in the DNA fingerprint or too small to be detected. The total number of detectable loci in broilers, using probe 33.6, was estimated as 62, of which 13 loci are on average scoreable and available for use. Poultry DNA fingerprints can be used for individual identification, linkage studies and as an aid in breeding programmes.     

DNA fingerprints of poultry

Human minisatellite probes cross-hybridize to DNA of several species of poultry (chicken, duck, turkey and goose), and detect high levels of polymorphism. The resulting DNA fingerprints are individual specific, and allow the discrimination even between closely related birds. The pattern of poultry DNA fingerprints is different from that of humans and other animals, having a higher average proportion of large DNA fragments. Pedigree analysis revealed a low number of allelic pairs of variable DNA fragments, indicating that most of the alleles are unresolved in the DNA fingerprint or too small to be detected. The total number of detectable loci in broilers, using probe 33.6, was estimated as 62, of which 13 loci are on average scoreable and available for use. Poultry DNA fingerprints can be used for individual identification, linkage studies and as an aid in breeding programmes.     

Minisatellite DNA markers in the chicken genome

This paper reports the detailed characterization of multilocus minisatellite DNA fingerprints in the chicken. Results are presented of DNA fingerprint segregation analyses carried out in three chicken pedigrees, calculating the number of detected loci, testing for Mendelian inheritance, and cosegregation among fingerprint bands. Two pedigrees (families 1 and 2) were analysed using the Jeffreys probes 33.6 and 33.15 only, and one pedigree (family 3) was analysed using 33.6, 33.15. 3′α-globin HVR and M13 protein III gene repeat. Mean band transmission frequencies in families 1 and 2 were near to the Mendelian expectation of 0.5 and no mutations were observed. Family 3 showed transmission frequencies slightly exceeding 0.5. Linkage among bands was higher than observed in some other avian species, with each allele represented by a mean of 1.48 HaeIII fragments. Cosegregation of heterozygous parental fragments representing distinguishable loci followed the expected binomial distribution. The number of minisatellites detectable by the four probes was estimated to be 217. The pattern of cosegregation among those minisatellite loci was tested against that expected for different levels of recombination through the use of a simulation model. We conclude that most minisatellites are unlinked and probably widely dispersed in the chicken genome.     

Minisatellite DNA fingerprints of salmonid fishes

The human minisatellite probes 33.6 and 33.15 cross-hybridized to DNA digests of Atlantic salmon, brown trout and rainbow trout revealing complex multi-banded patterns. These DNA fingerprints (in excess of 40 resolvable fragments in some cases) were highly polymorphic, individual specific and found to be stable, both somatically and in the germline. Pedigree analysis of an Atlantic salmon family confirmed that the minisatellite fragments showed Mendelian inheritance. With only a single occurrence of linkage and allelism being observed it is likely the minisatellite loci are widely distributed throughout the salmonid genome. The potential applications for both multi- and single locus minisatellite probes in salmonid research are discussed.     

Unequal crossingover between homologous chromosomes is not the major mechanism involved in the generation of new alleles at VNTR loci

To investigate the hypothesis that unequal exchange between homologous chromosomes is involved when new alleles are generated at VNTR loci, we used genetic linkage maps to identify flanking markers surrounding a VNTR marker locus. The minisatellite probe lambda MS1 was selected, as the hypervariable locus it detects undergoes spontaneous generation of new alleles in the germline at a rate of approximately 5%. Multipoint linkage analysis placed lambda MS1 within a cluster of polymorphic marker loci on chromosome 1p. Using the two closest flanking markers, CMM8 and YNZ2, we were able to characterize 12 new-allele events in terms of crossingover between the flanking markers. Statistical analysis of these data has allowed us to reject the model that assumes that events generating new alleles always involve unequal exchange between homologous chromosomes at meiosis.     

