Physical mapping of plant DNA sequences by simultaneous in situ hybridization of two differently labelled fluorescent probes

Genome ◽  
1991 ◽  
Vol 34 (3) ◽  
pp. 329-333 ◽  
Author(s):  
I. J. Leitch ◽  
A. R. Leitch ◽  
J. S. Heslop-Harrison
Keyword(s):  
In Situ Hybridization ◽  
Physical Mapping ◽  
Dna Sequences ◽  
Secale Cereale ◽  
Repeat Sequence ◽  
Tandem Repeat Sequence ◽  
Plant Dna ◽  
Multiple Labelling ◽  
Ribosomal Dna Sequence

Two haptens, biotin and digoxigenin, were used to label two highly repetitive plant DNA sequences: pTa71 (a clone containing a ribosomal DNA sequence from wheat, Triticum aestivum) and pSc119.2 (a clone containing a 120-bp tandem repeat sequence from rye, Secale cereale). These probes were simultaneously localized by in situ hybridization on chromosome spreads of rye, Secale cereale cv. Petkus Spring. The ability to localize two sequences simultaneously will be of major importance for physically ordering DNA sequences along plant chromosomes.Key words: physical mapping, DNA–DNA in situ hybridization, Secale, fluorescent mapping, multiple labelling.

Analysis and sorting of rye (Secale cereale L.) chromosomes using flow cytometry

Genome ◽  
2003 ◽  
Vol 46 (5) ◽  
pp. 893-905 ◽  
Author(s):  
M Kubaláková ◽  
M Valárik ◽  
J Bartoš ◽  
J Vrána ◽  
J Cíhalíková ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Physical Mapping ◽  
Secale Cereale ◽  
B Chromosomes ◽  
Addition Lines ◽  
Large Numbers ◽  
Secale Cereale L

Procedures for chromosome analysis and sorting using flow cytometry (flow cytogenetics) were developed for rye (Secale cereale L.). Suspensions of intact chromosomes were prepared by mechanical homogenization of synchronized root tips after mild fixation with formaldehyde. Histograms of relative fluorescence intensity obtained after the analysis of DAPI-stained chromosomes (flow karyotypes) were characterized and the chromosome content of the DNA peaks was determined. Chromosome 1R could be discriminated on a flow karyotype of S. cereale 'Imperial'. The remaining rye chromosomes (2R–7R) could be discriminated and sorted from individual wheat–rye addition lines. The analysis of lines with reconstructed karyotypes demonstrated a possibility of sorting translocation chromosomes. Supernumerary B chromosomes could be sorted from an experimental rye population and from S. cereale 'Adams'. Flow-sorted chromosomes were identified by fluorescence in situ hybridization (FISH) with probes for various DNA repeats. Large numbers of chromosomes of a single type sorted onto microscopic slides facilitated detection of rarely occurring chromosome variants by FISH with specific probes. PCR with chromosome-specific primers confirmed the identity of sorted fractions and indicated suitability of sorted chromosomes for physical mapping. The possibility to sort large numbers of chromosomes opens a way for the construction of large-insert chromosome-specific DNA libraries in rye.Key words: chromosome isolation, chromosome sorting, fluorescence in situ hybridization, repetitive DNA sequences, wheat-rye addition lines, B chromosomes, physical mapping.

Nonisotopic in situ hybridization and plant genome mapping: the first 10 years

Genome ◽  
1994 ◽  
Vol 37 (5) ◽  
pp. 717-725 ◽  
Author(s):  
Jiming Jiang ◽  
Bikram S. Gill
Keyword(s):  
In Situ Hybridization ◽  
Physical Mapping ◽  
Dna Sequences ◽  
Genome Mapping ◽  
Molecular Cytogenetics ◽  
Gene Families ◽  
Single Copy ◽  
Plant Genome ◽  
Metaphase Chromosomes

Nonisotopic in situ hybridization (ISH) was introduced in plants in 1985. Since then the technique has been widely used in various areas of plant genome mapping. ISH has become a routine method for physical mapping of repetitive DNA sequences and multicopy gene families. ISH patterns on somatic metaphase chromosomes using tandemly repeated sequences provide excellent physical markers for chromosome identification. Detection of low or single copy sequences were also reported. Genomic in situ hybridization (GISH) was successfully used to analyze the chromosome structure and evolution of allopolyploid species. GISH also provides a powerful technique for monitoring chromatin introgession during interspecific hybridization. A sequential chromosome banding and ISH technique was developed. The sequential technique is very useful for more precise and efficient mapping as well as cytogenetic determination of genomic affinities of individual chromosomes in allopolyploid species. A critical review is made on the present resolution of the ISH technique and the future outlook of ISH research is discussed.Key words: in situ hybridization, physical mapping, genome mapping, molecular cytogenetics.

