Physical mapping of rDNA sequences in four karyotypes of Ranunculus silerifolius (Ranunculaceae)

The chromosomal locations of the 18S-5.8S-26S rDNA and 5S rDNA sequences were examined in four cytotypes of Ranunculus silerifolius (the Matsuyama, Mugi, Otaru, and Karatsu types) using fluorescence in situ hybridization (FISH). Using the 18S-5.8S-26S rDNA probe, one pair of probe hybridization sites was detected by FISH in the interstitial region corresponding to the secondary constriction on the short arm of a satellite chromosome (chromosome pair 6) in all four karyotypes. FISH using 5S rDNA identified one pair of sites. The 5S rDNA locus was on different chromosomes in the four karyotypes: in the interstitial region of the short arm of the largest metacentric chromosome (chromosome pair 1) in the Matsuyama type, in the interstitial region of the short arm of the subtelocentric chromosome (pair 2) in the Mugi and Otaru types, and in the interstitial region of the short arm of the metacentric chromosome (pair 2) in the Karatsu type. This physical mapping of the 5S rDNA provides valuable information about karyotype evolution in R. silerifolius. Possible mechanisms of chromosome evolution are discussed.     

Distribution of highly repeated DNA sequences in species of the genus Lens Miller.

Multiple-target fluorescence in situ hybridization (FISH) was applied on mitotic chromosomes of seven Lens taxa using two highly repetitive sequences (pLc30 and pLc7) isolated from the cultivated lentil and the multigene families for the 18S-5.8S-25S (pTa71) and 5S rRNA (pTa794) from wheat simultaneously as probes. The number and location of pLc30 and pLc7 sites on chromosomes varied markedly among the species, whereas the hybridization pattern of 5S rDNA and 18S-5.8S-25S rDNA was less variable. In general, each species showed a typical FISH karyotype and few differences were observed among accessions belonging to the same species, except for the accessions of Lens odemensis. The most similar FISH karyotype to the cultivated lentil is that of Lens culinaris subsp. orientalis, whereas Lens nigricans and Lens tomentosus are the two species that showed the most divergent FISH patterns compared with all taxa for number and location of pLc30 and 18S-5.8S-25S rDNA sites.     

Molecular-cytogenetic studies of ribosomal genes and heterochromatin reveal conserved genome organization among 11 Quercus species

Genomes of 11 Quercus species were characterized using cytogenetic (Giemsa C-banding, fluorochrome banding), molecular-cytogenetic (fluorescence in situ hybridization, FISH, to ribosomal genes) and molecular (dot-blot for ribosomal gene-copy number assessment) techniques. Ribosomal genes are the first DNA sequences to be physically mapped in oaks, and the copy number of the 18S-5.8S-26 S rRNA genes is estimated for the first time. Oak karyotypes were analysed on the basis of DAPI banding and FISH patterns; five marker chromosomes were found. In addition, chromosomal organization of ribosomal genes with respect to AT- and GC-differentiated heterochromatin was studied. Fluorochrome staining produced very similar CMA/DAPI banding patterns, and the position and number of ribosomal loci were identical for all the species studied. The 18S-5.8S-26 S rRNA genes in oak complements were represented by a major locus at the subterminal secondary constriction (SC) of the only subtelocentric chromosome pair and a minor locus at paracentromeric SC of one metacentric pair. The only 5 S rDNA locus was revealed at the paracentromeric region of the second largest metacentric pair. A striking karyotypic similarity, shown by both fluorochrome banding and FISH patterns, implies close genome relationships among oak species no matter their geographic origin (European or American) or their ecophysiology (deciduous or evergreens). Dot-blot analysis gave preliminary evidence for different copy numbers of 18S-5.8S-26 S rRNA genes in diploid genomes of Q. cerris, Q. ilex, Q. petraea, Q. pubescens and Q. robur (2700, 1300, 2200, 4000 and 2200 copies, respectively) that was correlated with the size polymorphism of the major locus. Received: 26 February 1999 / Accepted: 16 March 1999     

