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 of B. oleracea has been analyzed based on a combination of rDNA sites, Cot-1 DNA fluorescent bands, chromosome lengths and arm ratios. __________ Translated from Journal of Wuhan University (Nat. Sci. Ed.), 2006, 52(2): 230–234 [译自: 武汉大学学报 (理学版)]     

白菜rDNA及C0t-1DNA的荧光原位杂交及其核型分析

以早熟白菜苔为实验材料,从其基因组DNA中分离出C0t-1DNA并用生物素标记作探针,25SrDNA用地高辛标记作探针,对有丝分裂中期相染色体进行双色荧光原位杂交。每对染色体上均显示出了特定的C0t-1DNA荧光原位杂交带型,5对染色体上显示出了25SrDNA荧光原位杂交带型。双色荧光原位杂交证实了C0t-1DNA与25SrDNA二者具有一致的染色体位置特征,表明基于rDNA及C0t-1 DNA的荧光原位杂交核型分析技术,优于目前普遍采用的只基于rDNA的荧光原位杂交核型分析方法。结合C0t-1 DNA与25SrDNA的荧光原位杂交带型和传统的染色体的形态学标记分析方法及白菜已公布的基于rDNA分布的核型分析结果,创建了一个精确的白菜核型。     

Comparative cytogenetics of giant trahiras Hoplias aimara and H. intermedius (Characiformes, Erythrinidae): chromosomal characteristics of minor and major ribosomal DNA and cross-species repetitive centromeric sequences mapping differ among morphologically identical karyotypes

Karyotype and cytogenetic characteristics of 2 species of giant trahiras, Hopliasintermedius, S?o Francisco river basin, and Hopliasaimara, Arinos river (Amazon basin), were examined by conventional (C-banding, Ag-NOR, DAPI/CMA(3) double-staining) and fluorescent in situ hybridization (FISH) with 5S, 18S rDNA probes and cross-species Cot-1 DNA probing. Both species invariably had diploid chromosome number 2n = 50 and identical karyotypes composed of 10 pairs of metacentric and 15 pairs of submetacentric chromosomes. On the other hand, staining with base-specific fluorochromes (CMA(3), DAPI) and FISH mapping of repetitive DNA sequences showed extensive interspecific differences: while the genome of H. aimara had one submetacentric pair bearing CMA(3)-positive (DAPI-negative) sites, that of H. intermedius had 4 such pairs; while FISH with a 5S rDNA probe showed one (likely homologous) signal-bearing pair, that with 18S rDNA displayed one signal-bearing pair in H. intermedius and 2 such pairs in H. aimara. Cross-species FISH probing with Cot-1 DNA prepared from total DNA of both species showed no signals of Cot-1 DNA from H. aimara on chromosomes of H. intermedius but reciprocally (Cot-1 DNA from H. intermedius on chromosomes of H. aimara) displayed signals on at least 4 chromosome pairs. Present findings indicate (i) different composition of repetitive sequences around centromeres, (ii) different NOR phenotypes and (iii) distinct taxonomic status of both giant trahira species.     

FISH-mapping of rDNAs and Arabidopsis BACs on pachytene complements of selected Brassicas.

To improve resolution of physical mapping on Brassica chromosomes, we have chosen the pachytene stage of meiosis where incompletely condensed bivalents are much longer than their counterparts at mitotic metaphase. Mapping with 5S and 45S rDNA sequences demonstrated the advantage of pachytene chromosomes in efficient physical mapping and confirmed the presence of a novel 5S rDNA locus in Brassica oleracea, initially identified by genetic mapping using restriction fragment length polymorphism (RFLP). Fluorescence in situ hybridization (FISH) analysis visualized the presence of the third 5S rDNA locus on the long arm of chromosome C2 and confirmed the earlier reports of two 45S rDNA loci in the B. oleracea genome. FISH mapping of low-copy sequences from the Arabidopsis thaliana bacterial artificial chromosome (BAC) clones on the B. oleracea chromosomes confirmed the expectation of efficient and precise physical mapping of meiotic bivalents based on data available from A. thaliana and indicated conserved organization of these two BAC sequences on two B. oleracea chromosomes. Based on the heterologous in situ hybridization with BACs and their mapping applied to long pachytene bivalents, a new approach in comparative analysis of Brassica and A. thaliana genomes is discussed.     

