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The development of a completely annotated sheep genome sequence is a key need for understanding the phylogenetic relationships and genetic diversity among the many different sheep breeds worldwide and for identifying genes controlling economically and physiologically important traits. The ovine genome sequence assembly will be crucial for developing optimized breeding programs based on highly productive, healthy sheep phenotypes that are adapted to modern breeding and production conditions. Scientists and breeders around the globe have been contributing to this goal by generating genomic and cDNA libraries, performing genome-wide and trait-associated analyses of polymorphism, expression analysis, genome sequencing, and by developing virtual and physical comparative maps. The International Sheep Genomics Consortium (ISGC), an informal network of sheep genomics researchers, is playing a major role in coordinating many of these activities. In addition to serving as an essential tool for monitoring chromosome abnormalities in specific sheep populations, ovine molecular cytogenetics provides physical anchors which link and order genome regions, such as sequence contigs, genes and polymorphic DNA markers to ovine chromosomes. Likewise, molecular cytogenetics can contribute to the process of defining evolutionary breakpoints between related species. The selective expansion of the sheep cytogenetic map, using loci to connect maps and identify chromosome bands, can substantially contribute to improving the quality of the annotated sheep genome sequence and will also accelerate its assembly. Furthermore, identifying major morphological chromosome anomalies and micro-rearrangements, such as gene duplications or deletions, that might occur between different sheep breeds and other Ovis species will also be important to understand the diversity of sheep chromosome structure and its implications for cross-breeding. To date, 566 loci have been assigned to specific chromosome regions in sheep and the new cytogenetic map is presented as part of this review. This review will also summarize the current cytogenomic status of the sheep genome, describe current activities in the sheep cytogenomics research sector, and will discuss the cytogenomics data in context with other major sheep genomics projects.

The proximal half of Bos taurus chromosome 27 (BTA27prox) delimited by microsatellite markers BM3507 and CSSM043 reveals complex rearrangements compared to its corresponding Homo sapiens chromosome (HSA) fragments. A comparative mapping approach combining somatic and radiation hybrid cell mapping techniques and related cytogenetic data resulted in an improved physical map for BTA27prox, which provides candidate genes for several important economic traits. The generated comprehensive map includes anchor loci for 103 genes and microsatellite markers. Mapping of genes proximal to BM3507 matching a region from 0.60 to 2.78 megabase pairs (Mb) of HSA8 confirmed recent sequence annotations on BTA27. Assignments of loci predicted to be on BTA27 to BTA1, BTA8, and BTA17 narrowed down evolutionary chromosome break points compared with corresponding chromosome segments in human. New physical anchors obtained in this study confirm in more detail the described evolutionary conservation between the proximal half of BTA27 and homologous segments of HSA4 and HSA8 and will contribute to the completion of the cattle DNA genome sequence.

A preliminary radiation hybrid (RH) map containing 50 loci on chromosome 7 of the domestic river buffalo Bubalus bubalis (BBU; 2n = 50) was constructed based on a comparative mapping approach. The RH map of BBU7 includes thirty-seven gene markers and thirteen microsatellites. All loci have been previously assigned to Bos taurus (BTA) chromosome BTA6, which is known for its association with several economically important milk production traits in cattle. The map consists of two linkage groups spanning a total length of 627.9 cR5,000. Comparative analysis of the BBU7 RH5,000 map with BTA6 in cattle gave new evidence for strong similarity between the two chromosomes over their entire length and exposed minor differences in locus order. Comparison of the BBU7 RH5,000 map with the Homo sapiens (HSA) genome revealed similarity with a large chromosome segment of HSA4. Comparative analysis of loci in both species revealed more variability than previously known in gene order and several chromosome rearrangements including centromere relocation. The data obtained in our study define the evolutionarily conserved segment on BBU7 and HSA4 to be between 3.5 megabases (Mb) and 115.8 Mb in the HSA4 (genome build 36) DNA sequence.

The coagulation factor IX gene (F9), the hypoxanthine phosphoribosyl transferase 1 gene (HPRT1), and the X-inactive specific transcript gene (XIST) were physically assigned in cattle to analyze chromosomal breakpoints on BTAX recently identified by radiation hybrid (RH) mapping experiments. Whereas the FISH assignment of XIST indicates a similar location on the q-arm of the human and cattle X chromosomes, the locus of HPRT1 supported the assumption of a chromosome rearrangement between the distal half of the q-arm of HSAX and the p-arm of BTAX identified by RH mapping. F9 previously located on the q-arm of BTAX was assigned to the p-arm of BTAX using RH mapping and FISH. The suggested new position of F9 close to HPRT1 supports the homology between HSAXq and BTAXp. The F9 locus corresponds with the gene order found in the homologous human chromosome segment. XIST was assigned on BTAXq23, HPRT1 and F9 were mapped to BTAXp22, and the verification of the location of F9 in a 5000 rad cattle-hamster whole genome radiation hybrid panel linked the gene to markers URB10 and HPRT1.

