A deletion in the bovine myostatin gene causes the double-muscled phenotype in cattle.

An exceptional muscle development commonly referred to as 'double-muscled' (Fig. 1) has been seen in several cattle breeds and has attracted considerable attention from beef producers. Double-muscled animals are characterized by an increase in muscle mass of about 20%, due to general skeletal-muscle hyperplasia-that is, an increase in the number of muscle fibers rather than in their individual diameter. Although the hereditary nature of the double-muscled condition was recognized early on, the precise mode of inheritance has remained controversial; monogenic (domainant and recessive), oligogenic and polygenic models have been proposed. In the Belgian Blue cattle breed (BBCB), segregation analysis performed both in experimental crosses and in the outbred population suggested an autosomal recessive inheritance. This was confirmed when the muscular hypertrophy (mh) locus was mapped 3.1 cM from microsatellite TGLA44 on the centromeric end of bovine chromosome 2 (ref. 5). We used a positional candidate approach to demonstrate that a mutation in bovine MSTN, which encodes myostatin, a member of the TGF beta superfamily, is responsible for the double-muscled phenotype. We report an 11-bp deletion in the coding sequence for the bioactive carboxy-terminal domain of the protein causing the muscular hypertrophy observed in Belgian Blue cattle.

Microsatellite mapping of the bovine roan locus: a major determinant of White Heifer disease.

In the Belgian Blue Cattle breed, coat color variation is mainly under the influence of a single autosomal locus, the roan locus, characterized by a pair of codominant alleles: r+ (black) and R (white). Heterozygous r+R animals have intermingled black and white hairs, yielding the "blue" phenotype typical of the breed. Major interest for the roan locus stems from its pleiotropic effect on fertility, owing to the critical role of the R allele in the determinism of White Heifer Disease. We describe the linkage mapping of the roan locus to bovine Chromosome (Chr) 5, in the interval between microsatellite markers BPI and AGLA293, with an associated lodscore of 11.2. Moreover, we map a candidate gene, the Steel locus coding for the mast cell growth factor, to bovine Chr 5.

The mh gene causing double-muscling in cattle maps to bovine Chromosome 2.

While the hereditary nature of the "double-muscling" phenotype (a generalized muscular hypertrophy documented in several cattle breeds) is well established, its precise segregation mode has remained controversial. Both monogenic models (autosomal dominant or recessive) and oligogenic models have been proposed. Using a panel of 213 bovine microsatellite markers, and an experimental pedigree obtained by backing "double-muscled (Belgian Blue) x conventional (Friesian)"1 dams to double-muscle sire, we have mapped a locus on bovine Chromosome (CHr) 2 that accounts for all the phenotypic variance in the backcross generation. This locus, referred to as mh (muscular hypertrophy), has been positioned with respects to a map composed of seven Chr 2-specific microsatellites, at 2 cM from the closet marker. This result confirms the validity in the Belgian Blue population of the monogenic model involving an autosomal mh locus, characterized by a wild-type "+" and a recessive "mh" allele, causing the double-muscling phenotype in the homozygous condition. The linkage relationship between the mh locus and the Chr 2 markers was confirmed in three informative pedigrees collected from the general Belgian Blue Cattle population, reinforcing the notice of genetic homogeneity of the double-muscling trait in this breed. This work paves the way towards marker-assisted selection for or against the double muscling trait, and towards positional cloning of the corresponding gene.

Transgenic mice harboring Aujeszky's disease virus gD gene.

Plasmid pMLP10 gp50.5 (gD) (Eloit et al., 1990) provided the sequence to integrate. The construct has been injected into the male pronucleus of one-cell mouse embryos (CBA/C5B1). A total of 1,567 microinjected embryos were transferred into the oviduct of pseudopregnant females (Hogan et al., 1986). PCR revealed that 3 out of 10 mice were born with an integrated construct; among them, 2 mice have integrated the construct in their gonads because 20 out of 107 (18.7%) of their offspring were also transgenic. This low percentage could be explained by germline mosaicism and/or differential mortality between transgenic and non-transgenic embryos.

On the use of DNA fingerprints for linkage studies in cattle.

To find a marker for the bovine "muscular hypertrophy" gene and for the "roan" locus, we have typed six cattle pedigrees totaling 540 animals for nine blood group systems, for 12 biochemical markers, for RFLPs at four loci, and with five probes revealing multilocus DNA fingerprints. Segregation analysis of the fingerprint bands showed that, in cattle, a fingerprint probe will reveal a mean of 7.6 clearly resolvable bands, behaving as simple, highly informative Mendelian entities characterized by a mean mutation rate of +/- 1/4500 gametes. For one of the bands, we observed a "mutation burst" generating germline mosaicism. Because some of the fingerprint bands were allelic or corresponded to clustered minisatellites, a mean of only 5.7 independent loci is explored per probe. Fingerprint bands revealed by different probes also show a clear propensity for close linkage, pointing toward nonrandom distribution of minisatellite sequences or the existence of minisatellite clusters. Although this reduces the power of fingerprints for linkage analysis substantially, we were able to demonstrate genetic linkage between fingerprint bands and at least three of the classical markers, to exclude the roan locus from 4.5 Morgans of the bovine genome with the DNA fingerprints and for an additional 2.5 Morgans with the classical markers, and to identify a solid candidate marker for the bovine muscular hypertrophy gene, yielding a lod score greater than or equal to 2.84 without any obliged recombinant.