Chromosomal organization of ribosomal genes and NOR-associated heterochromatin, and NOR activity in some populations of Allium commutatum Guss. (Alliaceae)

The position and the number of 18S-5.8S-26S and 5S rDNA loci, characterization of nucleolar organizing region (NOR)-associated heterochromatin and NOR activity assessment are given for six south-eastern Adriatic populations of Allium commutatum Guss. The karyotype characteristics were identical for all the populations studied, even those of distant islands. Diploid karyotypes (2 n = 16) always possessed two NOR-bearing chromosome pairs with pericentric and median secondary constrictions (SCs) on the short arm of the chromosomes VII and VIII. Fluorescent in situ hybridization (FISH) confirmed that these were the only sites of 18S-5.8S-26S rRNA genes. NOR-associated heterochromatin was of the constitutive character as shown after C-banding. Differential fluorochrome banding with Chromomycin A3 (CMA) and 4,6-diamidino-2-phenylindole (DAPI) revealed that this heterochromatin comprises both GC- and AT-rich DNA segments. Heteromorphism of C- and CMA-bands was noticed between homologous NOR-bearing chromosomes. The maximum number of four active NORs was correlated with the maximum number of four nucleoli in interphase. Variability of NOR-activity, expressed as number and size of silver stained NORs, existed between cells and between individuals of the same population. The different size of homologous and nonhomologous silver stained NORs was correlated with the extension of SCs. The only 5S rDNA locus was in an intercalary position on short arm of the chromosome VI, at the region of AT-rich constitutive heterochromatin. Dimorphism of C-bands and DAPI/Hoechst(H)-fluorescent bands was noticed between homologous chromosomes VI. © 2002 The Linnean Society of London, Botanical Journal of the Linnean Society , 2002, 139 , 99–108.     

Mapping of rDNA on the chromosomes of Eleusine species by fluorescence in situ hybridization

Mapping of rDNA sites on the chromosomes of four diploid and two tetraploid species of Eleusine has provided valuable information on genome relationship between the species. Presence of 18S-5.8S-26S rDNA on the largest pair of the chromosomes, location of 5S rDNA at four sites on two pairs of chromosomes and presence of 18S-5.8S-26S and 5S rDNA at same location on one pair of chromosomes have clearly differentiated E. multiflora from rest of the species of Eleusine. The two tetraploid species, E. coracana and E. africana have the same number of 18S-5.8S-26S and 5S rDNA sites and located at similar position on the chromosomes. Diploid species, E. indica, E. floccifolia and E. tristachya have the same 18S-5.8S-26S sites and location on the chromosomes which also resembled with the two pairs of 18S-5.8S-26S rDNA locations in tetraploid species, E. coracana and E. africana. The 5S rDNA sites on chromosomes of E. indica and E. floccifolia were also comparable to the 5S rDNA sites of E. africana and E. coracana. The similarity of the rDNA sites and their location on chromosomes in the three diploid and two polyploid species also supports the view that genome donors to tetraploid species may be from these diploid species.     

Physical mapping of 18S-5.8S-26S and 5S ribosomal RNA gene families in three important vetches (Vicia species) and their allied taxa constituting three species complexes

