Comparative physical mapping of the 5S and 18S-25S rDNA in nine wild Hordeum species and cytotypes

Absract  The physical locations of the 5S and 18S-25S rDNA sequences were examined in nine wild Hordeum species and cytotypes by double-target in situ hybridization using digoxigenin-labelled 5S rDNA and biotin-labelled 18S-25S rDNA as probes. H. vulgare ssp. spontaneum (2n=2x=14; I-genome) had a similar composition of 5S and 18S-25S rDNA to cultivated barley (H. vulgare ssp. vulgare, I-genome), with two major 18S-25S rDNA sites and minor sites on four of the other five chromosomes; three chromosomes had 5S rDNA sites. The closely related H. bulbosum (2x; also I-genome) showed only one pair of 5S rDNA sites and one pair of 18S-25S rDNA sites on different chromosomes. Four wild diploid species, H. marinum (X-genome), H. glaucum and H. murinum (Y-genomes) and H. chilense (H-genome), differed in the number (2–3 pairs), location, and relative order of 5S and the one or two major 18S-25S rDNA sites, but no minor 18S-25S rDNA sites were observed. H. murinum 4x had three chromosome pairs carrying 5S rDNA, while the diploid had only a single pair. Two other tetraploid species, H. brachyantherum 4x and H. brevisubulatum 4x (both considered to have H-type genomes), had minor 18S-25S rDNA sites, as well as the major sites. Unusual double 5S rDNA sites – two sites on one chromosome arm separated by a short distance – were found in the American H-genome species, H. chilense and H. brachyantherum 4x. The results indicate that the species H. brachyantherum 4x and H. brevisubulatum 4x have a complex evolutionary history, probably involving the multiplication of minor rDNA sites (as in H. vulgare sensu lato), or the incorporation of both I and H types of genome. The rDNA markers are useful for an investigation of chromosome evolution and phylogeny. Received: 9 February 1998 / Accepted: 14 July 1998     

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     

Detection and physical mapping of the 18S-5.8S-26S rDNA and the pKFJ660 probe with microsatellite sequences derived from the rice blast fungus (Magnaporthe grisea) in conifer species

Sequences homologous to the pKFJ660 probe, a fragment of DNA derived from the rice blast fungus (Magnaporthe grisea) carrying TC/AG repeat microsatellite sequences and 30 bp direct repeats were identified in the genome of Picea (spruce) and Pinus (pine) species by fluorescence in situ hybridization (FISH) and slot blot analyses. Slot blot analysis using the pKFJ660 probe revealed hybridization signals with genomic DNAs from various pine and spruce species. Further analyses indicated that the copy number of the (AG)30 motif was higher than 5 x 10(4) per plant genome for all plant samples tested, but the copy number of the sequences homologous to the whole pKFJ660 probe varies considerably among the 25 plant species tested. In situ hybridization of metaphase chromosomes from Pinus resinosa, P. banksiana and P. strobus showed the presence of sequences homologous to this probe on several chromosomes in a dispersed pattern. Major signals were observed on a few chromosomes indicating that some of these sequences are clustered in specific genomic locations. The locations of these repeats were compared to those of 18S-5.8S-26S rDNA in pine species. Chromosomal distribution of 18S-5.8S-26S rDNA varied among the three pine species (P. resinosa, P. banksiana and P. strobus) studied. Ribosomal DNA (rDNA) sites were identified on 14 to 20 chromosomes in these pine species.     

FISH mapping of the 5S and 18S-28S rDNA loci in different species of Glycine.

Wild germplasms are often the only significant sources of useful traits for crops, such as soybean, that have limited genetic variability. Before these germplasms can be effectively manipulated they must be characterized at the cytological and molecular levels. Modern soybean probably arose through an ancient allotetraploid event and subsequent diploidization of the genome. However, wild Glycine species have not been intensively investigated for this ancient polyploidy. In this article we determined the number of both the 5S and 18S-28S rDNA sequences in various members of the genus Glycine using FISH. Our results distinctly establish the loss of a 5S rDNA locus from the "diploid" (2n = 40) species and the loss of two from the (2n = 80) polyploids of GLYCINE: A similar diploidization of the 18S-28S rDNA gene family has occurred in G. canescens, G. clandestina, G. soja, and G. max (L.) Merr. (2n = 40). Although of different genome types, G. tabacina and G. tomentella (2n = 80) both showed two major 18S-28S rDNA loci per haploid genome, in contrast to the four loci that would be expected in chromosomes that have undergone two doubling events in their evolutionary history. It is evident that the evolution of the subgenus Glycine is more complex than that represented in a simple diploid-doubled to tetraploid model.     

