45S rDNA在小麦及其近缘物种染色体上的分布

将染色体C-分带和原位杂交技术相结合,系统研究了45S rDNA在栽培一粒小麦、野生二粒小麦、普通小麦、大麦、簇毛麦、硬簇麦、六倍体燕麦及鹅观草等物种染色体上的分布情况。这些物种染色体的次缢痕区都有45S rDNA位点, 某些非随体染色体上也有45S rDNA位点分布。以小麦—鹅观草1Rk^#1二体附加系为材料,通过顺序C分带-FISH技术首次将一个45S rDNA定位到1Rk^#1染色体短臂末端。     

Tissue culture triggers chromosome alterations, amplification, and transposition of repeat sequences in Allium fistulosum.

Structural alterations in nuclei and chromosomes of cells derived from callus culture of Allium fistulosum have been studied with fluorescent in situ hybridization (FISH) using 5S ribosomal DNA (rDNA), 45S rDNA, and 375-bp repeat probes. A high frequency of chromosome abnormalities was found to be caused by the loss of telomere-located 375-bp repeats, chromosome fusion, and subsequent breakage-fusion-bridge cycles. Products of chromosome fusions and monocentric and regularly shaped chromosomes showed additional 375-bp repeat and 45S rDNA clusters at unusual sites, suggesting dynamic copy-number changes and transposition of these repeats. Southern hybridization revealed no differences in the 375-bp repeat and 45S rDNA repeat array order or the degree of methylation between DNA isolated from leaves or tissue-culture cells. In addition, protruding, spike-like structures positive for 375-bp repeats were identified on the surface of different-sized nuclei. Transmission electron microscopy analysis revealed the accumulation of densely packed chromatin within spike-like structures. Because root calyptra cells showed similar structures, it is likely that heterochromatic spike-like structures are a feature of nondividing cells at the onset of programmed cell death.     

Evolution of 5S rDNA units and their chromosomal localization in Allium cepa and Allium schoenoprasum revealed by microdissection and FISH

Allium cepa and Allium schoenoprasum each possess 5S rDNA units of two different sizes. The evolution of the two repeat units and their chromosomal localization were investigated. A. cepa has 5S rDNA loci in the proximal and distal regions of the short arm of chromosome 7. When the proximal and distal segments of the short arm of chromosome 7 were microdissected separately, and used as templates for PCR, the short and long 5S rDNA fragments were amplified predominantly from the proximal and distal segments, respectively. The nucleotide sequence of the long 5S rDNA unit resulted from partial duplication of a non-transcribed spacer (NTS) and the insertion of a unique sequence. FISH using a probe consisting of the unique sequence demonstrated that the long unit was distally localized. In A. cepa, the long 5S rDNA unit is only present distally and the short unit is predominantly located proximally on the short arm of chromosome 7. In A. schoenoprasum, the NTSs of the two different-sized 5S rDNAs had quite different sequences. The two 5S rDNA loci were localized very close together in the interstitial region of chromosome 6. FISH, using long and short 5S rDNA unit probes with a competitor of a 120-bp sequence of the 5S rRNA gene, indicated that the long 5S rDNA unit was localized proximally and the short unit distally. Although the NTSs of the 5S rDNA of A. cepa and A. schoenoprasum had quite different nucleotide sequences, the long 5S rDNA units of A. cepa and A. schoenoprasum share a common 75-bp sequence. This sequence might act in the formation of the long 5S rDNA unit in Allium species.     

The specific isolation of complete 5S rDNA units from chromosome 1A of hexaploid, tetraploid, and diploid wheat species using PCR with head-to-head oriented primers.

The presence of 5S rDNA units on chromosome 1A of Triticum aestivum was shown by the development of a specific PCR test, using head-to-head oriented primers. This primer set allowed the amplification of complete 5S DNA units and was used to isolate SS-Rrna-A1 sequences from polyploid and diploid wheat species. Multiple-alignment and parsimony analyses of the 132 sequences divided the sequences into four types. The isolates from T. aestivum and the tetraploid species (T. dicoccoides, T. dicoccum, T durum, T. araraticum, and T timopheevi) were all of one type, which was shown to be closely related to the type mainly characteristic for T. urartu. The other two types were isolated exclusively from the diploid species T. monococcum, T aegilopoides, T. thaoudar, and T. sinskajae and the hexaploid species T. zhukovski. Triticum monococcum was the only species for which representatives of each of the four sequence types were found to be present. Further, we discuss the possible multicluster arrangement of the 5S-Rrna-A1 array.     

