J. D. Burzlaff

Mining the Mammalian Genome for Artiodactyl Systematics

A total of 7,806 nucleotide positions derived from one mitochondrial and eight nuclear DNA segments were used to provide a robust phylogeny for members of the order Artiodactyla. Twenty-four artiodactyl and two cetacean species were included, and the horse (order Perissodactyla) was used as the outgroup. Limited rate heterogeneity was observed among the nuclear genes. The partition homogeneity tests indicated no conflicting signal among the nuclear genes fragments, so the sequence data were analyzed together and as separate loci. Analyses based on the individual nuclear DNA fragments and on 34 unique indels all produced phylogenies largely congruent with the topology from the combined data set. In sharp contrast to the nuclear DNA data, the mtDNA cytochrome b sequence data showed high levels of homoplasy, failed to produce a robust phylogeny, and were remarkably sensitive to taxon sampling. The nuclear DNA data clearly support the paraphyletic nature of the Artiodactyla. Additionally, the family Suidae is diphyletic, and the nonruminating pigs and peccaries (Suiformes) were the most basal cetartiodactyl group. The morphologically derived Ruminantia was always monophyletic; within this group, all taxa with paired bony structures on their skulls clustered together. The nuclear DNA data suggest that the Antilocaprinae account for a unique evolutionary lineage, the Cervidae and Bovidae are sister taxa, and the Giraffidae are more primitive.

A molecular cytogenetic analysis of the tribe Bovini (Artiodactyla : Bovidae : Bovinae) with an emphasis on sex chromosome morphology and NOR distribution

Q-band comparisons were made among representative species of the four genera of the tribe Bovini (Bos, Bison, Bubalus, Syncerus) as well as to selected outgroup taxa representing the remaining two tribes of the subfamily Bovinae (nilgai, Boselaphini; eland, Tragelphini), the Bovidae subfamily Caprinae (domestic sheep) and the family Cervidae (sika deer and white- tailed deer). Extensive autosomal arm homologies were noted, but relatively few derivative character states were shared. Focus was then made on variation of the sex chromosomes and the chromosomal distribution of nucleolar organizer regions (NORs). Bovine BAC clones were used in molecular cytogenetic analyses to decipher rearrangements of the sex chromosomes, and a pocket gopher 28s ribosomal probe was used to map the chromosomal locations of nucleolar organizing regions (NORs). Some of the more noteworthy conclusions drawn from the comparative analysis were that: 1. The Bovidae ancestral X chromosome was probably acrocentric and similar to acrocentric X chromosomes of the Bovinae; 2. The domestic sheep acrocentric X is probably a deriative character state that unites non-Bovinae subfamilies; 3. Bos and Bison are united within the tribe Bovini by the presence of shared derivative submetacentric X chromosomes; 4. Sika and white- tailed deer X chromosomes differ by inversion from X chromosomes of the Bovinae; 5. The Bovini ancestral Y chromosome was probably a small acrocentric; 6. Bos taurus, B. gaurus and B. banteng share derivative metacentric Y chromosomes; 7. Syncerus and Bubalus are united by the acquisition of X-specific repetitive DNA sequence on their Y chromosomes; 8. Bovinae and Cervidae X chromosome centromere position varies without concomitant change in locus order. Preliminary data indicate that a knowledge of the chromosomal distribution of NORs among the Bovidae will prove to be phylogenetically informative.

A karyotypic analysis of nilgai, Boselaphus tragocamelus (Artiodactyla: Bovidae)

A combination of chromosomal banding and fluorescence in situ hybridization (FISH) was used to characterize the karyotype of Boselaphus tragocamelus (nilgai) relative to the domestic cattle standard karyotype. G-, Q- and C-band karyotypes of nilgai are presented, and the chromosomal complement of nilgai is determined to be 2n = 46 (female FN = 60, male FN = 59; NAA = 56), consistent with previous reports for the species. Comparisons with cattle identified extensive monobrachial homologies with some noteworthy exceptions. Chromosome 25 is centrically fused to 24, and chromosome 16 is acrocentric. Both appear to have additional pericentromeric material not seen in the equivalent cattle acrocentrics. This pericentromeric chromatin may be the result of de novo additions or translocation of pericentromeric material from chromosome 6, which is shown to be centrically fused to 13 but is only about two-thirds the length of cattle 6. Comparisons with cattle demonstrated that nilgai chromosome 17 has undergone a paracentric inversion and that chromosome 20 has two blocks of interstitial constitutive heterochromatin. The identities of both chromosomes were confirmed by chromosomal FISH. Furthermore, chromosomal banding and FISH were used to determine that autosome 14 has been fused to the ancestral X and Y of nilgai to form compound neo-X and -Y chromosomes. Additional FISH analyses were conducted to confirm other proposed chromosome homologies and to identify nucleolar organizing regions within the nilgai complement.