The most-important vetch species, Vicia narbonensis (narbon vetch, section Faba), Vicia villosa (hairy vetch, section Cracca) and Vicia sativa (common vetch, section Vicia) and their close relatives (often difficult to circumscribe into distinct taxa) constitute respectively, Narbonensis, Villosa and Sativa species complexes in the genus Vicia. The distribution of the 18S-5.8S-26S (18S-26S) and 5S ribosomal RNA (rRNA) gene families on the chromosomes of 19 (2n=2x=10,12,14) of the 24 species and subspecies belonging to the three species complexes, and Vicia bithynica (2n=12, section Faba) and Vicia hybrida (2n=12, section Hypechusa) was studied by fluorescence in situ hybridization (FISH) with pTa 71 (18S-26S rDNA) and pTa 794 (5S rDNA) DNA clones. Computer – aided chromosome analysis was performed on the basis of chromosome length, the arm-length ratio and the position of the hybridization signals. The positions of the four (2+2) signals of the two rRNA gene families were similar between each of the three, as well as two subspecies of V. narbonensis and Vicia johannis, respectively. Two major 18S-26S rDNA loci were found in the nucleolus organiser regions (NORs) of each of the species except V. hybrida, where it was present in two out of four SAT chromosomes. In addition to major NORs, two minor loci have been physically mapped at the centromeric regions of chromosomes of group 1 in Vicia amphicarpa, Vicia macrocarpa and V. sativa, and two NORs of group 5 in V. hybrida, and on the long arms of group 4 in V. bithynica. Two or four 5S rDNA loci, observed in the short arms of groups 2–4 and 5, and 18S-26S rDNA loci were located in different chromosomes of all the species within the Narbonensis and Villosa species complexes, and Vicia angustifolia of the Sativa species complex. In the remaining six species of the Sativa species complex, and V. bithynica and V. hybrida, the two or four 5S rDNA sites were present in chromosomes which harbor 18S-26S rRNA genes. The tandemly repeated 5S rDNA sites, located at the proximal part of the long arm of groups 3–5, were diagnostic for V. angustifolia, Vicia cordata, Vicia incisa, V. macrocarpa, Vicia nigra and V. sativa of the Sativa species complex. In V. amphicarpa of the same complex, the tandem repeats were located at the distal part of the long arms of group 3. Variability in the number, size and location of two ribosomal DNA probes could generally distinguish species within the Narbonensis and Sativa species complex, V. bithynica and V. hybrida. With respect to the four species of the Villosa species complex the karyotypes could not be identified individually on the basis of the distribution of two ribosomal gene families in three out of seven pairs of chromosomes. Received: 18 October 2000 / Accepted: 20 March 2001     

Trends in site-number change of rDNA loci during polyploid evolution in Sanguisorba (Rosaceae)

To elucidate the evolutionary dynamics of rDNA site number in polyploid plants, we determined 5S and 18S-5.8S-26S rDNA sites for ten species of Sanguisorba (2n=14, 28, 56) and a single species of each of three outgroup genera, Agrimonia (2n=28), Rosa (2n=14), and Rubus (2n=14) by the fluorescence in situ hybridization (FISH) method. We also estimated phylogenetic relationships among these species using matK chloroplast DNA (cpDNA) sequences, and reconstructed the evolutionary history of rDNA site number based on the maximum parsimony method. The 2n=14 and 2n=28 plants of all genera except Rosa carried two 5S rDNA sites, whereas Rosa and 2n=56 plants carried four sites. The 2n=14 plants had two 18S-5.8S-26S rDNA sites, whereas Sanguisorba annua and 2n=28 plants had four or six sites. Phylogenetic analysis showed that polyploidization from 2n=14 to 2n=28 has occurred once or three times in Sanguisorba and Agrimonia. The 5S rDNA sites duplicated during each ancestral polyploidization were evidently lost after each polyploidization. However, the duplicated 18S-5.8S-26S rDNA sites were all conserved after each polyploidization. Thus, the duplicated 5S rDNA sites tend to have been eliminated, whereas those of 18S-5.8S-26S rDNA tend to have been conserved in Sanguisorba. In the most parsimonious hypothesis, 2n=14 in S. annua is a secondary, putatively dysploid state, reduced from 2n=28.     

Comparative physical mapping of the 18S-5.8S-26S rDNA in three sorghum species.

The physical locations of the 18S-5.8S-26S rDNA sequences were examined in three sorghum species by fluorescence in situ hybridization (FISH) using biotin-labeled heterologous 18S-5.8S-26S rDNA probe (pTa71). Each 18S-5.8S-26S rDNA locus occurred at two sites on the chromosomes in Sorghum bicolor (2n = 20) and S. versicolor (2n = 10), but at four sites on the chromosomes of S. halepense (2n = 40) and the tetraploid S. versicolor (2n = 20). Positions of the rDNA loci varied from the interstitial to terminal position among the four accessions of the three sorghum species. The rDNA data are useful for investigation of chromosome evolution and phylogeny. This study excluded S. versicolor as the possible progenitor of S. bicolor.     