Ribosomal RNA genes in soybean and common bean: chromosomal organization, expression, and evolution

Ribosomal RNA (5S and 45S) genes were investigated by FISH in two related legumes: soybean [Glycine max (L.) Merr.] and common bean (Phaseolis vulgaris L.). These species are both members of the same tribe (Phaseoleae), but common bean is diploid while soybean is a tetraploid which has undergone diploidization. In contrast to ploidy expectations, soybean had only one 5S and one 45S rDNA locus whereas common bean had more than two 5S rDNA loci and two 45S rDNA loci. Double hybridization experiments with differentially labelled probes indicated that the soybean 45S and 5S rDNA loci are located on different chromosomes and in their distal regions. Likewise, the common bean 45S and 5S rDNA loci were on unique chromosomes, though two of the 5S rDNA loci were on the same chromosome. FISH analysis of interphase nuclei revealed the spatial arrangement of rDNA loci and suggested expression patterns. In both species, we observed one or more 5S rDNA hybridization sites and two 45S rDNA hybridization sites associated with the nucleolar periphery. The 45S rDNA hybridization patterns frequently exhibited gene puffs as de-condensed chromatin strings within the nucleoli. The other condensed rDNA sites (both 5S and 45S) were spatially distant from the nucleolus in nucleoplasmic regions containing heterochromatin. The distribution of rDNA between the nucleoplasm and the nucleoli is consistent with differential gene expression between homologous alleles and among homoeologous loci.     

Genetic Map of Diploid Wheat, Triticum Monococcum L., and Its Comparison with Maps of Hordeum Vulgare L

A genetic map of diploid wheat, Triticum monococcum L., involving 335 markers, including RFLP DNA markers, isozymes, seed storage proteins, rRNA, and morphological loci, is reported. T. monococcum and barley linkage groups are remarkably conserved. They differ by a reciprocal translocation involving the long arms of chromosomes 4 and 5, and paracentric inversions in the long arm of chromosomes 1 and 4; the latter is in a segment of chromosome arm 4L translocated to 5L in T. monococcum. The order of the markers in the inverted segments in the T. monococcum genome is the same as in the B and D genomes of T. aestivum L. The T. monococcum map differs from the barley maps in the distribution of recombination within chromosomes. The major 5S rRNA loci were mapped on the short arms of T. monococcum chromosomes 1 and 5 and the long arms of barley chromosomes 2 and 3. Since these chromosome arms are colinear, the major 5S rRNA loci must be subjected to positional changes in the evolving Triticeae genome that do not perturb chromosome colinearity. The positional changes of the major 5S rRNA loci in Triticeae genomes are analogous to those of the 18S-5.8S-26S rRNA loci.     

Cytogenetic and molecular identification of three Triticum aestivum-Leymus racemosus translocation addition lines

Chromosome 2C from Aegilops cylindrica has the ability to induce chromosome breakage in common wheat （Tritivum aestivum）. In the BC1F3 generation of the T. aestivum cv. Chinese Spring and a hybrid between T. aestivum-Leymus racemosus Lr.7 addition line and T. aestivum-Ae, cylindrica 2C addition line, three disomic translocation addition lines （2n = 44） were selected by mitotic chromosome C-banding and genomic in situ hybridization. We further characterized these T. aestivum-L, racemosus translocation addition lines, NAU636, NAU637 and NAU638, by chromosome C-banding, in situ hybridization using the A- and D-genome-specific bacterial artificial chromosome （BAC） clones 676D4 and 9M13; plasmids pAsl and pSc119.2, and 45S rDNA; as well as genomic DNA of L. racemosus as probes, in combination with double ditelosomic test cross and SSR marker analysis. The translocation chromosomes were designated as T3AS-Lr7S, T6BS-Lr7S, and T5DS-Lr7L. The translocation line T3AS-Lr7S was highly resistant to Fusarium head blight and will be useful germplasm for resistance breeding.     

5S rDNA genome regions of Lens species.

