F.A. Ponce de León

International system for standardized avian karyotypes (ISSAK): standardized banded karyotypes of the domestic fowl (Gallus domesticus).

The chicken genetic map is becoming very detailed. The genetic and physical maps need to be integrated in more detail. It is important to have a consensus banded karotype to permit this integration. An international committee met to develop a karyotype for the eight largest chromosomes and the Z and W chromosomes of the domestic fowl (Gallus domesticus). This map is presented in this report.

CLONING : NEW BREAKTHROUGHS LEADING TO COMMERCIAL OPPORTUNITIES

Research on cloning animals, again, came to the forefront of public attention in 1997. Most scientists involved in biomedical and agricultural research have emphasized the benefits, of which there are many, of cloning to the public. Basic studies on nuclear transfer have and will continue to contribute to our understanding of how genomic activation and cell cycle synchrony affect nuclear reprogramming and cloning efficiencies, specifically. Also, more basic information on actual mechanisms and specific factors in the oocyte causing nuclear reprogramming is forthcoming. As new molecular approaches in functional genomics are combined with nuclear transfer experiments, new genes involved in nuclear reprogramming will be found. The commercial potentials of products stemming from discoveries in cloning are vast. Cloning will be a more efficient, faster and more useful way of making transgenic fetuses for cell therapies, adult animals for protein production and organs for xenotransplantation. Clearly there are new opportunities in animal cloning technology that will produce many benefits to society.

A molecular cytogenetic analysis of X chromosome repatterning in the Bovidae: transpositions, inversions, and phylogenetic inference

Chromosomal homologies among the X chromosomes of species representative of eight bovid subfamilies and most of the recognized tribes were established using a combination of FISH and conventional G- and C-banding. Our analyses allowed for the delimitation of three X chromosome types represented, respectively, by cattle (Bovinae, tribe Bovini), the tragelaphines (Bovinae, tribe Tragelaphini), and a large assemblage comprising all the remaining subfamilies and their tribes (the Cephalophinae, Hippotraginae, Alcelaphinae, Antilopinae, Aepycerotinae, Peleinae, and Caprinae). The use of the bacterial artificial chromosome probe BAC 101 (which maps to Xp12 in cattle) and an Xp painting probe comprising sequences specific for the short arm of cattle Xp (Xp24→p12) allowed us to orient this region, which has moved as a conserved euchromatic block during the evolution of the bovid X chromosome. We show that the differences between the three chromosomal types are attributable to a transposition, two inversions, and heterochromatic additions/deletions. A paucity of comparative mapping data precludes the assignment of the sequences contained in cattle Xp to either the presumed conserved (XCR) or the recently added (XAR) region of the eutherian X chromosome, and the reasons for the retention of these sequences as an evolutionarily conserved unit in the intrachromosomal restructuring of the bovid X across lineages remain enigmatic.

Y-specific microsatellites reveal an African subfamily in taurine (Bos taurus) cattle.

Five cattle Y-specific microsatellites, totalling six loci, were selected from a set of 44 markers and genotyped on 608 Bos taurus males belonging to 45 cattle populations from Europe and Africa. A total of 38 haplotypes were identified. Haplogroups (Y1 and Y2) previously defined using single nucleotide polymorphisms did not share haplotypes. Nine of the 27 Y2-haplotypes were only present in African cattle. Network and correspondence analyses showed that this African-specific subfamily clustered separately from the main Y2-subfamily and the Y1 haplotypes. Within-breed genetic variability was generally low, with most breeds (78%) showing haplotypes belonging to a single haplogroup. AMOVA analysis showed that partitioning of genetic variation among breeds can be mainly explained by their geographical and haplogroup assignment. Between-breed genetic variability summarized via Principal Component Analysis allowed the identification of three principal components explaining 94.2% of the available information. Projection of principal components on geographical maps illustrated that cattle populations located in mainland Europe, the three European Peninsulas and Mediterranean Africa presented similar genetic variation, whereas those breeds from Atlantic Europe and British Islands (mainly carrying Y1 haplotypes) and those from Sub-Saharan Africa (belonging to Y2-haplogroup) showed genetic variation of a different origin. Our study confirmed the existence of two large Y-chromosome lineages (Y1 and Y2) in taurine cattle. However, Y-specific microsatellites increased analytical resolution and allowed at least two different Y2-haplotypic subfamilies to be distinguished, one of them restricted to the African continent.

Multiple paternal origins of domestic cattle revealed by Y-specific interspersed multilocus microsatellites

In this study, we show how Y-specific interspersed multilocus microsatellites, which are loci that yield several amplified bands differing in size from the same male individual and PCR reaction, are a powerful source of information for tracing the history of cattle. Our results confirm the existence of three main groups of sires, which are separated by evolutionary time and clearly predate domestication. These three groups are consistent with the haplogroups previously identified by Götherström et al. (2005) using five Y-specific segregating sites: Y1 and Y2 in taurine (Bos taurus) cattle and Y3 in zebu (Bos indicus) cattle. The zebu cattle cluster clearly originates from a domestication process that was geographically and temporally separated from that of taurine clusters. Our analyses further suggest that: (i) introgression of wild sire genetic material into domesticated herds may have a significant role in the formation of modern cattle, including the formation of the Y1 haplogroup; (ii) a putative domestication event in Africa probably included local Y2-like wild sires; (iii) the West African zebu cattle Y-chromosome may have partially originated from an ancient introgression of humped cattle into Africa; and (iv) the high genetic similarity among Asian zebu sires is consistent with a single domestication process.