Heterochromatin and rDNA patterns in Solanum species of the Morelloid and Dulcamaroid clades (Solanaceae)

The heterochromatin distribution and the position of 18-5.8-26S, and 5S rDNA loci were determined in 13 species of Solanum of the Morelloid and Dulcamaroid clades. The CMA/DAPI staining and FISH were employed. Two types of constitutive heterochromatin were determined: CMA⁺/DAPI^? associated to NOR and CMA⁺/DAPI^? distributed as terminal bands. In the Morelloid clade, CMA⁺/DAPI^? bands were found in five species while in the Dulcamaroid clade, only S. angustifidum presented this feature. In the Morelloid clade, two to four 18-5.8-26S rDNA loci occupied terminal positions and two rDNA 5S loci were found with variable positions (terminal, intercalary, and centromeric). In the Dulcamaroid clade, two terminal 18-5.8-26S rDNA loci were detected with the exception of S. salicifolium which possessed four such loci and two to four 5S rDNA loci. Solanum crispum is the only species possessing the 5S in synteny with 18-5.8-26S rDNA loci. Karyotype features chromosome banding pattern as well as the location of ribosomal genes which varied among the species, reflecting the chromosome differentiation and evolutionary divergence. The findings obtained contributed to the development of tools that can be used for establishing chromosomic homeologies among species and hence to clarify their taxonomic relationships.     

Phylogenetic and genomic relationships in Setaria italica and its close relatives based on the molecular diversity and chromosomal organization of 5S and 18S-5.8S-25S rDNA genes

We have analyzed the phylogenetic and genomic relationships in the genus Setaria Beauv. including diploid and tetraploid species, by means of the molecular diversity of the 5S rDNA spacer and chromosomal organization of the 5S and 18S-5.8S-25S rDNA genes. PCR amplification of the 5S rDNA sequences gave specific patterns. All the species studied here share a common band of about 340 bp. An additional band of an approximately 300-bp repeat unit was found for Setaria verticillata and the Chinese accessions of Setaria italica and Setaria viridis. An additional band of 450 bp was found in the sole species Setaria faberii. Fluorescent in situ hybridization was used for physical mapping of the 5S and 18S-5.8S-25S rDNA genes and showed that they are localized at two separate loci with no polymorphism of chromosome location among species. Two chromosome pairs carrying the 5S and 18S-5.8S-25S rDNA clusters can now be unambiguously identified using FISH. Phylogenetic trees based on the variation of the amplified 5S rDNA sequences showed a clear separation into four groups. The clustering was dependent on the genomic composition (genome A versus genome B) and confirmed the closest relationship of S. italica and S. viridis accessions from the same geographical region. Our results confirm previous hypotheses on the domestication centers of S. italica. They also show the wide difference between the A and B genomes, and even clarify the taxonomic position of S. verticillata. Received: 28 August 2000 / Accepted: 27 January 2001     

Cytogenetic studies in South American species of Serjania (Sapindaceae: Paullinieae)

Abstract

Serjania Mill. (Paullinieae) is considered the most important neotropical genus of Sapindaceae due to species number and its widespread distribution. In this study, 14 species belonging to three sections were analyzed using conventional staining, C/CMA/DAPI banding, and fluorescence in situ hybridization (FISH) with a 18S-5.8S-26S rDNA probe. New chromosome counts are reported for Serjania crassifolia, Serjania platycarpa, and Serjania regnellii, all with 2n = 24, which is remarkably constant for Serjania. The karyotypes are moderately asymmetric, and variations observed in A1 and A2 indices show resemblances between S. platycarpa, Serjania hebecarpa, and S. crassifolia, and between Serjania communis, Serjania gracilis, and S. regnellii. The banding pattern was homogeneous in Serjania. C/DAPI bands (AT-rich sites) were not clearly evidenced, but changes in the number and position of GC-rich sites (CMA bands) were observed. These segments were associated with 18S-5.8S-26S rDNA sites. The significance of the results is discussed in relation to chromosomal data available for the genus and in regard to the infrageneric treatment of Serjania.     