Development of microsatellite markers and comparative mapping for bovine chromosome 19

Previous research has mapped an ovulation rate quantitative trait locus (QTL) to bovine chromosome 19. In an effort to enhance comparative mapping information and develop additional markers for refined QTL mapping, microsatellite markers were developed in a targeted approach. A bovine bacterial artificial chromosome (BAC) library was screened for loci with either known or predicted locations on bovine chromosome 19. An average of 6.4 positive BAC were identified per screened locus. A total of 10 microsatellite markers were developed for five targeted loci with heterozygosity of 7-83% in a sample of reference family parents. The newly developed markers were typed on reference families along with four previously mapped marker loci and used to create a linkage map. Comparison of locus order between human and cattle provides support for previously observed rearrangement. One of the mapped loci myotubularin related protein 4 (MTMR4) potentially extends the proximal boundary of a conserved linkage group.     

Preparation of synthetic tandem-repetitive probes for DNA fingerprinting

DNA fingerprints are generated using probes that hybridize to hypervariable minisatellites, also known as variable number tandem repeat loci. Cloned minisatellites have served as the predominant source of DNA fingerprinting probes. A short segment within the repeat units of minisatellites, called the "core" sequence, is highly conserved within a family of related minisatellites, thereby allowing a single-cloned minisatellite to cross-hybridize to 20 to 40 other minisatellites. In this article, we describe a method for the synthetic preparation of polymeric core sequence probes for DNA fingerprinting. Unlike "monomeric" oligonucleotide probes, the polymeric probes mimic the tandem-repetitive structure of minisatellites, and thus each probe molecule can potentially form many sites of hybridization with a target minisatellite. The synthetic probes are cloned into plasmid DNA to provide a perpetual source of probe material.     

Genetic linkage study between the loci for Duchenne and Becker muscular dystrophy and nine X-chromosomal DNA markers

Summary A set of nine polymorphic loci defined by DNA probes was studied for linkage with the disease locus in ten families with a history of Duchenne muscular dystrophy (DMD), and three families with a history of Becker muscular dystrophy (BMD). The results confirm DMD and BMD linkage to all marker loci and suggest closer linkage of several probes than hitherto detected. This will be of practical interest for risk calculations in affected families.     

Large restriction fragments containing poly-TG are highly polymorphic in a variety of vertebrates.

Southern blots of genomic DNA from a variety of species digested by restriction endonucleases having a four-bp specificity, were probed with a bovine genomic clone consisting of seven tandem poly-TG stretches separated by a 29bp linker sequence. Highly variable DNA 'fingerprint' patterns were obtained in chicken, sheep, and horse, moderately variable DNA 'fingerprints' in mouse and man, and a monomorphic pattern in Drosophila. In chicken, horse and man a (TG)10 synthetic oligonucleotide probe gave results identical to those given by the bovine probe. Furthermore, in chicken the DNA fingerprint variation showed typical Mendelian inheritance and differed from the fingerprints obtained with Jeffreys 33.6 and M13 minisatellite probes. Thus, for a variety of vertebrate species, poly-TG-containing probes can uncover useful genetic variation.     

DNA fingerprint bands applied to linkage analysis with quantitative trait loci in chickens

Summary
An efficient approach to detect association between quantitative traits and bands of DNA fingerprint patterns uses intra-family tail analysis, which compares fingerprints of DNA mixes from individuals at the two tails of a phenotypic distribution. In analysis of 67 paternal half-sibs of a meat-type chicken family, of 57 sire bands generated by two probes, one sire-specific band (S6–6) was associated with abdominal fat deposition. The band effect was estimated by a linear model analysis to be 0–88 standard deviations, or about 30% of the family mean. The association between band S6–6 and abdominal fat was further examined by testing progeny of paternal half-sibs of the chickens which were used in the tail analysis, establishing genetic linkage between the DNA marker and a genetic locus affecting abdominal fat deposition.     

