The genome organizations of eight phylogenetically distinct species from five mammalian orders were compared in order to address fundamental questions relating to mammalian chromosomal evolution. Rates of chromosome evolution within mammalian orders were found to increase since the Cretaceous-Tertiary boundary. Nearly 20% of chromosome breakpoint regions were reused during mammalian evolution; these reuse sites are also enriched for centromeres. Analysis of gene content in and around evolutionary breakpoint regions revealed increased gene density relative to the genome-wide average. We found that segmental duplications populate the majority of primate-specific breakpoints and often flank inverted chromosome segments, implicating their role in chromosomal rearrangement.

We previously localized a quantitative trait locus (QTL) on chromosome 6 affecting milk fat and protein concentration to a 4-cM confidence interval, centered on the microsatellite BM143. We characterized the genes and sequence variation in this region and identified common haplotypes spanning five polymorphic sites in the genes IBSP, SPP1, PKD2, and ABCG2 for two sires heterozygous for this QTL. Expression of SPP1 and ABCG2 in the bovine mammary gland increased from parturition through lactation. SPP1 and all the coding exons of ABCG2 and PKD2 were sequenced for these two sires. The single nucleotide change capable of encoding a substitution of tyrosine-581 to serine (Y581S) in the ABCG2 transporter was the only polymorphism corresponding to the segregation status of all 3 heterozygous and 15 homozygous sires for the QTL in the Israeli and U.S. Holstein populations. The allele substitution fixed effects on the genetic evaluations of 335 Israeli sires were −341 kg milk, +0.16% fat, and +0.13% protein (F-value = 200). No other polymorphism gave significant effect for fat and protein concentration in models that also included Y581S. The allele substitution effects on the genetic evaluations of 670 cows, daughters of two heterozygous sires, were −226 kg milk, 0.09% fat, and 0.08% protein (F-value = 394), with partial dominance towards the 581S homozygotes. We therefore propose that Y581S in ABCG2 is the causative site for this QTL.

A second-generation 5000 rad radiation hybrid (RH) map of the cattle genome was constructed primarily using cattle ESTs that were targeted to gaps in the existing cattle–human comparative map, as well as to sparsely populated map intervals. A total of 870 targeted markers were added, bringing the number of markers mapped on the RH5000 panel to 1913. Of these, 1463 have significant BLASTN hits (E < e–5) against the human genome sequence. A cattle–human comparative map was created using human genome sequence coordinates of the paired orthologs. One-hundred and ninety-five conserved segments (defined by two or more genes) were identified between the cattle and human genomes, of which 31 are newly discovered and 34 were extended singletons on the first-generation map. The new map represents an improvement of 20% genome-wide comparative coverage compared with the first-generation map. Analysis of gene content within human genome regions where there are gaps in the comparative map revealed gaps with both significantly greater and significantly lower gene content. The new, more detailed cattle–human comparative map provides an improved resource for the analysis of mammalian chromosome evolution, the identification of candidate genes for economically important traits, and for proper alignment of sequence contigs on cattle chromosomes.

A new physical map of the bovine genome has been constructed by integrating data from genetic and radiation hybrid maps, and a new bovine BAC map, with the bovine genome draft assembly.

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Ten divergent homologs were identified using a subtractive bioinformatic analysis of 12,614 cattle placenta expressed sequence tags followed by comparative, evolutionary, and gene expression studies. Among the 10 divergent homologs, 8 have not been identified previously. These were named as follows: cattle cerebrum and skeletal muscle-specific transcript 1 (CSSMST1), cattle intestine-specific transcript 1 (CIST1), hepatitis A virus cellular receptor 1 amino-terminal domain-containing protein (HAVCRNDP), prolactin-related proteins 8, 9, and 11 (PRP8, PRP9, and PRP11, respectively) and secreted and transmembrane protein 1A and 1B (SECTM1A and SECTM1B, respectively). In addition, two previously known divergent genes were identified, trophoblast Kunitz domain protein 1 (TKDP1) and a new splice variant of TKDP4. Nucleotide substitution analysis provided evidence for positive selection in members of the PRP gene family, SECTM1A and SECTM1B. Gene expression profiles, motif predictions, and annotations of homologous sequences indicate immunological and reproductive functions of the divergent homologs. The genes identified in this study are thus of evolutionary and physiological importance and may have a role in placental adaptations. molecular evolution; bovine genome; comparative genomics THE PLACENTA DEMONSTRATES remarkable variation among eutherian mammals, and it is believed that the vast diversity of placental architectures seen in different mammalian lineages is the result of adaptive evolution (8, 47). Two major selective forces likely driving adaptation of the placenta are fetalmaternal conflict over nutrient resource allocation and maintenance of immunologic tolerance of the fetal semi-allograft (45,66). Cattle possess a synepitheliochorial placenta arranged as placentomes consisting of a fetal cotyledon interdigitating with a maternal caruncle. The cattle embryonal trophoblast contains granulated binucleate cells at the microvillar junction with cytoplasmic projections that penetrate the maternal uterine endometrium (70, 72). Ruminant-specific genes with expression localized to the trophoblast have been identified, including the interferon-genes (54) The expression of these genes at sites of direct contact between fetal and maternal cells suggests that they represent molecular adaptations mediating maternal-fetal interaction during pregnancy (8,55).Comparisons between the whole genome sequences of human, mouse, and rat revealed extensive lineage-specific expansions in gene families associated with reproduction, immunity, chemosensation, detoxification, and proteolysis (48, 52). The identification of orthologous relationships for ϳ80% of genes in the human and mouse genomes indicates that lineagespecific gene family expansion likely represents a major route of mammalian evolution. These lineage-specific paralogs are believed to represent adaptive innovations on the basis of evidence that positive selection has operated on a subset of proteins functioning in reproduction and immunity (48). It is likely that the ...

