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

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 (2R7R) could be discriminated and sorted from individual wheatrye 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.

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

Genome ◽

10.1139/g95-117 ◽

1995 ◽

Vol 38 (5) ◽

pp. 889-893 ◽

Cited By ~ 13

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.

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Highly repetitive sequences in B chromosomes of Secale cereale revealed by fluorescence in situ hybridization

Genome ◽

10.1139/g94-100 ◽

1994 ◽

Vol 37 (4) ◽

pp. 709-712 ◽

Cited By ~ 25

Author(s):

Angeles Cuadrado ◽

Nicolas Jouve

Keyword(s):

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Secale Cereale ◽

Fluorescent In Situ Hybridization ◽

Repetitive Sequences ◽

5S Rdna ◽

B Chromosomes ◽

Multigene Families ◽

Hybridization Probes

An analysis of the presence and distribution of the rye and wheat repeated sequences in rye B chromosomes was carried out by fluorescent in situ hybridization. Probes used consisted of three highly repetitive sequences from rye (pSc119.2, pSc74, and pSc34) and the multigene families for the 25S–5.8S–18S and 5S rDNA from wheat (pTa71 and pTa794, respectively). pSc74 and pSc119.2 showed hybridization signals in the telomeric regions of rye B chromosomes. The remaining DNA clones did not hybridize to the B chromosomes.Key words: Secale cereale, rye, repetitive DNA, fluorescence in situ hybridization, B chromosomes.

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Telomere length analysis by fluorescence in situ hybridization and flow cytometry (Flow-FISH)/Telomerlängen-Messung mittels Fluoreszenz-in-situ-Hybridisierung und Durchflusszytometrie (Flow-FISH)

LaboratoriumsMedizin ◽

10.1515/labmed.2004.047 ◽

2004 ◽

Vol 28 (4) ◽

pp. 307-316

Author(s):

Ulrike Hartmann ◽

Fabian Beier ◽

Tim H. Brümmendorf

Keyword(s):

Flow Cytometry ◽

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Telomere Length ◽

Length Analysis

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Correlation of assessment of plasma cells by flow cytometry and detection of cytogenomic abnormalities by fluorescence in situ hybridization in plasma cell neoplasms

International Journal of Laboratory Hematology ◽

10.1111/j.1751-553x.2011.01323.x ◽

2011 ◽

pp. no-no

Author(s):

G. LU ◽

X. X. ZHANG ◽

M. J. YOU ◽

W. CHEN

Keyword(s):

Flow Cytometry ◽

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Plasma Cell ◽

Plasma Cells ◽

Plasma Cell Neoplasms

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Physical mapping of barley genes using an ultrasensitive fluorescence in situ hybridization technique

Genome ◽

10.1139/g03-084 ◽

2004 ◽

Vol 47 (1) ◽

pp. 179-189 ◽

Cited By ~ 29

Author(s):

J L Stephens ◽

S E Brown ◽

N L.V Lapitan ◽

D L Knudson

Keyword(s):

In Situ Hybridization ◽

Hordeum Vulgare ◽

Fluorescence In Situ Hybridization ◽

Linkage Map ◽

Physical Mapping ◽

Physical Map ◽

Primary Objective ◽

Hybridization Technique ◽

Hordeum Vulgare L

The primary objective of this study was to elucidate gene organization and to integrate the genetic linkage map for barley (Hordeum vulgare L.) with a physical map using ultrasensitive fluorescence in situ hybridization (FISH) techniques for detecting signals from restriction fragment length polymorphism (RFLP) clones. In the process, a single landmark plasmid, p18S5Shor, was constructed that identified and oriented all seven of the chromosome pairs. Plasmid p18S5Shor was used in all hybridizations. Fourteen cDNA probes selected from the linkage map for barley H. vulgare 'Steptoe' × H. vulgare 'Morex' (Kleinhofs et al. 1993) were mapped using an indirect tyramide signal amplification technique and assigned to a physical location on one or more chromosomes. The haploid barley genome is large and a complete physical map of the genome is not yet available; however, it was possible to integrate the linkage map and the physical locations of these cDNAs. An estimate of the ratio of base pairs to centimorgans was an average of 1.5 Mb/cM in the distal portions of the chromosome arms and 89 Mb/cM near the centromere. Furthermore, while it appears that the current linkage maps are well covered with markers along the length of each arm, the physical map showed that there are large areas of the genome that have yet to be mapped.Key words: Hordeum vulgare, barley, physical mapping, FISH, cDNA, genetics, linkage, chromosome, BACs.

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