Genetic and physical mapping of the natural resistance-associated macrophage protein 1 (NRAMP1) in chicken

The chicken natural resistance-associated macrophage protein 1 (NRAMP1) gene has been mapped by linkage analysis by use of a reference panel to develop the chicken molecular genetic linkage map and by fluorescence in situ hybridization. The chicken homolog of the murine Nramp1 gene was mapped to a linkage group located on Chromosome (Chr) 7q13, which includes three genes (CD28, NDUSF1, and EF1B) that have previously been mapped either to mouse Chr 1 or to human Chr 2q. Physical mapping by pulsed-field gel electrophoresis revealed that NRAMP1 is tightly linked to the villin gene and that the genomic organization (gene order and presence of CpG islands) of the chromosomal region carrying NRAMP1 is well conserved between the chicken and mammalian genomes. The regions on mouse Chr 1, human Chr 2q, and chicken Chr 7q that encompass NRAMP1 represent large conserved chromosomal segments between the mammalian and avian genomes. The chromosome mapping of the chicken NRAMP1 gene is a first step in determining its possible role in differential susceptibility to salmonellosis in this species.

Bovine Y chromosome microsatellite polymorphisms

Thirty-eight bovine Y chromosome (BTAY) microsatellites (MS) were assessed for polymorphisms in DNA samples obtained from 17 unrelated bulls. Thirty-three of these microsatellites are new and were used for the construction of a first generation radiation hybrid map for BTAY (Liu et al., 2002). Five MS had been previously reported and were used as positive controls. Fourteen out of 38 MS were found to be polymorphic; the remaining 24 were uninformative among the animals tested. The number of hemizygous loci per MS within individual ranged from two to over 20. Seven MS presented smear- or ladder-like bands, a unique feature for Y chromosome multi-copy hemizygous MS loci. The locus length variance, within individual, ranged from 2 to 42 bp corresponding to the MS with the minimum and maximum number of loci observed, respectively. Within the 14 polymorphic MS, the five pseudoautosomal MS, on average, were more polymorphic (35.3%) than the nine Y-specific MS (19.6%). Haplotypes resulting from combinations of these polymorphic loci will provide a powerful tool for future studies on the origin of domestic cattle and the evolution of bovid species.

An integrated genetic and physical map of the bovine X chromosome

Genotypic data for 56 microsatellites (ms) generated from maternal full sib families nested within paternal half sib pedigrees were used to construct a linkage map of the bovine X Chromosome (Chr) (BTX) that spans 150 cM (ave. interval 2.7 cM). The linkage map contains 36 previously unlinked ms; seven generated from a BTXp library. Genotypic data from these 36 ms was merged into an existing linkage map to more than double the number of informative BTX markers. A male specific linkage map of the pseudoautosomal region was also constructed from five ms at the distal end of BTXq. Four informative probes physically assigned by fluorescence in situ hybridization defined the extent of coverage, confirmed the position of the pseudoautosomal region on the q-arm, and identified a 4.1-cM marker interval containing the centromere of BTX.

Genomic structure and transcript variants of the bovine DAZL gene

The Deleted in AZoospermia Like (DAZL) gene is a member of the DAZ family and encodes an RNA-binding protein that is expressed in prenatal and postnatal germ cells of males and females. In the human, there are five highly-related members in the DAZ family, four (DAZ1–4) on the Y chromosome and one (DAZL) on an autosome (HSA3). Mutations in these genes have been linked to severe spermatogenic failure and infertility in men. In the present study, we have cloned and characterized the bovine DAZL (bDAZL) gene. The full-length bDAZL cDNA is predicted to encode a protein of 295 amino acids with an RNA recognition motif. The deduced protein sequence of bDAZL is 96 and 97% similar to human and mouse DAZL, respectively. Fluorescence in situ hybridization (FISH) maps bDAZL to the distal region on BTA1q. The bDAZL gene consists of 11 exons and 10 introns. A bDAZL pseudogene was identified on BTA16. Expression analysis of bDAZL in 13 different tissues by RT-PCR shows that two transcripts, variant 1 (2,996 bp) and variant 2 (1,373 bp), of the bDAZL gene are detected only in testis mRNA. The variants probably result from alternative RNA splicing as variant 1 contains an additional 1,623-bp insertion in the 3′ UTR. Our results lay the groundwork for possible single nucleotide polymorphism (SNP) and functional studies of the DAZL gene in cattle.

Molecular characterization of the bovine chromodomain Y-like genes.

The human chromodomain protein, Y-like (CDYL) gene family consists of three members, one on the Y chromosome (CDY) and two on autosomes (CDYL and CDYL2). Studies in the human and mouse showed that genes in the CDYL family are abundantly expressed in testis and play an important role in spermatogenesis. In this study, we have characterized the bovine CDYL (bCDYL) and CDYL2 (bCDYL2) genes. We found that bCDYL and bCDYL2 are very similar to the human orthologues at both mRNA (79% and 85%) and protein (89% and 93%) levels. However, the similarity between the bCDYL and bCDYL2 proteins is low (41%). The bCDYL gene is composed of nine exons, and the bCDYL2 has seven exons. The bCDYL and bCDYL2 genes were mapped by radiation hybrid mapping to bovine chromosomes (BTA) 24 and 18 respectively. The bCDYL gene has four transcript variants that produce four protein isoforms. RT-PCR expression analysis in 12 bovine tissues showed that bCDYL variant 2 was expressed in the testis only, bCDYL variants 1, 3 and 4 were expressed predominantly in the testis and at very low or undetectable levels in the remaining tissues and bCDYL2 was expressed ubiquitously. Examination of bovine testis with in situ hybridization revealed that the bCDYL and bCDYL2 transcripts were found mainly in spermatids, though the amounts of transcripts varied among genes/variants. In addition, antisense transcripts were detected in bCDYL variants 2/3 and 4, as well as in the bCDYL2 gene.