James E. Womack

Genome-Wide Survey of SNP Variation Uncovers the Genetic Structure of Cattle Breeds

A survey of genetic diversity of cattle suggests two domestication events in Asia and selection by husbandry. Not Just Dinner on Legs Several thousand years ago, human beings realized the virtues of domesticating wild animals as easy meat. Soon other possibilities became apparent, and as revealed in a series of papers in this issue, early pastoralists became selective about breeding for wool, leather, milk, and muscle power. In two papers, Gibbs et al. report on the bovine genome sequence (p. 522; see the cover, the Perspective by Lewin, and the Policy Forum by Roberts) and trace the diversity and genetic history of cattle (p. 528), while Chessa et al. (p. 532) survey the occurrence of endogenous retroviruses in sheep and map their distribution to historical waves of human selection and dispersal across Europe. Finally, Ludwig et al. (p. 485) note the origins of variation in the coat-color of horses and suggest that it is most likely to have been selected for by humans in need of good-looking transport. The imprints of domestication and breed development on the genomes of livestock likely differ from those of companion animals. A deep draft sequence assembly of shotgun reads from a single Hereford female and comparative sequences sampled from six additional breeds were used to develop probes to interrogate 37,470 single-nucleotide polymorphisms (SNPs) in 497 cattle from 19 geographically and biologically diverse breeds. These data show that cattle have undergone a rapid recent decrease in effective population size from a very large ancestral population, possibly due to bottlenecks associated with domestication, selection, and breed formation. Domestication and artificial selection appear to have left detectable signatures of selection within the cattle genome, yet the current levels of diversity within breeds are at least as great as exists within humans.

A genetic linkage map of the bovine genome

A cattle genetic linkage map was constructed which marks about 90% of the expected length of the cattle genome. Over 200 DNA polymorphisms were genotyped in cattle families which comprise 295 individuals in full sibling pedigrees. One hundred and seventy–one loci were found linked to one other locus. Twenty nine of the 30 chromosome pairs are represented by at least one of the 36 linkage groups. Less than a 50 cM difference was found in the male and female genetic maps. The conserved loci on this map show as many differences in gene order compared to humans as is found between humans and mice. The conservation is consistent with the patterns of karyotypic evolution found in the rodents, primates and artiodactyls. This map will be important for localizing quantitative trait loci and provides a basis for further mapping.

Association of a lysine-232/alanine polymorphism in a bovine gene encoding acyl-CoA:diacylglycerol acyltransferase (DGAT1) with variation at a quantitative trait locus for milk fat content

DGAT1 encodes diacylglycerol O-acyltransferase (EC 2.3.1.20), a microsomal enzyme that catalyzes the final step of triglyceride synthesis. It became a functional candidate gene for lactation traits after studies indicated that mice lacking both copies of DGAT1 are completely devoid of milk secretion, most likely because of deficient triglyceride synthesis in the mammary gland. Our mapping studies placed DGAT1 close to the region of a quantitative trait locus (QTL) on bovine chromosome 14 for variation in fat content of milk. Sequencing of DGAT1 from pooled DNA revealed significant frequency shifts at several variable positions between groups of animals with high and low breeding values for milk fat content in different breeds (Holstein–Friesian, Fleckvieh, and Braunvieh). Among the variants was a nonconservative substitution of lysine by alanine (K232A), with the lysine-encoding allele being associated with higher milk fat content. Haplotype analysis indicated the lysine variant to be ancestral. Two animals that were typed heterozygous (Qq) at the QTL based on marker-assisted QTL-genotyping were heterozygous for the K232A substitution, whereas 14 animals that are most likely qq at the QTL were homozygous for the alanine-encoding allele. An independent association study in Fleckvieh animals confirmed the positive effect of the lysine variant on milk fat content. We consider the nonconservative K232A substitution to be directly responsible for the QTL variation, although our genetic studies cannot provide formal proof.