自身基因组原位杂交揭示植物基因组重复DNA沿染色体的分布

重复DNA沿染色体的分布是认识植物基因组的组织和进化的要素之一。本研究采用一种改良的基因组原位杂交程序,对基因组大小和重复DNA数量不同的6种植物进行了自身基因组原位杂交(self-genomic in situ hybridization,self-GISH)。在所有供试物种的染色体都观察到荧光标记探针DNA的不均匀分布。杂交信号图型在物种间有明显的差异,并与基因组的大小相关。小基因组拟南芥的染色体几乎只有近着丝粒区和核仁组织区被标记。基因组相对较小的水稻、高粱、甘蓝的杂交信号分散分布在染色体的全长,但在近着丝粒区或近端区以及某些异染色质臂的分布明显占优势。大基因组的玉米和大麦的所有染色体都被密集地标记,并在染色体全长显示出强标记区与弱标记或不标记区的交替排列。此外,甘蓝染色体的所有近着丝粒区和核仁组织区、大麦染色体的所有近着丝粒区和某些臂中间区还显示了增强的信号带。大麦增强的信号带带型与其N-带带型一致。水稻自身基因组原位杂交图型与水稻Cot-1DNA在水稻染色体上的荧光原位杂交图型基本一致。研究结果表明,自身基因组原位杂交信号实际上反映了基因组重复DNA序列对染色体的杂交,因而自身基因组原位杂交技术是显示植物基因组中重复DNA聚集区在染色体上的分布以及与重复DNA相关联的染色质分化的有效方法。     

Karyotyping of Brassica napus L. Based on C0t-1 DNA Banding by Fluorescence In Situ Hybridization

In order to precisely recognize and karyotype Brassica napus L. chromosomes, C0t-1 DNA was extracted from its genomic DNA, labeled with biotin-1 1-dUTP and in situ hybridized. The hybridized locations were detected by Cy3-conjugated streptavidin. Specific fluorescence in situ hybridization （FISH） signal bands were detected on all individual chromosome pairs. Each chromosome pair showed specific banding patterns. The B. napus karyotype has been constructed, for the first time, on the basis of both Cot-1 DNA FISH banding patterns and chromosome morphology.     

Cloning of DNA sequences localized on proximal fluorescent chromosome bands by microdissection in Pinus densiflora Sieb. & Zucc.

Japanese red pine, Pinus densiflora, has 2n=24 chromosomes, of which most carry chromomycin A3 (CMA) and 4',6-diamidino-2-phenylindole (DAPI) bands at their centromere-proximal regions. It was proposed that these regions contain highly repetitive DNA. The DNA localized in the proximal fluorescent bands was isolated and characterized. In P. densiflora, centromeric and neighboring segments of the somatic chromosomes were dissected with a manual micromanipulator. The centromeric DNA was amplified from the DNA contained in dissected centromeric segments by degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR) and a cloned DNA library was constructed. Thirty-one clones carrying highly repetitive DNA were selected by colony hybridization using Cot-1 DNA from this species as a probe, and their chromosomal localization was determined by fluorescent in situ hybridization (FISH). Clone PDCD501 was localized to the proximal CMA band of 20 chromosomes. This clone contained tandem repeats, comprising a 27 bp repeat unit, which was sufficient to provide the proximal FISH signal, with a 52.3% GC content. The repetitive sequence was named PCSR (proximal CMA band-specific repeat). Clone PDCD159 was 1700 bp in length, with a 61.7% AT content, and produced FISH signals at the proximal DAPI band of the remaining four chromosomes. Four clones hybridized strongly to the secondary constriction and gave weak signals at the centromeric region of several chromosomes. Clone PDCD537, one of the four clones, was homologous to the 26S rRNA gene. A PCR experiment using microdissected centromeric regions suggested that the centromeric region contains 18S and 26S rDNA. Another 24 clones hybridized to whole chromosome arms, with varying intensities and might represent dispersed repetitive DNA.     