Genomic organization, sequence interrelationship, and physical localization using in situ hybridization of two tandemly repeated DNA sequences in the genus Olea

Genome ◽  
1998 ◽  
Vol 41 (4) ◽  
pp. 527-534 ◽  
Author(s):  
Andreas Katsiotis ◽  
Marianna Hagidimitriou ◽  
Alexandra Douka ◽  
Polydefkis Hatzopoulos
Keyword(s):  
In Situ Hybridization ◽  
Dna Sequences ◽  
Genomic Library ◽  
Repeat Sequence ◽  
Repeated Dna ◽  
Repeated Dna Sequences ◽  
Young Leaves ◽  
Partial Genomic Library ◽  
Tandemly Repeated Dna

Two tandemly repeated DNA sequences, the 81-bp family and pOS218, have been isolated from a Sau3AI Olea europaea ssp. sativa partial genomic library. Sequencing of the 81-bp element showed the monomer to be between 78 and 84 bases long and to contain 51-58% adenine and thymidine residues. Comparison between the monomers revealed heterogeneity of the sequence primary structure. The clone pOS218 is 218 bases long, and sequence comparison between the two elements revealed that an internal region of the pOS218 repeated DNA sequence had 79% homology to the 81 bp repeat sequence. A breakage-reunion mechanism, involving the CAAAA sequence, could be responsible for the derivation of pOS218 from the 81 bp family element. By using double target in situ hybridization, co-localization of the two sequences on Olea chromosomes was observed. The sequences were present at DAPI stained heterochromatic regions, as major or minor sites having a subtelomeric or interstitial location. Methylation studies using two sets of isoschizomers, Sau3AI-MboI and MspI-HpaII, demonstrated that most cytosine residues in the GATC sites and the internal cytosine in the CCGG sites of both elements were methylated in O. europaea ssp. sativa. No major difference in methylation was apparent between DNA extracted from young leaves or from callus of O. europaea ssp.sativa. Both elements are also present in Olea chrysophylla, Olea oleaster, and Olea africana, but are absent from other Oleaceae genera, including Phillyrea, Forsythia, Ligustrum, Parasyringa, and Jasminum.Key words: in situ hybridization, methylation, Oleaceae, phylogenetic relationships, repeated sequences.

Refined physical mapping of the Sec-1 locus on the satellite of chromosome 1R of rye (Secale cereale)

Genome ◽  
1995 ◽  
Vol 38 (5) ◽  
pp. 889-893 ◽  
Author(s):  
W. Busch ◽  
R. G. Herrmann ◽  
R. Martin
Keyword(s):  
In Situ Hybridization ◽  
Genetic Mapping ◽  
Fluorescence In Situ Hybridization ◽  
Physical Mapping ◽  
Secale Cereale ◽  
Secondary Constriction ◽  
Heterologous Hybridization ◽  
Secale Cereale L ◽  
Chromosome 1R

The Sec-1 locus (ω-secalin) of rye (Secale cereale L.) was mapped in the satellite of the short arm of chromosome 1R using fluorescence in situ hybridization and a genomic probe called pSec2B. Sec-1 is located in the middle of the satellite at the junction of the proximal euchromatic and the distal heterochromatic regions. Double hybridization experiments using rDNA and pSec2B showed that the NOR spans over the secondary constriction of the short arm of chromosome 1R and that there is a clearly visible gap between the NOR and Sec-1. Heterologous hybridization of pSec2B to barley visualized the B-hordein locus on chromosome 1H.Key words: fluorescence in situ hybridization, physical mapping, genetic mapping, secalin, rye, B-hordein, rDNA.