Major cytogenetic landmarks and karyotype analysis inDaucus carota and other Apiaceae

Karyotype analysis provides insights into genome organization at the chromosome level and into chromosome evolution. Chromosomes were marked for comparative karyotype analysis using FISH localization of rDNA genes for the first time in Apioideae species including taxa of economic importance and several wild Daucus relatives. Interestingly, Daucus species did not vary in number of rDNA loci despite variation in chromosome number (2n = 18, 20, 22, and 44) and previous publications suggesting multiple loci. All had single loci for both 5S and 18S-25S (nucleolar organizing region) rDNA, located on two different chromosome pairs. The 5S rDNA was on the short arm of a metacentric chromosome pair in D. crinitus (2n = 22) and D. glochidiatus (2n = 44) and on the long arm of a metacentric pair in other Daucus species, suggesting possible rearrangement of this chromosome. For other Apiaceae, from two (Apium graveolens), to three (Orlaya grandiflora), to four (Cuminum cyminum) chromosomes had 18S-25S rDNA sites. Variability for number and position of the 5S rDNA was also observed. FISH signals enabled us to identify 20-40% of the chromosome complement among species examined. Comparative karyotype analysis provides insights into the fundamental aspects of chromosome evolution in Daucus.     

Karyotype analysis in Hyacinthella dalmatica (Hyacinthaceae) reveals vertebrate-type telomere repeats at the chromosome ends.

Chromosome analysis of three different populations of Hyacinthella dalmatica (Lallem.) Trinajsti?, an endemic species of the coastal region of southeastern Europe, showed a unique chromosome number, 2n = 2x = 20, and bimodal karyotype with one large and nine smaller pairs of chromosomes. Staining with fluorochromes CMA3 (chromomycin A3) and DAPI (4,6-diamidino-2-phenylindole) revealed heterochromatic regions associated with NORs, centromeres, and several interstitial heterochromatic bands on the longest chromosome pair. Double-target FISH with two ribosomal DNA probes revealed one locus of 5S rRNA genes in the pericentromeric region of chromosome pair 3 and one locus of 18S-5.8S-26S rRNA genes on the short arm of chromosome pair 4 in all plants and populations analyzed. Southern hybridization analysis and FISH experiments demonstrated that the distal ends of H. dalmatica chromosomes contain the vertebrate telomere (5'-TTAGGG-3') repeat type rather than the Arabidopsis (5'-TTTAGGG-3') heptamer, and so suggest that this Asparagales species along with Aloe and Othocallis contains the vertebrate-type telomere repeat.     

Chromosomal localization of 5S and 18S rDNA in five species of subgenus Strobus and their implications for genome evolution of Pinus

BACKGROUND AND AIMS: Studying the genome structure of pines has been hindered by their large genomes and uniform karyotypes. Consequently our understanding of the genome organization and evolutionary changes in different groups of pines is extremely limited. However, techniques are now available that can surmount these difficulties. The purpose of this study was to exploit some of these techniques to characterize the genome differentiation between the two subgenera of Pinus: Pinus and Strobus. METHODS: Double-probe fluorescence in-situ hybridization (FISH) was used to localize the 5S and 18S rDNA loci on chromosomes of five species from the subgenus Strobus: P. bungeana, P. koraiensis, P. armandii, P. wallichiana and P. strobus. * KEY RESULTS: The rDNA FISH pattern varied considerably among the five species, with P. bungeana being the most distinct. By comparing the results obtained with those of previous rDNA FISH studies of members of the subgenus Pinus, several general features of rDNA loci distribution in the genus Pinus can be discerned: (a) species of subgenus Strobus generally have more rDNA loci than species of subgenus Pinus, correlating with their larger genomes in the subgenus Strobus; (b) there is a clear differentiation in 5S and 18S rDNA loci linkage patterns between the two subgenera; (c) variations in the rDNA FISH pattern correlate with phylogenetic relationships among species within the subgenus; (d) P. bungeana has fewer 18S rDNA sites than other pines investigated to date, but they give intense signals, and may reflect the primary distribution of the 18S-25S rDNA loci in the genus. CONCLUSIONS: The stable differentiation in rDNA FISH pattern between the subgenera suggests that chromosomal rearrangements played a role in the splitting of the two subgenera, and transpositional events rather than major structural changes are likely responsible for the variable rDNA distribution patterns among species of the same subgenus with conserved karyotypes.     