Linkage relationships among 20 genetic markers in cattle. Evidence for linkage between two pairs of blood group systems: B-Z and S-F/V respectively.

Five bovine paternal half-sib pedigrees for a total of 527 individuals were typed for six blood group systems: A, B, F/V, L, S, Z; for nine biochemical polymorphisms: ADA, MPI, PGM-3(slow), NP, Gc, Pi2, Tf, Ptf1 and Ptf2; and for restriction fragment length polymorphisms at five autosomal loci: Tg, GH, LDLr, BoLA-DQ and BoLA-DY. Two of the pedigrees were informative for segregation at the 'muscular hypertrophy' locus, and one was informative at the coat colour determining 'roan' locus. Linkage analysis was performed between all markers. Linkage was demonstrated between the S and F/V blood group systems (z = 3.11), adding one locus to the previously identified linkage group VII (LGVII) [Pi-2 and S], the most likely order being Pi2-S-F/V with maximum likelihood recombination rates of 0.208 and 0.211. Also shown to be linked were the blood group systems B and Z (z = 5.7, theta = 0.245). We confirmed the observation previously made by Andersson et al. (1988) of a high recombination rate between class II genes DQ and DY, suggesting either a larger physical distance between those genes than expected from comparative data, or the presence of a 'recombinational hotspot' in the bovine major histocompatibility complex. No linkage was found either with the 'muscular hypertrophy' locus, or with the 'roan' locus. However, these two loci could be excluded from respectively 1.7 and 2.5 Morgans of the bovine genome.

Analysis of genetic variation in the Belgian Blue cattle breed using DNA sequence polymorphism at the growth hormone, low density lipoprotein receptor, alpha-subunit of glycoprotein hormones and thyroglobulin loci.

New DNA sequence polymorphisms were identified at four bovine autosomal loci: growth hormone, low density lipoprotein receptor, alpha-subunit of glycoprotein hormones and thyroglobulin. Assuming independent assortment between these polymorphisms, the probabilities to be heterozygous at these four loci are 0.48, 0.36, 0.10 and 0.77 respectively, within the Belgian Blue Cattle breed (BBCB). Nucleotide diversity was estimated, showing that animals from the BBCB are heterozygous for 1/1450 nucleotides, a value significantly lower than the 1/500 value found in man. Moreover, we have estimated that the mutation rate at the cytosines of CG dinucleotides is about 10 times higher than that for other nucleotides.

Use of DNA bar codes to resolve a canine paternity dispute.

The DNA fingerprinting method was used to resolve a canine paternity dispute. During the same estrus, a Shih Tzu bitch was inseminated by 2 dogs--a Shih Tzu and a Coton de Tulear. Because both breeds are alike phenotypically, it was difficult to decide whether the pups were purebred or of mixed breeding. The DNA bar codes indicated unambiguously that the 2 sires had fathered one pup each, thus documenting superfecundation.

DNA fingerprinting in domestic animals using four different minisatellite probes.

Four probes known to allow DNA fingerprinting in the human (M13, Jeffreys' core sequence, the human alpha globin hypervariable region [HVR], and a mouse probe related to the Drosophila Per gene) were checked for their ability to reveal "genetic bar codes" in cattle, horses, pigs, dogs, chickens, and a European cyprinid fish, the barbel (Barbus barbus L.). Individual-specific patterns were obtained in cattle using M13, Jeffreys' core sequence, and the alpha globin HVR, in horses, dogs, and pigs using M13, Jeffreys' core sequence, and the Per probe, and in chicken and fish using the four different probes. Although we observed a considerable heterogeneity in the extent of interindividual variation, depending on the particular probe-species combination, the fingerprints are polymorphic enough to be used efficiently in animal identification, paternity testing, and as a source of genetic markers for linkage analysis. These markers should substantially accelerate the mapping of genes affecting economically important traits.

Genetic variation of the bovine thyroglobulin gene studied at the DNA level.

The bovine thyroglobulin gene has been analysed for variation using restriction endonucleases. Six independent restriction fragment length polymorphisms have been identified. One of these results most probably from a 2.5-kb deletion, the others being compatible with point mutations. We determined that an individual taken at random within the Belgian White and Blue breed is, on average, heterozygous for one out of 1700 nucleotides within the thyroglobulin gene.

Genetic linkage between the bovine plasma protease inhibitor 2 (Pi-2) and S blood group loci.

A linkage relationship has been detected between the bovine plasma protease inhibitor 2 (Pi-2) and S blood group loci by linkage study within a single pedigree. Using the sequential lodscore test, the recombination fraction (theta) with maximum likelihood has been estimated at 0.200 +/- 0.043, with a maximum lodscore value of 3.466 at theta = 0.200.

Allozymic variation as an estimate of heterozygosity in Belgian pig breeds.

An electrophoretic survey of 13 enzymes corresponding to 19 loci has been carried out in Belgian Landrace and Pietrain pig breeds. Four of these enzymes have been shown to exhibit electrophoretically detectable polymorphism. The average heterozygosity per locus was found to be 0.066 in the Belgian Landrace and 0.028 in the Pietrain pigs.