Cytogenetic alignment of the bovine chromosome 13 genome map by fluorescence in-situ hybridization of human chromosome 10 and 20 comparative markers

A bovine bacterial artificial chromosome (BAC) library was screened for the prescence of six genes (IL2RA, VIM, THBD, PLC-II, CSNK2A1 and TOP1) previously assigned to human chromosomes 10 or 20 (HSA10 or HSA20). Four of the genes were found repesented in the bovine BAC library by at least one clone. The identified BAC clones were used as probes in single-color fluorescence in-situ hybridization (FISH) to determine the chromosomal band location of each gene. As predicted by the human/bovine comparative map and comparative chromosome painting analysis, the four genes mapped to bovine chromosome 13 (BTA13). Dual-color FISH was then used to integrate these four type I markers into the existing BTA13 genome map. These FISH results anchor the BTA13 genome map from bands 14–23, and confirm the presence of a conserved HSA10 homologous synteny group on BTA13 centromeric to a HSA20 homologous segment.

Physical mapping of the endothelin receptor type B to bovine chromosome 12

kinase [1], is widely expressed in hematopoietic cells and is considered to be involved in the signal transductions from the B cell antigen receptor [4], high affinity IgE receptors [5] and T cell receptor [6]. The present study with FISH revealed that SYK gene resides on porcine Chr 14q14. A representative chromosome spread showing hybridization signals on Chr 14q14 was shown in Fig. 1. In this study, 142 chromosome spreads having signals were examined to locate the gene on chromosomes. One-hundred and thirty-two spreads of them showed signals on porcine Chr 14q14, whereas the rest showed no specific signal localization on porcine chromosomes. The ACTA1, ACTN2, LPL, ATA, PLAU, DAO, UBC, and GGT genes have been localized on porcine Chr 14 [7,8], where the SYK gene has been found in the present study. As for human chromosomes, the ACTA1 and ACTN2 genes are localized on Chr 1; LPL gene, on Chr 8; ATA and PLAU genes, on Chr 10; DAO and UBC genes, on Chr 12; and GGT gene, on Chr 22 [9]. Since the human SYK gene has been assigned to Chr 9q22 [3], our findings of SYK gene on porcine Chr 14q14 provide evidence that a part of porcine Chr 14, especially around q14 region, has homology with human Chr 9. Recently, ZOO-FISH analysis has been increasingly used to determine the correspondence of chromosomal regions between species. Rettenberger and associates [10] reported that porcine Chr 14 had homology with human Chrs 1, 8, 10, 12, and 22, whereas other groups reported that porcine Chr 14 had homology with human Chrs 8, 10, 12, and 22 [11,12]. The inconsistency of the correspondence between porcine Chr 14 and human Chr 1 in the reports is possibly owing to the limit of resolution in ZOO-FISH analysis. The correspondence between porcine Chr 14 and human Chr 9 observed in our study but not observed in the ZOO-FISH has revealed the limit of resolution in ZOO-FISH analysis. Therefore, admitting that the ZOO-FISH analysis is a useful tool for generating comparative maps, the precise comparative maps should be constructed by FISH analysis with individual DNA sequences as probes.

Physical assignment of microsatellite-containing BACs to bovine chromosomes

Here we report the physical assignment of 40 microsatellite markers by fluorescence in situ hybridization to 13 different bovine chromosomes. This information will be valuable in providing physically anchored landmarks for the construction of contigs throughout the bovine genome. It also is useful for the purpose of integrating the linkage maps of these chromosomes to their physical maps and determining the physical coverage of these linkage groups.