Towards interbreed IBD fine mapping of the mh locus: Double-muscling in the Asturiana de los Valles breed involves the same locus as in the Belgian Blue cattle breed

The Spanish ``Asturiana' cattle breed is characterized by the segregation of a genetically determined muscular hypertrophy referred to as double-muscling or ``culones'. We demonstrate by linkage analysis that this muscular hypertrophy involves the mh locus previously shown to cause double-muscling in the Belgian Blue cattle breed, pointing towards locus homogeneity of this trait across both breeds. Moreover, using a twopoint and multipoint maximum likelihood approach, we show that flanking microsatellite markers are in linkage disequilibrium with the mh locus in both breeds albeit with different alleles. Finally, we discuss how allelic homogeneity across breeds might be exploited to achieve efficient genetic fine-mapping of the mh locus. Received: 13 September 1996 / Accepted: 20 January 1997     

An online catalogue of AFLP markers covering the potato genome

An AFLP marker catalogue is presented for gene mapping within cultivated potato. The catalogue is comprised of AFLP fingerprint images of 733 chromosome-specific AFLP markers which are mapped relative to 220 RFLP loci, isozyme loci, morphological characteristics and disease resistance traits. Use of the catalogue is based on identification of common AFLP markers which are visually recognized on autoradiogram images as co-migrating bands in fingerprints generated from different genotypes. Images of AFLP fingerprints combined with detailed information on the genomic location of all AFLP markers are available at URL: http://www.spg.wau.nl/pv/aflp/catalog.htm. It is demonstrated that the comparison of autoradiogram images and subsequent identification of common AFLP markers solely are efficient means for alignment of linkage groups and mapping target genes.     

Mouse DNA ''fingerprints'': analysis of chromosome localization and germ-line stability of hypervariable loci in recombinant inbred strains.

Human minisatellite probes cross-hybridize to mouse DNA and detect multiple variable loci. The resulting DNA "fingerprints" vary substantially between inbred strains but relatively little within an inbred strain. By studying the segregation of variable DNA fragments in BXD recombinant inbred strains of mice, at least 13 hypervariable loci were defined, 8 of which could be regionally assigned to mouse chromosomes. The assigned loci are autosomal, dispersed and not preferentially associated with centromeres or telomeres. One of these minisatellites is complex, with alleles 90 kb or more long and with internal restriction endonuclease cleavage sites which produce a minisatellite "haplotype" of multiple cosegregating fragments. In addition, one locus shows extreme germ-line instability and should provide a useful system for studying more directly the rates and processes of allelic variation of minisatellites.     

The PiGMaP consortium linkage map of the pig (Sus scrofa)

A linkage map of the porcine genome has been developed by segregation analysis of 239 genetic markers. Eighty-one of these markers correspond to known genes. Linkage groups have been assigned to all 18 autosomes plus the X Chromosome (Chr). As 69 of the markers on the linkage map have also been mapped physically (by others), there is significant integration of linkage and physical map data. Six informative markers failed to show linkage to these maps. As in other species, the genetic map of the heterogametic sex (male) was significantly shorter (16.5 Morgans) than the genetic map of the homogametic sex (female) (21.5 Morgans). The sex-averaged genetic map of the pig was estimated to be 18 Morgans in length. Mapping information for 61 Type I loci (genes) enhances the contribution of the pig gene map to comparative gene mapping. Because the linkage map incorporates both highly polymorphic Type II loci, predominantly microsatellites, and Type I loci, it will be useful both for large experiments to map quantitative trait loci and for the subsequent isolation of trait genes following a comparative and candidate gene approach.     

DNA fingerprinting in cattle using oligonucleotide probes

Oligonucleotide probes specific for simple tandem repeat sequences produce individual specific DNA fingerprints in man and all animal species tested so far. Here 11 different synthetic probes were hybridized to bovine genomic DNAs which had been digested with the restriction endonucleases HinfI, AluI and HaeIII. Two of these probes gave DNA fingerprint patterns which were analysed for three German breeds. Different parameters were calculated, such as the average number of bands per individual or the probability of finding identical fingerprints in two unrelated individuals. The number of polymorphic bands varies from 11 to 23 in the different breeds and the probability of finding the same banding pattern in two unrelated individuals ranges from 1.5 x 10(-7) to 2.4 x 10(-7). Hence this DNA fingerprinting procedure allows precise identification of individuals. It is also a useful additional method for paternity testing in cattle.