Results of fluorescence in situ hybridization (FISH) of Bos taurus and B. indicus Y chromosomes using the bovine locus-specific Y probes BC1.2 and λES6.0 and region-specific probes of B. indicus and B. taurus Y chromosomes, which were generated by microdissection and DOP-PCR, indicate that the Y chromosomes of B. indicus (BIN Y) and B. taurus (BTA Y) differ by a pericentric inversion. Parts of the short and long arms of the Y chromosome in B. taurus and the distal half of the Y chromosome in B. indicus were microdissected, amplified by DOP-PCR, biotinylated, and rehybridized in situ to the corresponding metaphase chromosomes to test the chromosome fragment specificity of the DNA probes. The region-specific painting probes were used for hybridization to metaphase chromosomes of the other species. The DNA painting probes BTA Yp12 and BTA Yq12.1-ter derived from BTA Y hybridized to the distal and proximal halves of BIN Y, respectively. Complex hybridization signals on BTA Yq12.1→qter were generated with the DNA probe BIN Yqcen-centr (centromere-central) after FISH. The results demonstrate that BTA Yp is homologous to the distal half of BIN Y and that BTA Yq corresponds to the proximal part of BIN Yq. Hybridization of the Y chromosome-specific DNA probes λES6.0 to BTA Yp12→p11 and near to the telomere of BIN Y and BC1.2 to BTA Yq12→q13 and to the telomere of BIN Y indicate an opposite orientation of the homologous chromosome fragments BTA Yp and of the distal half of BIN Yq.

The gene BCL2L2 encoding protein BCL2-like 2, alias BCL-W is involved in regulation of programmed cell death. It represents a member of the BCL-2 gene family comprising nearly 20 proteins, which govern the mitochondria-dependent pathway for apoptosis (Reed, 2000). BCL2-like 2 belongs to the subfamily of anti-apoptotic proteins (Denisov et al., 2003). Enforced expression of BCL2L2 in human lymphoid and myeloid cells has been shown to contribute to reduced cell apoptosis under cytotoxic conditions (Gibson et al., 1996). Expression analysis of BCL2L2 in Alzheimer disease brain indicates neuroprotective properties of the gene (Zhu et al., 2004). Studies of the orthologous gene in mice demonstrated an essential role in adult spermatogenesis (Ross et al., 1998). The chromosome locus for BCL2L2 in human was found on HSA14q11.2→q12. In ruminants, the role of anti-apoptotic and pro-apoptotic genes of the BCL-2 gene family was recently examined by functional and positional analysis of five BCL-2 genes in sheep including BCL2L2 (Lyahyai et al., 2005). Structure, function and chromosomal location of most members of the BCL-2 gene family in cattle are unknown. Here, we report the physical mapping of bovine BCL2L2 by FISH and PCR typing in a somatic cell hybrid panel.

Tetradysmelia is an inherited defect observed in Holstein cattle (Kuhn et al., 2002) characterized by complete lack or extremely severe reduction of all limb constituents distal of scapula and pelvic girdle. Due to the marked similarity in phenotype between fibroblast growth factor (Fgf10)–/– knockout mice (Min et al., 1998) and tetradysmelia calves, FGF10 was a strong candidate gene for the bovine defect. In humans FGF10 is localized on HSA5p13→p12 in close vicinity to the growth hormone receptor (GHR) gene. Bovine GHR was genetically mapped to the middle part of BTA20 (Barendse et al., 1997). Comparative mapping (Everts-van der Wind et al., 2004) confirmed the chromosomal homology between HSA5 and the centromeric and middle part of BTA20, indicating that FGF10 should be located in the middle part of BTA20. Multipoint likelihood homozygosity mapping, however, excluded the middle part of BTA20 as locus for tetradysmelia (Kuhn et al., 2002). Proof of the assumed genomic localization of FGF10 in the middle part of BTA20 unambiguously excludes FGF10 as the causal gene underlying the genetic defect tetradysmelia in cattle despite its strong candidacy.

TPD52 belongs to the D52 gene family. D52 proteins act as signaling molecules, which may play a role as regulators of cell proliferation (Nourse et al., 1998). It is also suggested that the gene product acts differently in normal and tumor affected tissues and between fetal and adult tissues (e.g. Byrne et al., 1995; Tiacci et al., 2005). Chen et al. (1996) identified overexpression of the gene in tumor derived cell lines from multiple cancers. TPD52 in human and mouse is located on HSA8q21 and MMU3A1-A2, respectively. A bovine partial gene sequence of TPD52 was identified with the strategy of comparative mapping by annotation and sequence similarity (COMPASS) and according to the gene assignment in human a locus was predicted on Bos taurus (BTA) chromosome 14 (Ma et al., 1998). Several independent investigations give experimental proof that BTA14 is associated with different quantitative traits influencing milk production (Khatkar et al., 2004). The physical assignment of the bovine TPD52 gene by somatic cell and radiation cell hybrid mapping techniques presented in this report will enhance our knowledge about loci in trait associated BTA14 chromosome segments. Materials and methods