A medium-density genetic linkage map of the bovine genome

A cattle genetic linkage map was constructed which covers more than 95 percent of the bovine genome at medium density. Seven hundred and forty six DNA polymorphisms were genotyped in cattle families which comprise 347 individuals in full sibling pedigrees. Seven hundred and three of the loci are linked to at least one other locus. All linkage groups are assigned to chromosomes, and all are orientated with regards to the centromere. There is little overall difference in the lengths of the bull and cow linkage maps although there are individual differences between maps of chromosomes. One hundred and sixty polymorphisms are in or near genes, and the resultant genome-wide comparative analyses indicate that while there is greater conservation of synteny between cattle and humans compared with mice, the conservation of gene order between cattle and humans is much less than would be expected from the conservation of synteny. This map provides a basis for high-resolution mapping of the bovine genome with physical resources such as Yeast and Bacterial Artificial Chromosomes as well as providing the underpinning for the interpolation of information from the Human Genome Project.USDA-MARC family and data for validating this family. P. Creighton, C. Skidmore, T. Holm, and A. Georgoudis provided some validation data for the BOVMAP families. R. Fries, S. Johnson, S. Solinas Toldo, and A. Mezzelani kindly made some of their FISH assignments available before publication. We wish to thank all those researchers who kindly sent us probes and DNA primers.

Sequencing and automated whole-genome optical mapping of the genome of a domestic goat ( Capra hircus )

We report the ∼2.66-Gb genome sequence of a female Yunnan black goat. The sequence was obtained by combining short-read sequencing data and optical mapping data from a high-throughput whole-genome mapping instrument. The whole-genome mapping data facilitated the assembly of super-scaffolds >5× longer by the N50 metric than scaffolds augmented by fosmid end sequencing (scaffold N50 = 3.06 Mb, super-scaffold N50 = 16.3 Mb). Super-scaffolds are anchored on chromosomes based on conserved synteny with cattle, and the assembly is well supported by two radiation hybrid maps of chromosome 1. We annotate 22,175 protein-coding genes, most of which were recovered in the RNA-seq data of ten tissues. Comparative transcriptomic analysis of the primary and secondary follicles of a cashmere goat reveal 51 genes that are differentially expressed between the two types of hair follicles. This study, whose results will facilitate goat genomics, shows that whole-genome mapping technology can be used for the de novo assembly of large genomes.

A whole-genome radiation hybrid panel for bovine gene mapping

Although the idea of irradiation and fusion gene transfer was published more than 20 years ago (Goss and Harris 1975) and employed in an isolated mapping experiment ten years later (Willard et al. 1985), it was not systematically employed as a human gene mapping tool until resurrected by Cox and associates (1990) for constructing a high-resolution map of human Chr 21. Wholegenome radiation hybrid (WG-RH) mapping utilizing irradiated diploid human cells rather than single chromosome hybrids was revived by Walter and colleagues (1994) and has subsequently become a major tool for high-resolution mapping of the human genome. As reviewed by McCarthy (1996), panels of human radiation hybrids (RH) have been effectively utilized to integrate linkage and physical maps, to anchor or order large insert contigs, and to construct expressed sequence tag (EST) maps that already contain more than 12,000 entries and are growing rapidly. RH mapping has not been effectively utilized in constructing maps of other mammalian species, with the exception of the mouse (Schmitt et al. 1996). This situation is destined to change, however, owing to the potential of the technique for integrating linkage and physical maps. It is an especially powerful tool for comparative gene mapping, since chromosomal order can be established for expressed genes that are usually conserved between species but often recalcitrant to linkage mapping for lack of allelic variation. The bovine donor cell l used in constructing this panel were a normal diploid fibroblast culture established from an Angus bull, JEW38. Nearly confluent flasks were trypsinized and suspended in Gibco DMEM without supplements. Approximately 10 7 cells were irradiated at room temperature in 10 ml suspension medium in a T75 flask. A cobalt 60 source delivered 185 rad/min for a total dose of 5000 rad. Attached cells were removed with trypsin, and all cells were suspended in ca/mg-free Hanks balanced salt solution (HBSS), pH 8.0, at 106 cells/ml. One-half ml (5 x 105 cells) was removed as control and 4.5 ml used for fusion. The recipient Chinese hamster TKfibroblast line A23 was kindly provided by David Cox (Stanford University School of Medicine). These cells were also suspended in HBSS at 106/ml, and 0.7 ml of this suspension was removed as control and 9 ml used for fusion. JEW38 (4.5 x 10 6 cells) and A23 (9 x 10 6 cells) were thoroughly mixed, pelleted, and resuspended. One-half ml PEG (Boehringer Mannheim polyethylene glycol 1500 in 50% sterile solution) was slowly added with constant mixing. After 2 min, 10 ml HBSS, pH 8.0, was also added slowly with gentle mixing. Cells were pelleted, then resuspended in 5 ml HBSS, pH 8.0, for 15 min at 37~ Each control line was exposed to PEG by the same process. The fusion suspension was mixed into Gibco DMEM to 10% FBS plus HAT (Sigma) plus 5 x 10 -7 M ouabain to a total volume of 90 ml. Ten ml of this mixture was dispensed to each of nine 100-mm plates (approximately 1.5 x 105 cells/plate). Controls were mixed in the same solution and plated identically. All were incubated at 37~ in 5% CO 2. All JEW38 control cells were dead on day 7, while one A23 colony, apparently a TK revertant, sur-