Karyotyping of Brassica amphidiploids using 5S and 25S rDNA as chromosome markers

Species of Brassica have small, morphologically similar chromosomes, which makes karyotyping difficult using conventional cytogenetic methods. Molecular cytogenetic methods, like fluorescence in situ hybridisation (FISH) have the potential to improve karyotyping through the use of chromosome- or genome-specific markers. Simultaneous application of more than one DNA probe can greatly enrich the results obtained compared with separate single target FISH experiments. This paper demonstrates the use of multicolour fluorescence in situ hybridisation with 5S and 25S rDNA for karyotyping three amphidiploid species: B. napus, B. juncea and B. carinata. Using this method, it was possible to identify eight out of nineteen pairs of chromosomes in B. napus, ten out of eighteen pairs in B. juncea and six out of sixteen pairs in B. carinata. Additionally, rDNA sites allow the determination of the genomic origin of all marked chromosomes in B. napus and B. juncea.     

Chromosomal localization and characterization of rDNA loci in the Brassica A and C genomes.

Using fluorescence in situ hybridization, we located ribosomal DNA loci on prometaphase chromosomes of the diploid species Brassica rapa and Brassica oleracea and their amphidiploid Brassica napus. Based on comparisons of chromosome morphology and hybridization patterns, we characterized the individual B. napus rDNA loci according to their presumed origins in the Brassica A and C genomes. As reported in other studies, the sum of rDNA loci observed on B. rapa (AA genome) and B. oleracea (CC genome) chromosomes was one greater than the total number of loci seen in their amphidiploid B. napus (AACC). Evidence is presented that this reduction in B. napus rDNA locus number results from the loss of the smallest A genome rDNA site in the amphidiploid.     

A and C genome distinction and chromosome identification in brassica napus by sequential fluorescence in situ hybridization and genomic in situ hybridization

The two genomes (A and C) of the allopolyploid Brassica napus have been clearly distinguished using genomic in situ hybridization (GISH) despite the fact that the two extant diploids, B. rapa (A, n = 10) and B. oleracea (C, n = 9), representing the progenitor genomes, are closely related. Using DNA from B. oleracea as the probe, with B. rapa DNA and the intergenic spacer of the B. oleracea 45S rDNA as the block, hybridization occurred on 9 of the 19 chromosome pairs along the majority of their length. The pattern of hybridization confirms that the two genomes have remained distinct in B. napus line DH12075, with no significant genome homogenization and no large-scale translocations between the genomes. Fluorescence in situ hybridization (FISH)-with 45S rDNA and a BAC that hybridizes to the pericentromeric heterochromatin of several chromosomes-followed by GISH allowed identification of six chromosomes and also three chromosome groups. Our procedure was used on the B. napus cultivar Westar, which has an interstitial reciprocal translocation. Two translocated segments were detected in pollen mother cells at the pachytene stage of meiosis. Using B. oleracea chromosome-specific BACs as FISH probes followed by GISH, the chromosomes involved were confirmed to be A7 and C6.     

Linkage of the evolutionarily-related serum albumin and alpha-fetoprotein genes within q11-22 of human chromosome 4

Albumin and alpha-fetoprotein are structurally related serum proteins, having a similar gene structure and, conceivably, a common evolutionary origin. To test their relative arrangement in the human genome, the serum albumin and alpha-fetoprotein genes were mapped by in situ hybridization of cloned human albumin or alpha-fetoprotein cDNA to human mitotic chromosome preparations. Analysis of cells hybridized with the serum albumin probe showed that 39% of cells exhibited grains on the proximal portion of the long arm of chromosome 4 (bands q11-22), with these grains comprising 30% of all labeled sites throughout these mitoses. Similarly, in cells hybridized with the alpha-fetoprotein probe, 39% of cells were observed to contain silver grains on 4q11-22, these grains constituting 20% of all labeled sites in these cells. These results demonstrate chromosomal localization and linkage of the serum albumin and alpha-fetoprotein genes within bands q11-22 of the long arm of human chromosome 4.     

