H. A. Ansari

Resolving ambiguities in the karyotype of domestic sheep (Ovis aries).

The unambiguous identification of ovine chromsomes has become essential for the mapping of the sheep genome, which predominantly consists of telocentric chromosomes of gradually decreasing size. Nucleolus organizer regions (NORs) and Robertsonian fusions have been used here as the cytological and morphological markers, respectively, to define the banding pattern of eight paris of sheep telocentric chromosomes that have an ambiguous identification status. Five Robertsonian chromosomes involving most of the ambiguous chromosomes as well as normal prometaphase chromosomes were stained sequentially and separately by QFQ, GTG, and Ag-NOR methodologies. The prometaphase banding patterns of the ambiguous chromosomes 4, 6, 8, 9, 21, 24, 25 and 26 are represented schematically. For providing an accurate image of the banding pattern, a system of shading has been employed to show the relative intensity of bands in a given chromosome. The results presented here will facilitate the regional mapping of the sheep genome, extend the information on cytogenetic homology with other bovids, and substantially accelerate the comparative mapping studies in Bovidae.

A Lineage-Specific Centromeric Satellite Sequence in the Genus Trifolium

We report the molecular structure, genomic organization, chromosomal distribution and evolutionary dynamics of TrR350, a satellite DNA isolated from the forage legume white clover (Trifolium repens L.; 2n = 4x= 32). The basic repeating unit is an A+T rich 350 bp HindIII fragment with a complex dimeric structure consisting of an internal direct repeat of 156 bp packed between unrelated flanking sequences. Each 156 bp repeat has a conserved 24 bp motif repeating at two places. Most of the 24 bp short repeating units enclose a pentanucleotide CAAAA motif, presumed to be involved in breakage-reunion mechanism of tandemly repeating arrays. The dimers share high sequence homology among themselves while monomers within dimers show significant sequence divergence. Genomic Southern hybridization and/or fluorescence in situ hybridization (FISH) on 17 Trifolium species/subspecies revealed that it is a lineage-specific repeat confined to several species within the section Lotoidea originating in the Mediterranean region. The uniform length of the basic repeating unit and the centromeric localization in most of the species harbouring it reflects its extensive conservation in the lineage. However, the HindIII restriction profile in seven species also indicated independent evolution of this repeat.

The linkage map of sheep chromosome 6 compared with orthologous regions in other species

The genetic linkage map of sheep Chromosome (Chr) 6 has been extended to include 35 loci with the addition of 11 RFLP and 12 microsatellite loci. The sex-averaged linkage map now spans 154 cM from phosphodiesterase cyclic GMP beta polypeptide (PDE6B) to OarCP125, an anonymous sheep microsatellite. The male and female map lengths, at 180 cM and 132 cM respectively, did not differ significantly. The physical assignment of PDE6B to Chr 6q33-qter orientates the linkage map on sheep Chr 6 with PDE6B near the telomere and OarCP125 towards the centromere. The order and genetic distances between loci are similar for the sheep Chr 6 and cattle Chr 6 maps, except for the position of the casein genes. The sheep Chr 6 linkage map is also comparable to portions of human Chr 4, mouse Chrs 5 and 3, and pig Chr 8. The synteny between sheep Chr 6 and human Chr 4 has been extended from PDE6B (4pl6.3) to epidermal growth factor (EGF, 4q25-q27). However, a region from platelet-derived growth factor receptor α polypeptide (PDGFRA) to bone morphogenetic protein 3 (BMP3), which spans 19 cM on sheep Chr 6, appears to be inverted with respect to the human and mouse loci. Other differences in the gene order between sheep, pig, and mouse suggest more complex rearrangements.