Characterization of 65 bovine microsatellites

Microsatellites or simple sequence repeat (SSR) polymorphisms are used widely in the construction of linkage maps in many species. High levels of polymorphism coupled with the ease of analysis of the polymerase chain reaction (PCR) have resulted in this type of maker being one of the most widely used for genetic analysis. In this paper we describe 58 polymorphic bovine microsatellites that were isolated from insert size selected bovine genomic libraries. Primer sequences, number of alleles, and heterozygosity levels in cattle reference families are reported. Chromosomal locations for 47 of these microsatellites as well as for 7 previously described systems derived from entries in the Genbank or EMBL databases have been determined. The markers map to 24 syntenic or chromosomal locations. Polymorphic bovine microsatellites were estimated to occur, on average, every 320 kb, and there is no evidence of clustering in the genome. Thirty of the bovine-derived microsatellite systems gave specific and polymorphic products in sheep, adding to the number of useful markers in that species.

Physically mapped, cosmid-derived microsatellite markers as anchor loci on bovine chromosomes

To identify physical and genetic anchor loci on bovine chromosomes, 13 cosmids, obtained after the screening of partial bovine cosmid libraries with the (CA)n microsatellite motif, were mapped by fluorescence in situ hybridization (FISH). Eleven cosmid probes yielded a specific signal on one of the bovine chromosomes and identified the following loci: D5S2, D5S3, D6S3, D8S1, D11S5, D13S1, D16S5, D17S2, D19S2, D19S3, D21S8. Two cosmids produced centromeric signals on many chromosomes. The microsatellite-containing regions were subcloned and sequenced. The sequence information revealed that the two centromeric cosmids were derived from bovine satellites 1.723 and 1.709, respectively. A cosmid located in the subtelomeric region of Chromosome (Chr) 17 (D17S2) had features of a chromosome-specific satellite. Primers were designed for eight of the nonsatellite cosmids, and seven of these microsatellites were polymorphic with between three and eight alleles on a set of outbred reference families. The polymorphic and chromosomally mapped loci can now be used to physically anchor other bovine polymorphic markers by linkage analysis. The microsatellite primers were also applied to DNA samples of a previously characterized panel of somatic hybrid cell lines, allowing the assignment of seven microsatellite loci to defined syntenic groups. These assignments confirmed earlier mapping results, revealed a probable case of false synteny, and placed two formerly unassigned syntenic groups on specific chromosomes.

Sequencing the Mouse Y Chromosome Reveals Convergent Gene Acquisition and Amplification on Both Sex Chromosomes

We sequenced the MSY (male-specific region of the Y chromosome) of the C57BL/6J strain of the laboratory mouse Mus musculus. In contrast to theories that Y chromosomes are heterochromatic and gene poor, the mouse MSY is 99.9% euchromatic and contains about 700 protein-coding genes. Only 2% of the MSY derives from the ancestral autosomes that gave rise to the mammalian sex chromosomes. Instead, all but 45 of the MSYs genes belong to three acquired, massively amplified gene families that have no homologs on primate MSYs but do have acquired, amplified homologs on the mouse X chromosome. The complete mouse MSY sequence brings to light dramatic forces in sex chromosome evolution: lineage-specific convergent acquisition and amplification of X-Y gene families, possibly fueled by antagonism between acquired X-Y homologs. The mouse MSY sequence presents opportunities for experimental studies of a sex-specific chromosome in its entirety, in a genetically tractable model organism.

Standardization of cattle karyotype nomenclature: Report of the committee for the standardization of the cattle karyotype

The purpose of this paper is to publish a table which correlates the previous nomenclature with marker genes mapped on cattle chromosomes. This table also presents the human correspondences and the chromosome measurements expressed as relative lengths.