The length variability of the nontranscribed spacer (NTS) of the 5S rDNA repeats was analyzed in species of the genus Lens by means of PCR amplification. The NTS ranged from approximately 227 to approximately 952 bp. The polymorphism detected was higher than previous NTS polymorphisms described in this genus. Three NTS length variants from Lens culinaris subsp. culinaris and 2 from Lens culinaris subsp. orientalis were sequenced. The culinaris NTS fragment lengths were 239, 371, and 838 bp, whereas the orientalis ones were 472 bp and 506 bp, respectively. As a result of sequence similarities, 2 families of sequences were distinguished, 1 including the sequences of 838 and 506 bp, and others with the sequences of 239, 371, and 472 bp. The 1st family was characterized by the presence of a repeated sequence designated A, whereas the 2nd family showed a single A sequence and other repeated sequences designated B, C, and D. The presence of an (AT)n microsatellite was also observed in the 2nd family of sequences. The fragments, which included the 239-bp and 838-bp NTS sequences, as well as the intergenic spacer (IGS) of the 18S-5.8S-26S ribosomal DNA also from L. culinaris subsp. culinaris, were used to localize the nucleolar organizer region (NOR) and the 5S rDNA loci in the chromosomes of several species of the genus Lens by means of fluorescence in situ hybridization (FISH). The selective hybridization of the 2 NTS probes allowed us to distinguish between different 5S rDNA chromosomal loci.     

Molecular and cytological characterization of 5S rDNA in Oryza species: genomic organization and phylogenetic implications.

We investigated the molecular characteristics and chromosomal organization of 5S rDNA in the genus Oryza, including diploid and tetraploid species. A phylogenetic tree of Oryza species was constructed based on the non-transcribed spacer sequences of 5S rDNA, and some novel relationships were discovered. Specifically, comparative sequence analysis of 5S rDNA in several wild rice species showed unique characteristics inconsistent with the model of concerted evolution: (1) multiple distinct 5S rDNA types were detected within a species, leading to intraspecific divergence of 5S rDNA; (2) multiple identical 5S rDNA types were shared among species, resulting in interspecies clustering of 5S rDNA types; and (3) intraspecific nucleotide diversity was detected within a 5S rDNA class. Our results obtained by fluorescence in situ hybridization revealed that each rice species studied contained only one 5S rDNA locus with two hybridization sites, which were located on either chromosome 7 or chromosome 11. These results suggest that different 5S rDNA classes within the rice genome were arranged together and that one pair of 5S rDNA loci from a diploid progenitor of the tetraploid species might have been lost during evolution. Taken together, our data show that 5S rDNA in rice species is more informative at the gene level than at the chromosome level.     

Localization of the 5S rRNA genes and evidence for diversity in the 5S rDNA region of maize

The 5S rRNA genes of maize are located in the long arm of chromosome 2 (88% of the distance from the centromere to the end) and organized in a 320-bp repeat. Genomic blots of maize DNA digested with the restriction enzymes BamHI and MspI reveal ladders of bands in multiples of 320 bp. Analysis of 5S rDNA from genomic clones reveals that the ladders are due to both modification and divergence of the 5s rDNA nucleotide sequence.     

Molecular cytogenetic analysis of the Appenine endemic cyprinid fish Squalius lucumonis and three other Italian leuciscines using chromosome banding and FISH with rDNA probes

Karyotype and other chromosomal characteristics of the Appenine endemic cyprinid fish, Toscana stream chub Squalius lucumonis, were analysed using conventional banding and FISH with 45S and 5S rDNA probes. The diploid chromosome number (2n = 50) and karyotype characteristics including pericentromeric heterochromatic blocks and GC-rich CMA₃-positive sites corresponding to both positive Ag-NORs and 45S rDNA loci on the short arms of a single medium-sized submetacentric chromosome pair were consistent with those found in most European leuciscine cyprinids. On other hand, 5S rDNA FISH in the Toscana stream chub and three other Italian leuciscines, S. squalus, Rutilus rubilio and Telestes muticellus, revealed a species-specific hybridization pattern, i.e. signals on four (S. lucumonis), three (S. squalus and R. rubilio) and two (T. muticellus) chromosome pairs. Whereas all the species shared the 5S rDNA loci on the largest subtelocentric chromosome pair, a “leuciscine” cytotaxonomic marker, S. lucumonis showed both classes of rDNA loci tandem aligned on the short arms of chromosome pair No. 12. The present findings suggest that the observed high variability of 5S rDNA loci provides a powerful tool for investigation of karyotype differentiation in karyologically conservative leuciscine fishes.     