Experimental evidence for the ancestry of allotetraploid Trifolium repens and creation of synthetic forms with value for plant breeding

BackgroundWhite clover (Trifolium repens) is a ubiquitous weed of the temperate world that through use of improved cultivars has also become the most important legume of grazed pastures world-wide. It has long been suspected to be allotetraploid, but the diploid ancestral species have remained elusive. Putative diploid ancestors were indicated by DNA sequence phylogeny to be T. pallescens and T. occidentale. Here, we use further DNA evidence as well as a combination of molecular cytogenetics (FISH and GISH) and experimental hybridization to test the hypothesis that white clover originated as a hybrid between T. pallescens and T. occidentale.ResultsT. pallescens plants were identified with chloroplast trnL intron DNA sequences identical to those of white clover. Similarly, T. occidentale plants with nuclear ITS sequences identical to white clover were also identified. Reciprocal GISH experiments, alternately using labeled genomic DNA probes from each of the putative ancestral species on the same white clover cells, showed that half of the chromosomes hybridized with each probe. F1 hybrids were generated by embryo rescue and these showed strong interspecific chromosome pairing and produced a significant frequency of unreduced gametes, indicating the likely mode of polyploidization. The F1 hybrids are inter-fertile with white clover and function as synthetic white clovers, a valuable new resource for the re-incorporation of ancestral genomes into modern white clover for future plant breeding.ConclusionsEvidence from DNA sequence analyses, molecular cytogenetics, interspecific hybridization and breeding experiments supports the hypothesis that a diploid alpine species (T. pallescens) hybridized with a diploid coastal species (T. occidentale) to generate tetraploid T. repens. The coming together of these two narrowly adapted species (one alpine and the other maritime), along with allotetraploidy, has led to a transgressive hybrid with a broad adaptive range.

Molecular and cytogenetic evidence for an allotetraploid origin of Trifolium dubium (Leguminosae)

Suckling clover, Trifolium dubium Sibth., is a European grassland legume that has spread to many parts of the world. The present work shows that it is an allotetraploid (2n = 4x = 30) combining the genomes of T. campestre Schreb. (2n = 2x = 14) and T. micranthum Viv. (2n = 2x = 16), two diploid species of similar geographic distribution. T. dubium has two nuclear ITS sequences that closely match those of T. campestre and T. micranthum. Genomic in situ hybridisation using genomic DNA of T. campestre and T. micranthum as probes has differentiated the ancestral sets of chromosomes in T. dubium cells. Comparative fluorescence in situ hybridisation analyses of 5S and 18S-26S rDNA loci were also consistent with an allotetraploid structure of the T. dubium genome. A marked preponderance of ITS repeats from T. campestre over those from T. micranthum indicated that concerted evolution has resulted in partial homogenisation of these sequences by depletion of the T. micranthum-derived 18S-26S rDNA repeats. In parallel with this, the epigenetic phenomenon of nucleolar dominance has been observed in T. dubium such that the chromatin associated with the 18S-26S rDNA loci derived from T. campestre is decondensed (transcriptionally active), whilst that from T. micranthum remains highly condensed throughout the cell cycle. T. dubium, therefore, appears to have arisen by way of hybridisation between forms of the diploid species T. campestre and T. micranthum accompanied by chromosome doubling. The observed genomic changes in rDNA resulting from interspecific hybridisation provide evidence for the process of genome diploidisation in T. dubium.

Resolving ambiguities in the karyotype of domestic sheep (Ovis aries). II. G-, Q-, and R-banded idiograms, and chromosome-specific molecular markers.

Internally consistent G-, Q- and R-banded karyotypes and idiograms for sheep chromosomes at the 422-band level of resolution are presented. These were derived by sequential Q- to G-staining, and sequential Q- to R-staining of prometaphase spreads prepared from sheep with normal and Robertsonian chromosomes. The fused chromosomes served as stable morphological markers. To minimise confusion due to chromosomal nomenclature, we have listed chromosome-specific (reference) molecular markers that have been mapped byin situ hybridization to sheep chromosomes. The use of molecular markers in conjunction with the sequential Q- to G- and sequential Q- to R-banded karyotypes and iodiograms provided here will elimiate ambiguities in identifying and numbering sheep chromosomes and will facilitate their comparison with cattle chromosomes.

Eco-geographically divergent diploids, Caucasian clover (Trifolium ambiguum) and western clover (T. occidentale), retain most requirements for hybridization.