中间偃麦草(Thinopyrum intermedium)18S-5.8S-26S rDNA位点在染色体上的分布及对其核ITS序列异质性的分析(英文)

中间偃麦草(Thinopyrum intermedium(Host)Barkworth et Dewey)是禾本科小麦族植物中的一个异源六倍体物种,是重要的牧草植物,在小麦的抗病育种中发挥了重要作用。利用荧光原位杂交(FISH)技术,在体细胞中期染色体上,对18S-5.8S-26S rDNA位点进行了物理定位,发现该物种有3～4对染色体携带18S-5.8S-26S rDNA主位点。结合基因组原位杂交(GISH)分析,证明中间偃麦草的St基因组中有一对同源染色体短臂末端携带一个主位点,其余2～3对主位点位于E基因组染色体上。对不同来源的材料研究表明:18S-5.8S-26S rDNA位点的数目(包括主位点和小位点)、位置、拷贝数在不同收集材料之间的差异较大,甚至在同一个体的不同细胞中也存在差异。讨论了rDNA物理作图数据在分析系统发育问题中的局限性。结合中间偃麦草的三个可能的二倍体基因组供体(Th.bessarabicum、Th. elongatum和Pseudoroegneria stipifolia)rDNA位点分析的结果,对中间偃麦草进化过程中rDNA位点的变化进行了分析,同时,对其中一份材料的核ITS序列进行了克隆、测序和系统发育分析,发现在中间偃麦草中,ITS序列具有很高的异质性。     

Ribosomal, telomeric and heterochromatin sequences localization in the karyotype of Anemone hortensis

The karyotype of the Mediterranean species Anemone hortensis L. (Ranunculaceae) was characterized with emphasis on heterochromatin distribution and localization of ribosomal (18S−5.8S−26S and 5S rDNA) and telomeric repeats (TTTAGGG). Diploid chromosome complement, 2 n  = 2 x  = 16, common to all investigated populations, consisted of three acrocentric, one meta-submetacentric and four metacentric chromosomes ranging in size from 6.34 to 10.47 µm. Fluorescence in situ hybridization (FISH) with 18S and 5S rDNA probes revealed two 18S−5.8S−26S rDNA loci on a satellite and secondary constriction of acrocentric chromosome pair 2 and terminally on acrocentric chromosome pair 3, and two 5S rDNA loci in the pericentromeric region of meta-submetacentric chromosome pair 4 and in the proximity of the 18S−5.8S−26S rDNA locus on chromosome pair 2. The only GC-rich heterochromatin, as revealed by fluorochrome Chromomycin A3 staining, was that associated with nucleolar organizer regions, whereas AT-rich heterochromatin, stained with 4,6-diamino-2-phenylindole (DAPI), was distributed intercalarly and terminally on the long arm of all three acrocentric chromosomes, and terminally on chromosomes 4 and 5. FISH with Arabidopsis -type telomeric repeats (TTTAGGG) as a probe revealed two classes of signals, small dot-like and large bands, at chromosome termini exclusively, where they corresponded to terminal DAPI-stained heterochromatin. Heteromorphism of chromosome pair 4, which refers to terminal DAPI bands and FISH signals, was observed in populations of Anemone hortensis . Chromosome pairing during meiosis was regular with formation of localized chiasmata proximal to the centromere.  © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society , 2006, 150 , 177–186.     

rDNA (18S-28S and 5S) colocalization and linkage between ribosomal genes and (TTAGGG)(n) telomeric sequence in the earthworm,Octodrilus complanatus (Annelida: Oligochaeta: Lumbricidae), revealed by single- and double-color FISH

Spermatogonial and metaphase I chromosomes of the lumbricid earthworm Octodrilus complanatus (Annelida: Oligochaeta) were examined using fluorescent in situ hybridization (FISH) with three repetitive DNA probes-5S rDNA, 18S-26S rDNA, and (TTAGGG)(n). Single-color FISH consistently mapped one chromosome pair per spread using either 5S rDNA or 18S-26S rDNA as probes. Simultaneous (18S-26S)-5S and (18S-26S)-(TTAGGG)(n) FISH demonstrated that repeated units of the two ribosomal families were overlapped and closely associated with telomeric sequences.     