FISH mapping of 18S-28S and 5S ribosomal DNA, (GATA)n and (TTAGGG)n telomeric repeats in the periwinkle Melarhaphe neritoides (Prosobranchia, Gastropoda, Caenogastropoda)

Spermatocyte chromosomes of Melarhaphe neritoides (Mollusca, Prosobranchia, Caenogastropoda) were studied using fluorescent in situ hybridization (FISH) with four repetitive DNA probes (18S rDNA, 5S rDNA, (TTAGGG)n and (GATA)n). Single-colour FISH consistently mapped one chromosome pair per spread using either 18S or 5S rDNA as probes. The telomeric sequence (TTAGGG)n hybridized with termini of all chromosomes whereas the (GATA)n probe did not label any areas. Simultaneous 18S-5S rDNA and 18S-(TTAGGG)n FISH demonstrated that repeated units of the three multicopy families are closely associated on the same chromosome pair.     

Heterochromatin patterns and ribosomal DNA loci distribution in diploid and polyploid Crotalaria species (Leguminosae, Papilionoideae), and inferences on karyotype evolution

Most Crotalaria species display a symmetric karyotype with 2n = 16, but 2n = 14 is found in Chrysocalycinae subsection Incanae and 2n = 32 in American species of the section Calycinae. Seven species of the sections Chrysocalycinae, Calycinae, and Crotalaria were analyzed for the identification of heterochromatin types with GC- and AT-specific fluorochromes and chromosomal location of ribosomal DNA loci using fluorescent in situ hybridization (FISH). A major 45S rDNA locus was observed on chromosome 1 in all the species, and a variable number of minor ones were revealed. Only one 5S rDNA locus was observed in the species investigated. Chromomycin A(3) (CMA) revealed CMA(+) bands colocalized with most rDNA loci, small bands unrelated to ribosomal DNA on two chromosome pairs in Crotalaria incana, and CMA(+) centromeric bands that were quenched by distamycin A were detected in species of Calycinae and Crotalaria sections. DAPI(+) bands were detected in C. incana. The results support the species relationships based on flower specialization and were useful for providing insight into mechanisms of karyotype evolution. The heterochromatin types revealed by fluorochromes suggest the occurrence of rearrangements in repetitive DNA families in these heterochromatic blocks during species diversification. This DNA sequence turnover and the variability in number/position of rDNA sites could be interpreted as resulting from unequal crossing over and (or) transposition events. The occurrence of only one 5S rDNA locus and the smaller chromosome size in the polyploids suggest that DNA sequence losses took place following polyploidization events.     

Removal of repetitive sequences from FISH probes using PCR-assisted affinity chromatography

The vast majority of probes used in fluorescence in situ hybridization (FISH) contain repetitive DNA. This DNA is usually competed out of a hybridization reaction by the addition of an unlabeled blocking agent, Cot-1 DNA. We have successfully removed repetitive DNA from two complex FISH probe sets: a degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) single human chromosome library and genomic DNA. The procedure involved hybridizing in solution a DOP-PCR-amplifiable probe set with a 50-fold excess of biotin-labeled Cot-1 DNA, and capturing the Cot-1 DNA-containing hybrids using streptavidin magnetic particles, followed by purification and reamplification of the unbound fraction. Probes were checked for depletion of repeats by hybridization to chromosomes without Cot-1 DNA. Results showed hybridization patterns comparable to those achieved with untreated probes hybridized with Cot-1 DNA. Received: 21 January 1997 / Accepted: 2 April 1997     

Multicolor fluorescence in situ hybridization with combinatorial labeling probes enables a detailed karyotype analysis of Larix principis-rupprechtii