Ribosomal RNA Multigene Loci: Nomads of the Triticeae Genomes

The nucleolus organizing regions (NORs) on the short arms of chromosomes 1A(m) and 5A(m) of diploid wheat, Triticum monococcum L., are at the most distal loci in the linkage maps of these two chromosome arms. This distal location differs from the interstitial location of the Nor loci on chromosome arms 1BS of tetraploid Triticum turgidum L. and hexaploid T. aestivum L., 5DS of T. aestivum and diploid Ae. tauschii Coss., and 5HS of barley. Moreover, the barley 5HS locus is at a different location than the 5DS locus. However, other markers, including the centromeres, are colinear. These findings showed that the major Nor loci have repeatedly changed position in the chromosome arms during the radiation of species in the tribe Triticeae without rearrangements of the linkage groups. It is suggested that Nor loci may change position via dispersion of minor loci, that are shown here to exist in the T. monococcum genome, magnification of gene copy numbers in these minor loci, and subsequent deletion of the original major loci. Implications of these findings for the use of rRNA nucleotide sequences in phylogenetic reconstructions are pointed out.     

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.     

Microheterogeneity of the primary structure of the short repeated 5S DNA unit in diploid wheat Triticum monococcum L

Nucleotide sequences of two 5S rRNA genes located in repeated 327 bp long units were determined in diploid wheat Triticum monococcum. They were compared with sequences of 5S rRNA genes of Tr. monococcum and Tr. aestivum which were earlier determined. The differences were revealed in two localizations of the nucleotide sequence in 5S DNA coding regions of Tr. monococcum and - in nine localizations in nontranscribed spacer. It was established that the nucleotide sequence of 5S rRNA gene cloned in pTm5S9 plasmid and 5S DNA coding region in Tr. aestivum have significant homology. Diploid wheat Tr. monococcum was supposed to have 5S rRNA genes with different functional activity within one multigene family.     

Constitutive heterochromatin, 5S and 18S rDNA genes in Apareiodon sp. (Characiformes, Parodontidae) with a ZZ/ZW sex chromosome system

Karyotype, sex chromosome system and cytogenetics characteristics of an unidentified species of the genus Apareiodon originating from Piquiri River (Paraná State, Brazil) were investigated using differential staining techniques (C-banding and Ag-staining) and fluorescent in situ hybridization (FISH) with 5S and 18S rDNA probes. The diploid chromosome number was 2n = 54 with 25 pairs of meta- (m) to submetacentric (sm) and 2 pairs of subtelocentric (st) chromosomes. The major ribosomal rDNA sites as revealed by Ag-staining and FISH with 18S rDNA probe were found in distal region of longer arm of st chromosome pair 26, while minor 5S sites were observed in the interstitial sites on chromosome pairs 2 (smaller cluster) and 7 (larger one). The C-positive heterochromatin had pericentromeric and telomeric distribution. The heteromorphic sex chromosome system consisted of male ZZ (pair 21) and female middle-sized m/st Z/W chromosomes. The pericentric inversion of heterochromatinized short arm of ancestral Z followed by multiplication of heterochromatin segments is hypothesized for origin of W chromosome. The observed karyotype and chromosomal markers corresponded to those found in other species of the genus.     

Assessment of genome organization among diploid species (2n = 2x = 14) belonging to primary and tertiary gene pools of pearl millet using fluorescent in situ hybridization with rDNA probes.