Genomic characterization and physical mapping of two fucosyltransferase genes in Medicago truncatula

Genome ◽  
2005 ◽  
Vol 48 (1) ◽  
pp. 168-176 ◽  
Author(s):  
Alexandra Castilho ◽  
Margarida Cunha ◽  
Ana Rita Afonso ◽  
Leonor Morais-Cecílio ◽  
Pedro S Fevereiro ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Medicago Truncatula ◽  
Physical Mapping ◽  
Dna Sequences ◽  
Genomic Organization ◽  
Rrna Genes ◽  
Fucosyltransferase Gene ◽  
Gene Coding ◽  
Genomic Characterization

Fucosyltransferases catalyse fucose transfer onto oligosaccharides. Two fucosylated structures have been identified in plants: the α1,4-fucosylated Lewis-a epitope and the α1,3-fucosylated core. Here we report the cloning, genomic characterization, and physical mapping of two genes encoding proteins similar to α1,4-fucosyltransferase (EC 2.4.1.65, MtFUT1) and α1,3-fucosyltransferase (EC 2.4.1.214, MtFUT2) in Medicago truncatula. Analysis of the genomic organization of the fucosyltransferase genes in M. truncatula, revealed the presence of two genomic variants of the MtFUT1 gene coding sequence, one containing a single intron and the other intronless, whereas in MtFUT2, the gene coding region is interrupted by four introns. Using for the first time fluorescence in situ hybridization (FISH) to physically map fucosyltransferase genes in plants, this study reveals a high genomic dispersion of these genes in Medicago. The MtFUT1 genes are mapped on chromosomes 4, 7, and 8, colocalizing on three of the five MtFUT2 loci. Chromosomes 1 and 5 carry the additional MtFUT2 loci. Moreover, the intensity of the FISH signals reveals marked differences in the number of gene copies per locus for both genes. Simultaneous mapping of rRNA genes on chromosome 5 shows that several MTFUT2 gene loci are inserted within the rDNA array. Insertions of coding DNA sequences into the rDNA repeats were never reported to date.Key words: core α1,3-fucosyltransferase gene, α1,4-fucosyltransferase gene, genomic organization, in situ hybridization, Medicago truncatula.

Fluorescent in situ hybridization and C-banding analyses of highly repetitive DNA sequences in the heterochromatin of rye (Secale montanum Guss.) and wheat incorporating S. montanum chromosome segments

Genome ◽  
1995 ◽  
Vol 38 (4) ◽  
pp. 795-802 ◽  
Author(s):  
Angeles Cuadrado ◽  
Nicolás Jouve
Keyword(s):  
In Situ Hybridization ◽  
Repetitive Dna ◽  
Dna Sequences ◽  
Secale Cereale ◽  
Fluorescent In Situ Hybridization ◽  
Fish Analysis ◽  
C Banding ◽  
Translocation Breakpoints ◽  
Secale Montanum

The molecular characterization of C-banded regions of Secale montanum Guss. by means of in situ hybridization was performed in order to provide new information about their chromosome structure relative to cultivated rye, Secale cereale L. Accurate identification of individual chromosomes was achieved using simultaneous and (or) successive fluorescent in situ hybridization (FISH) and C-banding. FISH identification was performed using total rye DNA, three highly repetitive rye DNA sequences (pSc119.2, pSc74, and pSc34), and the ribosomal RNA probes pTa71 (18S, 5.8S, and 26S rDNA) and pTa794 (5S rDNA). FISH was also used to identify the chromosome segment involved in two spontaneous translocation lines recovered from a 'Chinese Spring' – S. montanum wheat–rye addition line. FISH analysis revealed the exact translocation breakpoints and allowed the identification of the transferred rye segments. The value of this type of analysis is discussed.Key words: Secale cereale, Secale montanum, rye, repetitive DNA, fluorescence in situ hybridization.

DNA sequence mapping in interphase and metaphase chromosomes by fluorescence in situ hybridization

1992 ◽  
Vol 50 (1) ◽  
pp. 496-497
Author(s):  
Barbara Trask ◽  
Susan Allen ◽  
Anne Bergmann ◽  
Mari Christensen ◽  
Anne Fertitta ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Dual Band ◽  
Nick Translation ◽  
Metaphase Chromosomes ◽  
Band Pass ◽  
Texas Red ◽  
Fluorescent Spot

Using fluorescence in situ hybridization (FISH), the positions of DNA sequences can be discretely marked with a fluorescent spot. The efficiency of marking DNA sequences of the size cloned in cosmids is 90-95%, and the fluorescent spots produced after FISH are ≈0.3 μm in diameter. Sites of two sequences can be distinguished using two-color FISH. Different reporter molecules, such as biotin or digoxigenin, are incorporated into DNA sequence probes by nick translation. These reporter molecules are labeled after hybridization with different fluorochromes, e.g., FITC and Texas Red. The development of dual band pass filters (Chromatechnology) allows these fluorochromes to be photographed simultaneously without registration shift.

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