Intraspecific variation of 18S-5.8S-26S rDNA sites revealed by FISH and RFLP in wild oat, Avena agadiriana

The tetraploid species Avena agadiriana that was first described in 1985 is distributed on the Atlantic coastal strip south of Casablanca in Morocco. Five accessions of this species (M55, M59, M60, M71 and M74) were compared by using FISH and RFLP analysis of 18S-5.8S-26S rDNA. The FISH data indicated that three pairs of major hybridization sites of the rDNA were located on satellite chromosomes in accessions M55, M59, M60 and M71. Accession M74, however, had only two pairs of major sites of hybridization. A pair of the major rDNA sites in M71 was very small and closely located at the terminal region of Nor-ST (Nucleolar organizing region of subtelocentrics) chromosomes. RFLP analysis of the rDNA sequence fragments identified differences among M55, M71 and M74, whilst M59 and M60 were the same with regard to the four restriction enzyme fragments utilized. M74 always lost single rDNA fragments in four restriction enzyme digests. The RFLP data made it possible to distinguish M55 from M59 and M60 in the northern Haut-Atlas Mountains group. A unique 20 kb EcoRI fragment characterized M71. Thus, a combination of FISH and RFLP analysis of rDNA was a good tool for inferring intraspecific evolutionary relationship of A. agadiriana.     

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。     

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

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.     

18S-25S rDNA variation in tissue culture of some Gentiana L. species

18S-25S rDNA of intact plants and tissue cultures of G. acaulis, G. punctata and G. lutea have been investigated by using blot-hybridization. The decrease of rDNA amount was found in the callus cultures as compared with the plants. In contrast to other species, G. lutea showed intragenome heterogeneity of rRNA genes as well as qualitative rDNA changes in tissue culture, in particular appearance of altered repeats. The relationship between the peculiarities of rRNA gene structure and their rearrangements in in vitro culture was suggested.     

Concerted evolution of 18–5.8–26S rDNA repeats in Nicotiana allotetraploids

We review and extend data showing concerted evolution of parental 18–5.8–26S nuclear ribosomal DNA (18–26S rDNA) gene families in three natural Nicotiana allotetraploids ( N. tabacum , N. rustica and N. arentsii , each 2 n  = 4 x  = 48) and one synthetic N. tabacum line (Th37, ♀ N. sylvestris (2 n  = 24) × ♂ N. tomentosiformis (2 n  = 24)). The origin of the gene families was analysed by sequence polymorphisms in the intergenic spacer (IGS) region and the number of chromosomal loci by fluorescence in situ hybridization (FISH). FISH revealed that the number and locations of 18–26S rDNA in the natural allopolyploids was the sum of those found in the diploid progenitors. However, the rDNA restriction patterns showed polymorphisms in the IGS that were not additive, suggesting that parental rDNA clusters were partially ( N. tabacum, N. rustica ) or completely ( N. arentsii ) overwritten by hybrid-specific units. Thus the Nicotiana allotetraploids show evidence of concerted evolution, including both intralocus and interlocus gene conversion. A feral N. tabacum collected in Bolivia had a higher proportion of unconverted parental rDNA units than cultivated tobacco varieties, suggesting either that rDNA homogenization is accelerated by inbreeding or multiple origins of tobacco. There is no evidence for the elimination of N. sylvestris- derived rDNA units in the synthetic Th37 tobacco line as occurred in natural tobacco, although several novel rDNA unit variants were found in most but not all the hybrid plants. Factors that may control the occurrence and extent of rDNA homogenization are discussed for allopolyploids in Nicotiana and other taxa.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 82 , 615–625.     

Molecular Cytogenetics ofMusaSpecies, Cultivars and Hybrids: Location of 18S-5.8S-25S and 5S rDNA and Telomere-like Sequences

The physical sites of 18S-5.8S-25S and 5S rRNA genes and telomericsequences in theMusaL. genome were localized by fluorescentinsituhybridization on mitotic chromosomes of selected lines.A single major intercalary site of the 18S-5.8S-25S rDNA wasobserved on the short arm of the nucleolar organizing chromosomein each genome. AA and BB genome diploids had a single pairof sites, triploids had three sites while a tetraploid hybridhad four sites. The probe is useful for quick determinationof ploidy, even using interphase nuclei from slowly growingtissue culture material. Variation in the intensity of signalswas observed among heterogeneousMusalines indicating variationin the number of copies of the 18S-5.8S-25S rRNA genes. Eightsubterminal sites of 5S rDNA were observed in Calcutta 4 (AA)while Butohan 2 (BB) had six sites; some were weaker in bothgenotypes. Triploid lines showed six to nine major sites of5S rDNA of widely varying intensity and near the limit of detection.The diploid hybrids had five to nine sites of 5S rDNA whilethe tetraploid hybrid had 11 sites. The telomeric sequence wasdetected as pairs of dots at the ends of all the chromosomesanalysed but no intercalary sequences were seen. The molecularcytogenetic studies ofMusausing repetitive and single copy DNAprobes should yield insight into the genome and its evolutionand provide data forMusabreeders, as well as generating geneticmarkers inMusa.Copyright 1998 Annals of Botany Company Genome evolution, nucleolar organizing regions, telomeres,in situhybridization, genetic markers, banana, plantain.     