comparative genomics ͉ radiation hybrid ͉ map integration D etailed whole-genome maps are a major currency for comparative and functional genomics (1). With the human, rat, and mouse genomes completely sequenced (2-6), eight mammalian genomes to follow with Ͼ6-fold coverage, and eight others to be sequenced in draft form (www.genome.gov͞10002154), greater insights into mammalian genome evolution and function can be obtained because of the improved accuracy and resolution of comparative maps (1). We previously reported two expressed sequence tag (EST)-based 5,000-rad cattle radiation hybrid (RH) maps containing 768 and 1,463 anchor points in the human genome, corresponding to Ϸ45% and 65% of comparative human genome sequence coverage, respectively (7, 8). These maps, collectively known as the Illinois-Texas 5,000-rad radiation hybrid panel (IL-TX RH 5,000 ) maps, facilitated the identification of genes responsible for double-muscling (9, 10), chondrodysplasia (11), and two major quantitative trait loci for milk production (12, 13).Recently, Itoh et al. (14) constructed a cattle-human comparative RH map consisting of 5,593 EST and microsatellite markers. This map contains 1,716 anchor points between the human and cattle genomes, of which most are ESTs, and was reported to have Ϸ72% comparative coverage of the human genome. A significant drawback of EST-based comparative maps is the uneven marker distribution that often results in relatively large gaps in comparative coverage. These gaps significantly complicate comparative genome analysis, especially around the evolutionary breakpoint regions. To overcome this limitation, we initiated the development of highresolution RH maps that use the sequenced ends of bacterial artificial chromosome (BAC) inserts as comparatively anchored markers (15). The BAC-end sequences (BESs) selected for mapping were linked by sequence similarity to the human and mouse genome sequences and were evenly spaced in the reference genomes at Ϸ1-Mbp intervals. The approach resulted in the construction of detailed comparative maps of BTA15 and BTA29, with Ϸ86% comparative coverage of HSA11 (15). Furthermore, the study revealed the presence of chromosome breakpoints that were ''reused'' in mammalian evolution, results that were recently confirmed in a more detailed multispecies analysis (1). Herein, we report extension of our methodology to mapping the whole cattle genome. The new map has Ϸ91% comparative coverage of the human genome sequence and contains single-linkage groups for all cattle autosomes. In addition, we used the new high-resolution cattle-human comparative map to study the evolution of centromeres and telomeres, conduct an analysis of gene content within evolutionary breakpoints, and demonstrate the power of the map for proofing the order of contigs in the physical map. MethodsMarker Selection and Mapping Strategy. BESs from the cattle male CHORI-240 (http:͞͞bacpac.chori.org͞bovine240.htm) BAC library were downloaded from GenBank and repeat-masked by using REPEATMASKER software (...

As a step toward the goal of adding the cattle genome to those available for multispecies comparative genome analysis, 40,224 cattle BAC clones were end-sequenced, yielding 60,547 sequences (BAC end sequences, BESs) after trimming with an average read length of 515 bp. Cattle BACs were anchored to the human and mouse genome sequences by BLASTN search, revealing 29.4% and 10.1% significant hits (E < e-5), respectively. More than 60% of all cattle BES hits in both the human and mouse genomes are located within known genes. In order to confirm in silico predictions of orthology and their relative position on cattle chromosomes, 84 cattle BESs with similarity to sequences on HSA11 were mapped using a cattle–hamster radiation hybrid (RH) panel. Resulting RH maps of BTA15 and BTA29 cover ∼85% of HSA11 sequence, revealing a complex patchwork shuffling of segments not explained by a simple translocation followed by internal rearrangements. Overlay of the mouse conserved syntenies onto HSA11 revealed that segmental boundaries appear to be conserved in all three species. The BAC clone-based comparative map provides a foundation for the evolutionary analysis of mammalian karyotypes and for sequencing of the cattle genome.