Two contrasting forms of Pennisetum belonging to the primary and tertiary gene pools were assessed for genomic organization using in situ hybridization with rDNA probes (18S-5.8S-25S and 5S) on metaphase and interphase cell nuclei. The primary gene pool is represented by diploid (2n = 2x = 14) cultivated pearl millet (Pennisetum glaucum) and its close wild relatives (Pennisetum violaceum and Pennisetum mollissimum). Pennisetum schweinfurthii (2n = 2x = 14) was taken as representative of the tertiary gene pool, owing to its diploid status and its similarity to the accessions of the primary gene pool in chromosome number. Using the 18S-5.8S-25S probe, we observed two sites of distribution in the four species but at different locations. Within the primary gene pool, signals were detected on the telomeric part of the short arm of chromosome pair VI and at the nucleolar organizing region (NOR) of the satellited chromosome pair (VII). Signals were observed at the NOR of the two satellited chromosome pairs (I and IV) of P. schweinfurthii. The 5S probe was detected at the telomeric part of the short arm of metacentric chromosome pair IV of the three species of the primary gene pool, while it occured in an intercalary position on the short arm of chromosome pair II of P. schweinfurthii. These results showed a chromosomal similarity of rDNA sequence locations within the primary gene pool and are in agreement with the high genetic identity between wild and cultivated forms of pearl millet previously revealed by allozyme studies. Implications of genomic organization for genetic resource enhancement are discussed. Key words : Pennisetum, in situ hybridization, rDNA probes, genomic organization.     

Detection of 5S and 25S rRNA genes in Sinapis alba, Raphanus sativus and Brassica napus by double fluorescence in situ hybridization

Different ribosomal RNA (5S and 25S) genes were investigated simultaneously by fluorescence in situ hybridization (FISH) in Sinapis alba, Raphanus sativus and Brassica napus. The chromosomes of S. alba carried four 5S and six 25S gene sites, and those of R. sativus four sites of each gene, respectively. These two species have one chromosome pair with both rDNA genes; the two are closely located on a short arm of S. alba, while in R. sativus one is distal on the short arm (5S) and the other more proximal on the long arm (25S). In B. napus we have confirmed 12sites of 25S rDNA. The detection of 5S rDNA genes revealed 14 signals on 12 chromosomes. Of these, six chromosomes had signals for both rDNA genes. The FISH with 5S rDNA probes detected two sites closely adjacent in four chromosomes of B napus. These results are discussed in relation to a probable homoeologous chromosome pair in B. oleracea. Received: 20 July 1999 / Accepted: 8 October 1999     

A direct repeat sequence associated with the centromeric retrotransposons in wheat.

A high-density BAC filter of Triticum monococcum was screened for the presence of a centromeric retrotransposon using the integrase region as a probe. Southern hybridization to the BAC digests using total genomic DNA probes of Triticum monococcum, Triticum aestivum, and Hordeum vulgare detected differentially hybridizing restriction fragments between wheat and barley. The fragments that hybridized to genomic DNA of wheat but not to that of barley were subcloned. Fluorescence in situ hybridization (FISH) analysis indicated that the clone pHind258 hybridized strongly to centromeric regions in wheat and rye and weakly to those in barley. The sequence of pHind258 was homologous to integrase and long terminal repeats of centromeric Ty3-gypsy retrotransposons of cereal species. Additionally, pHind258 has a pair of 192-bp direct repeats. FISH analysis indicated that the 192-bp repeat probe hybridized to centromeres of wheat and rye but not to those of barley. We found differential FISH signal intensities among wheat chromosomes using the 192-bp probe. In general, the A-genome chromosomes possess strong FISH signals, the B-genome chromosomes possess moderate signals, and the D-genome chromosomes possess weak signals. This was consistent with the estimated copy numbers of the 192-bp repeats in the ancestral species of hexaploid wheat.     

Karyotyping of Brassica oleracea L. based on rDNA and Cot-1 DNA fluorescence in situ hybridization

To explore an effective and reliable karyotyping method in Brassica crop plants,Cot-1 DNA was isolated from Brassica oleracea genome,labeled as probe with Biotin-Nick Translation Mix kit,in situ hybridized to mitotic spreads,and where specific fluorescent bands showed on each chromosome pair.25S and 5S rDNA were labeled as probes with DIG-Nick Translation Mix kit and Biotin-Nick Translation Mix kit,respectively,in situ hybridized to mitotic preparations,where 25S rDNA could be detected on two chromosome pairs and 5S rDNA on only one.Cot-1 DNA contains rDNA and chromosome sites identity between Cot-1 DNA and 25S rDNA was determined by dual-colour fluorescence in situ hybridization.All these showed that the karyotyping technique based on a combination of rDNA and Cot-1 DNA chromosome landmarks is superior to all but one.A more exact karyotype ofB.oleracea has been analyzed based on a combination of rDNA sites,Cot-1 DNA fluorescent bands,chromosome lengths and arm ratios.