Physical mapping of 5S and 18S-25S rDNA and repetitive DNA sequences in Aegilops umbellulata.

An accurate physical map of the location of the 5S and the 18S-5.8S-25S rRNA genes and a repetitive DNA sequence has been produced on Aegilops umbellulata Zhuk., (2n = 2x = 14) chromosomes by in situ hybridization. Chromosome morphology together with the hybridization pattern of pSc119.2, a DNA sequence from rye, allowed identification and discrimination of different chromosomes; pSc119.2 hybridizes with all Ae. umbellulata chromosomes at the telomeres, except for the short arm of chromosome 6U, and shows intercalary sites on the long arms of chromosomes 6U and 7U. The 5S and 18S-25S rDNA have been mapped physically only on the short arms of chromosomes 1U and 5U. On chromosome 1U the order of the genes is 5S rDNA subterminal and 18S-25S rDNA more proximal, while on chromosome 5U the position of the genes is reversed. The relative order of the genes, together with the hybridization pattern of the pSc119.2, is useful in identifying whole chromosomes or chromosome segments from Ae. umbellulata in recombinant or addition lines with wheat. The data help link the physical organization of chromosomes to the genetic map. Other members of the Triticeae vary in the presence and order of the 5S and 18S-25S rDNA sequences on groups 1 and 5, indicating multiple and complex evolutionary rearrangements of the chromosome arms.     

Physical localization of the 18S-5.8S-26S rDNA and sequence analysis of ITS regions in Thinopyrum ponticum (Poaceae: Triticeae): implications for concerted evolution

Fluorescence in situ hybridization was used in Thinopyrum ponticum, a decaploid species, and its related diploid species, to investigate the distribution of the 18S-5.8S-26S rDNA. The distribution of rDNA was similar in all three diploid species (Th. bessarabicum, Th. elongatum and Pseudoroegneria stipifolia). Two pairs of loci were observed in each somatic cell at metaphase and interphase. One pair was located near the terminal end and the other in the interstitial regions of the short arms of one pair of chromosomes. However, all of the major loci in Th. ponticum were located on the terminal end of the short arms of chromosomes, and one chromosome had only one major locus. The maximum number of major loci detected on metaphase spreads was 20, which was the sum of that of its progenitors. The interstitial loci that exist in the possible diploid genome donor species were probably 'lost' during the evolutionary process of the decaploid species. A number of minor loci were also detected on whole regions of two pairs of homologous chromosomes. These results suggested that the position of rDNA loci in the Triticeae might be changeable rather than fixed. Positional changes of 18S-5.8S-26S rDNA loci between Th. ponticum and its candidate genome donors indicate that it is almost impossible to find a genome in the polyploid species that is completely identical to that of its diploid donors. The possible evolutionary significance of the distribution of the rDNA is also discussed. Internal transcribed spacer (ITS) regions of nuclear DNA in Th. ponticum were investigated by PCR amplification and sequencing. The sequence data from five positive clones selected at random, together with restriction site analysis, indicated that the ITS repeated units are nearly homogeneous in this autoallodecapolypoid species. Combined with in situ hybridization results, the data led to the conclusion that the ITS region has experienced interlocus as well as intralocus concerted evolution. Phylogenetic analyses showed that the sequences from Th. ponticum have concerted to the E genome repeat type.     

The use of double fluorescence in situ hybridization to physically map the positions of 5S rDNA genes in relation to the chromosomal location of 18S-5.8S-26S rDNA and a C genome specific DNA sequence in the genus Avena.

A physical map of the locations of the 5S rDNA genes and their relative positions with respect to 18S-5.8S-26S rDNA genes and a C genome specific repetitive DNA sequence was produced for the chromosomes of diploid, tetraploid, and hexaploid oat species using in situ hybridization. The A genome diploid species showed two pairs of rDNA loci and two pairs of 5S loci located on both arms of one pair of satellited chromosomes. The C genome diploid species showed two major pairs and one minor pair of rDNA loci. One pair of subtelocentric chromosomes carried rDNA and 5S loci physically separated on the long arm. The tetraploid species (AACC genomes) arising from these diploid ancestors showed two pairs of rDNA loci and three pairs of 5S loci. Two pairs of rDNA loci and 2 pairs of 5S loci were arranged as in the A genome diploid species. The third pair of 5S loci was located on one pair of A-C translocated chromosomes using simultaneous in situ hybridization with 5S rDNA genes and a C genome specific repetitive DNA sequence. The hexaploid species (AACCDD genomes) showed three pairs of rDNA loci and six pairs of 5S loci. One pair of 5S loci was located on each of two pairs of C-A/D translocated chromosomes. Comparative studies of the physical arrangement of rDNA and 5S loci in polyploid oats and the putative A and C genome progenitor species suggests that A genome diploid species could be the donor of both A and D genomes of polyploid oats. Key words : oats, 5S rDNA genes, 18S-5.8S-26S rDNA genes, C genome specific repetitive DNA sequence, in situ hybridization, genome evolution.     