Ribosomal DNAs: an exception to the conservation of gene order in rice genomes

rDNA (18S-5.8S-25S rDNA) and 5S rDNA loci were visualized on the chromosomes of six species of the genus Oryza by fluorescence in situ hybridization (FISH) and the labeled rice chromosomes were identified based on their condensation patterns. As a result, the chromosomes harboring rDNA and/or 5S rDNA loci were determined in the complement for all the known rice genomes. Variation in the location of the rDNA loci indicated the transpositional nature of the rDNAs in the genus Oryza, as also suggested in Triticeae and Allium. Comparative analysis of the locations of rDNA loci among rice, maize and wheat revealed that variability in the physical location of the rDNA loci was characteristic of the genus Oryza and also of the genera of Gramineae. This variability in the location of the rDNA loci between evolutionarily related species is in sharp contrast to the conservation of the general order of genes in their genomes.     

Physical mapping of rRNA genes in Medicago sativa and M. glomerata by fluorescent in situ hybridization

Fluorescent in situ hybridization (FISH) was applied to diploid and tetraploid subspecies of alfalfa (Medicago sativa L.) to investigate the distribution of rRNA genes and to utilize the sites of 18S-5.8S-25S rDNA and 5S rDNA sequences as markers for studying the genome evolution within the species. Medicago glomerata Balb., the species considered to be the ancestor of alfalfa, was included in this study in order to obtain more information on the phylogenetics of alfalfa. Simultaneous in situ hybridization was performed with the probes pTa71 and pXVI labeled with digoxigenin and biotin, respectively. In the diploid taxa, M. glomerata, M. sativa ssp. coerulea Schmalh and ssp. falcata Arcangeli, the 18S-5.8S-25S rDNA sequences were mapped to two sites corresponding to the secondary constrictions of the nucleolar chromosome pair, while 5S rDNA appeared to be distributed in two pairs of sites. Chromosomes carrying 5S loci could be distinguished on the basis of their morphological characteristics. The number of rDNA sites detected in the tetraploid M. sativa ssp. falcata and ssp. sativa (L.) L. & L. were twice the number found in the respective diploid ssp. falcata and ssp. coerulea. The results of this study show that the distribution of ribosomal genes was maintained during the evolutionary steps from the primitive diploid to the cultivated alfalfa. Modifications of the number of rRNA loci were not observed. The importance of in situ hybridization for improving karyotype analysis in M. sativa L. is discussed.     

Nuclear DNA content,base composition,heterochromatin and rDNA in Picea omorika and Picea abies

Two closely related spruces, Picea abies and Picea omorika, a Balkan paleoendemic species, often share habitats, yet never hybridize in nature. The present study adresses their characteristics such as nuclear DNA content, base composition, heterochromatin and rDNA pattern. The genome size of P. abies was 10% larger than that of P. omorika when assessed by flow cytometry, respectively 2C=37.2 pg and 33.8 pg; although when estimated as total chromosome length it was virtually the same. The heterochromatin Chromomycin-A (CMA)/ DAPI fluorochrome banding patterns of both P. abies and P. omorika are given here for the first time. Simultaneous FISH (fluorescent in situ hybridization) using 18S-26S and 5S rDNA probes revealed 16 18S rDNA sites in P. omorika, 12 18S rDNA sites in P. abies, and a single 5S rDNA locus in both species. The genomes have about 41% GC. The number and position of CMA/DAPI bands and rDNA loci provide good chromosome markers to clarify the karyotypes of the two species. Received: 18 October 2000 / 14 June 2001