The chromosomes (2n = 2x = 24) of Larix principis-rupprechtii are composed of six pairs of large metacentrics and six pairs of medium-sized submetacentrics. The identification of homologous pairs is hampered by their high degree of similarity at the morphological level in each group. As one of the most extensively used methods in molecular cytogenetics producing chromosome landmarks, fluorescence in situ hybridization (FISH) has significantly facilitated karyotype construction, especially in species with morphologically similar chromosomes. This study developed a simple but effective use of combinatorial labeling probes to distinguish chromosomes of Larix principis-rupprechtii by multicolor FISH. Three highly repetitive sequences in Larix were selected: 25S rDNA hybridized at all of the secondary constrictions of two pairs of metacentrics and the largest pair of submetacentrics; 5S rDNA hybridized at subtelomeric sites of one pair of metacentrics that also harboured 25S rDNA on different arms; LPD family sequences are tandem repeats hybridized at proximal regions of 22 chromosomes. The three different probes were labeled with only two different labels, hybridized to metaphase chromosomes of Larix principis-rupprechtii, simultaneously visualized, and unequivocally distinguished in a single FISH experiment. These multicolor FISH marks largely improved the karyotype analysis of Larix principis-rupprechtii.     

Comparative analysis of rDNA distribution in chromosomes of various species of Brassicaceae

BACKGROUND AND AIMS: The Brassicaceae family encompasses numerous species of great agronomic importance, belonging to such genera, as Brassica, Raphanus, Sinapis and Armoracia. Many of them are characterized by extensive intraspecific diversity of phenotypes. The present study focuses on the polymorphism of number, appearance and chromosomal localization of ribosomal DNA (rDNA) sites and, when possible, in relation to polyploidy, in 42 accessions of Brassica species and ten accessions of Diplotaxis, Eruca, Raphanus and Sinapis species. METHODS: Chromosomal localization of ribosomal DNA was carried out using dual colour fluorescence in situ hybridization (FISH) with 5S rDNA and 25S rDNA sequences as probes on enzymatically digested root-tip meristematic cells. KEY RESULTS: Loci for 5S and 18S-5.8S-25S rDNA were determined for the first time in six taxa, and previously unreported rDNA constellations were described in an additional 12 accessions. FISH revealed frequent polymorphism in number, appearance and chromosomal localization of both 5S and 25S rDNA sites. This phenomenon was most commonly observed in the A genome of Brassica, where it involves exclusively pericentromeric sites of 5S and 25S rRNA genes. The intraspecific polymorphism was between subspecies/varieties or within a variety or cultivar (i.e. interindividual). CONCLUSIONS: The number of rDNA sites can differ up to 5-fold in species with the same chromosome number. In addition to the eight previously reported chromosomal types with ribosomal genes, three new variant types are described. The extent of polymorphism is genome dependent. Comparing the A, B and C genomes revealed the highest rDNA polymorphism in the A genome. The loci carrying presumably inactive ribosomal RNA genes are particularly prone to polymorphism. It can also be concluded that there is no obvious polyploidization-related tendency to reduce the number of ribosomal DNA loci in the allotetraploid species, when compared with their putative diploid progenitors. The observed differences are rather caused by the prevailing polymorphism within the diploids and allotetraploids. This would make it difficult to predict expected numbers of rDNA loci in natural polyploids.     

Flow cytometric quantification of human chromosome specific repetitive DNA sequences by single and bicolor fluorescent in situ hybridization to lymphocyte interphase nuclei