Physical mapping of 5S and 18S-26S ribosomal RNA gene families inAllium victorialis var.platyphyllum

A physical map of the 5S and 18S–26S rRNA genes was determined using bi-color fluorescencein situ hybridization technique inA. victorialis var.platyphyllum. 5S rRNA genes were positioned in the intercalary regions of the short arms in homologous chromosomes 6. Two major loci of the 18S-26S rRNA genes were detected in the secondary constrictions flanking with a pair of satellite and terminal region of short arm in chromosome 4. And two additional minor loci were heterotype, representing one signal on the terminal region of the short arm in one homolog of chromsome 2, and other on one homolog of chromosome 6 with linked 5S rRNA loci. In addition chromomycin A₃ (CMA,) fluorescent banding method was used to identify the relation between Nucleolus Organizer Region (NOR) sites and CMA, positive heterochromatin sites. In homologous chromosome 4 showing 18S–26S rDNA hybridization signals revealed also distinct CMA, positive band.     

5.8S rDNA variability in Allium species belonging to the third evolutionary group

Sequence analysis of 5.8S rDNA in 67 accessions of the subgenus Allium and six other subgenera belonging to the third evolutionary group of Allium genus (Friesen et al., 2006) was performed. Nucleotide substitutions in 5.8S rDNA sequences of Allium accessions were identified and studied for the first time. The probable secondary structure of 5.8S rRNA was constructed. It was shown that mutations in 5.8S rDNA do not involve conserved motifs, and they did not significantly affect the secondary structure of the RNA molecule in Allium accessions.     

Phylogenetic position of yeast-like symbiotes from three rice planthoppers (Homoptera: Delphacidae ) based on 18S rDNA and ITS-5.8S rDNA sequences

为研究水稻3种主要害虫灰飞虱Laodelphax striatellus、褐飞虱Nilaparvata lugens和白背飞虱Sogatella furcifera体内类酵母共生菌( yeast-like symbiotes,YLS)的种属地位及与寄主的进化关系,测定了其体内YIS的18SrDNA及ITS-5.8S rDNA的全长序列.基于3种稻飞虱体内YLS的18S rDNA序列比对表明,褐飞虱YLS和白背飞虱YLS的一致性比其与灰飞虱YLs的高(褐飞虱YLS和白背飞虱YLS为98.91％,灰飞虱YLS和褐飞虱YLS为95.74％,灰飞虱YLS和白背飞虱YLS为96.02％),而基于ITS-5.8S rDNA序列比对,灰飞虱YLS和白背飞虱YLS的一致性比其与褐飞虱YLS的要高(白背飞虱YLS和灰飞虱YLS为99.57％,灰飞虱YLS和褐飞虱YIS为91.91％,白背飞虱YLS和褐飞虱YLS为90.46％).基于真菌18S rDNA和ITS-5.8S rDNA的系统发育树均表明,3种稻飞虱体内YLS与其他已知真菌进化关系较远.本研究证实了昆虫真菌类共生菌与寄主形成了长期的进化关系,从而形成了不同于已知真菌的分类地位.     

Characterization of 18S-ITS1-5.8S rDNA in eleven species in Soleidae: implications for phylogenetic analysis

Wildfire is one of the most important global agents of disturbance affecting terrestrial and riparian vegetation. Post-fire vegetation changes can alter stream resource pathways and cause channel reorganization and sediment-laden debris flows. Yet, little is known about macroinvertebrate community recovery following wildfire and debris flows and how these communities fit into the broader stream community mosaic. We examined the effects of wildfire and debris flows on relative resource availability and macroinvertebrate assemblages at 31 streams in Idaho, USA using a space-for-time study design. Wildfire and debris flows had no apparent effects on resource standing crop. However, macroinvertebrate communities among unburned, burned, and debris flow streams were quite different. Compared to unburned streams, biomass and density were higher at streams which experienced debris flows ~ 10 years post fire, but exhibited the near-complete absence of macroinvertebrates at streams with more recent debris flows. Stream macroinvertebrate communities impacted by debris flows were distinct compared to unburned and burned streams which did not experience debris flows. When found, differences in macroinvertebrate biomass, density, richness, and community structures were largely due to the incidence of debris flows. Debris flows removed the riparian vegetation, slowing its recovery, cascading to affect macroinvertebrate community structure into the long term.