BACKGROUND AND AIMS DNA sequence similarities and hybridization patterns in Trifolium (clovers) section Trifoliastrum suggest that rapid radiation from a common ancestral source led to this complex of diverse species distributed across Europe, western Asia and North Africa. Two of the most geographically and ecologically divergent of these species are the rhizomatous T. ambiguum from high altitudes in eastern Europe and western Asia and the stoloniferous T. occidentale from sea level in western Europe. Attempts were made to hybridize these species to ascertain whether, despite this separation, gene flow could be achieved, indicating the retention of the genetic factors necessary for hybridization. METHODS Three F(1) hybrids formed after embryo rescue were described, characterized by conventional and molecular cytogenetics, subjected to fertility tests and progeny generations were developed. RESULTS AND CONCLUSIONS Partially fertile hybrids between Trifolium ambiguum and T. occidentale were obtained for the first time. The F(1) hybrids produced seeds after open-pollination, and also produced triploid progeny in backcrosses to T. occidentale from the functioning of unreduced gametes in the hybrids. These plants were fertile and produced progeny with T. occidentale and with T. repens. Meiotic chromosome pairing in the F(1) showed six to eight bivalents per pollen mother cell, indicating pairing between the parental genomes. A chromosome-doubled form of one hybrid, produced using colchicine, showed some multivalents, indicative of interspecific chromosome pairing. The hybrid plants were robust and combined phenotypic characteristics of both species, having stolons, thick roots and a few rhizomes. Results show that despite separation by the entire breadth of Europe, the speciation process is incomplete, and these taxa have partially retained most of the genetic compatibilities needed for hybridization (possibly except for endosperm development, which was not tested). The fertile progeny populations could lead to new clover breeding strategies based on new hybrid forms.

Assignment of five loci from human chromosome 8q onto sheep chromosome 9

Using a chromosomally characterized minipanel of sheep x hamster cell hybrids, five new loci, including carbonic anhydrase II (CA2), calbindin 1 (28 kDa) (CALB1), corticotropin releasing hormone (CRH), cytochrome P450 11B subfamily XIB (steroid-11-beta-hydroxylase), polypeptide 1 (CYP11B1), and interleukin 7 (IL7), have been assigned to sheep chromosome 9. A homolog of CA2 was detected on sheep chromosome 1. CRH was regionally localized to sheep 9q23-->q28 by in situ hybridization. This study assigns chromosome 9 as the sheep equivalent of cattle chromosome 14 and indicates that CALB1, CYP11B1, and IL7, which have not been mapped on the cattle genome, are likely to be present on cattle chromosome 14. It also shows by comparative genome analysis that a large segment of human chromosome 8q is highly conserved in sheep chromosome 9 and cattle chromosome 14. Based on these data, we propose that sheep chromosome 9 be recognised as the equivalent of cattle chromosome 14.

New Robertsonian translocation chromosomes in domestic sheep (Ovis aries)

We report two new Robertsonian centric fusions, designated t4 and t5, in New Zealand Romney sheep. Q- and G-banding studies show that sheep chromosomes 5 and 8 are fused in t4 and that chromosomes 8 and 22 are fused in t5. The presence of large blocks of centromeric heterochromatin in t4 and t5 suggests that the fusions may be of recent origin.

Four human Chromosome 3q and four human Chromosome 21 loci map onto sheep Chromosome 1q

Eight new loci have been assigned to sheep Chromosome (Chr) 1q by use of a chromosomally characterized minipanel of sheep x hamster cell hybrids. Four loci, which have been mapped to the distal region of human Chr 3q, are ceruloplasmin (CP), sucrase isomaltase (SI), glucose transporter 2 (GLUT2), and ectopic viral integration site 1 (EVI1). The other four loci, on human Chr 21, include interferon alpha receptor (IFNAR); interferon inducible protein p78, murine (MX1); collagen type VI, alpha 1 (COL6A1); and S100 protein, beta polypeptide (S100B). All of these loci, except GLUT2 and MX1, have been mapped onto bovine Chr 1 or are syntenic with loci on this chromosome. The in situ localization of transferrin (TF) to sheep Chr 1q42-q45 confirms our previous assignment of this locus and independently anchors the eight new syntenic loci to sheep Chr 1q.