Fluorescent in situ hybridization allows for rapid and precise detection of specific nucleic acid sequences in interphase and metaphase cells. We applied fluorescent in situ hybridization to human lymphocyte interphase nuclei in suspension to determine differences in amounts of chromosome specific target sequences amongst individuals by dual beam flow cytometry. Biotinylated chromosome 1 and Y specific repetitive satellite DNA probes were used to measure chromosome 1 and Y polymorphism amongst eight healthy volunteers. The Y probe fluorescence was found to vary considerably in male volunteers (mean fluorescence 169, S.D. 35.6). It was also detectable in female volunteers (mean fluorescence 81, S.D. 10.7), because 5-10% of this repetitive sequence is located on autosomes. The Y probe fluorescence in males was correlated with the position of the Y chromosome cluster in bivariate flow karyotypes. When chromosome 1 polymorphism was studied, one person out of the group of eight appeared to be highly polymorphic, with a probe fluorescence 26% below the average. By means of fluorescent in situ hybridization on a glass slide and bivariate flow karyotyping, this 26% difference was found to be caused by a reduction of the centromere associated satellite DNA on one of the homologues of chromosome 1. The simultaneous hybridization to human lymphocyte interphase nuclei of biotinylated chromosome 1 specific repetitive DNA plus AAF-modified chromosome Y specific DNA was detected by triple beam flow cytometry. The bicolor double hybridized nuclei could be easily distinguished from the controls. When the sensitivity of this bicolor hybridization is improved, this approach could be useful for automatic detection of numerical chromosome aberrations, using one of the two probes as an internal control.     

Nucleolar dominance does not occur in root tip cells of allotetraploid Brassica species.

Using in situ hybridization and silver staining methods, the numbers of active and inactive rDNA loci have been established for three allotetraploid species of Brassica (B. napus, B. carinata, and B. juncea) and their diploid ancestors (B. campestris, B. nigra, and B. oleracea). The allotetraploid species have chromosome numbers equal to the sum of the numbers in their diploid relatives, but have fewer rDNA loci. All species investigated have lower numbers of active NORs (AgNORs, nucleolar organizer regions) compared with the numbers of rDNA sites revealed by in situ hybridization. The number of active rDNA loci of the allotetraploid species is equal to the number of AgNORs in their diploid ancestors, indicating the absence of nucleolar dominance in amphidiploid Brassica species, at least in root meristematic cells.     

Chromosome markers in the tetraploid wheat Aegilops ventricosa analysed by in situ hybridization

 Three lines of the tetraploid wheat Aegilops ventricosa Tausch (2n=4x=28), which contains good resistance to eyespot, were analysed using fluorescent in situ hybridization. Probes used included rDNA, cloned repeated sequences from wheat and rye, simple-sequence repeats (SSRs) and total genomic DNA. The banding patterns produced could be used to distinguish most chromosome arms and will aid in the identification of Ae. ventricosa chromosomes or chromosome segments in breeding programmes. All lines had a single major 18S-25S rDNA site, the nucleolar organizing region (NOR) in chromosome 5N and several minor sites of 18S-25S rDNA and 5S rDNA. A 1NL.3DL, 1NS.3DS translocation was identified, and other minor differences were found between the lines. Received: 11 August 1998 / Accepted: 28 November 1998     

Characterization of rDNAs and tandem repeats in the heterochromatin of Brassica rapa

We describe the morphology and molecular organization of heterochromatin domains in the interphase nuclei, and mitotic and meiotic chromosomes, of Brassica rapa, using DAPI staining and fluorescence in situ hybridization (FISH) of rDNA and pericentromere tandem repeats. We have developed a simple method to distinguish the centromeric regions of mitotic metaphase chromosomes by prolonged irradiation with UV light at the DAPI excitation wavelength. Application of this bleached DAPI band (BDB) karyotyping method to the 45S and 5S rDNAs and 176 bp centromere satellite repeats distinguished the 10 B. rapa chromosomes. We further characterized the centromeric repeat sequences in BAC end sequences. These fell into two classes, CentBr1 and CentBr2, occupying the centromeres of eight and two chromosomes, respectively. The centromere satellites encompassed about 30% of the total chromosomes, particularly in the core centromere blocks of all the chromosomes. Interestingly, centromere length was inversely correlated with chromosome length. The morphology and molecular organization of heterochromatin domains in interphase nuclei, and in mitotic and meiotic chromosomes, were further characterized by DAPI staining and FISH of rDNA and CentBr. The DAPI fluorescence of interphase nuclei revealed ten to twenty conspicuous chromocenters, each composed of the heterochromatin of up to four chromosomes and/or nucleolar organizing regions.