Physical mapping of wheat-Aegilops longissima breakpoints in mildew-resistant recombinant lines using FISH with highly repeated and low-copy DNA probes

Genome ◽  
1999 ◽  
Vol 42 (5) ◽  
pp. 1013-1019 ◽  
Author(s):  
Marco Biagetti ◽  
Francesca Vitellozzi ◽  
Carla Ceoloni
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Common Wheat ◽  
Dna Sequences ◽  
Dna Probes ◽  
Genetic Maps ◽  
Chromosome Engineering ◽  
Aegilops Longissima ◽  
Copy Dna

Fluorescence in situ hybridization (FISH) with multiple probes, consisting of highly repeated DNA sequences (pSc119.2 and pAs1) and of a low-copy, 3BS-specific RFLP sequence (PSR907), enabled determination of the physical position of the wheat-alien breakpoints (BPs) along the 3BS and 3DS arms of common wheat recombinant lines. These lines harbour 3SlS Aegilops longissima segments containing the powdery mildew resistance gene Pm13. In all 3B recombinants, the wheat-Aegilops longissima physical BPs lie within the interval separating the two most distal of the three pSc119.2 3BS sites. In all such recombinants a telomeric segment, containing the most distal of the pSc119.2 3BS sites, was in fact replaced by a homoeologous Ae. longissima segment, marked by characteristic pSc119.2 hybridization sites. Employment of the PSR907 RFLP probe as a FISH marker allowed to resolve further the critical region in the various 3B recombinant lines. Three of them, like the control common wheat, exhibited between the two most distal pSc119.2 sites a single PSR907 FISH site, which was missing in a fourth recombinant line. The amount of alien chromatin can thus be estimated to represent around 20% of the recombinant arm in the three former lines and a maximum of 27% in the latter. A similar physical length was calculated for the alien segment contained in three 3D recombinants, all characterized by the presence of the Ae. longissima pSc119.2 sites distal to the nearly telomeric pAs1 sites of normal 3DS. Comparison between the FISH-based maps and previously developed RFLP maps of the 3BS-3SlS and 3DS-3SlS arms revealed substantial differences between physical and genetic map positions of the wheat-alien BPs and of molecular markers associated with the critical chromosomal portions.Key words: wheat-alien recombinants, chromosome engineering, fluorescence in situ hybridization, highly repeated and low-copy DNA probes, physical versus genetic maps.

scholarly journals Applications of fluorescence in situ hybridization (FISH) in genome research

2020 ◽  
Vol 17 (3) ◽  
pp. 393-410
Author(s):  
Hoang Thi Nhu Phuong ◽  
Huynh Thi Thu Hue ◽  
Cao Xuan Hieu
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Dna Sequences ◽  
Dna Probes ◽  
Dna Repeats ◽  
Genome Research ◽  
Multicolor Fish ◽  
Vector Systems ◽  
Assembly Sequences

Fluorescence in situ hybridization (FISH) technique enables the direct detection of DNA sequences inintact cellular materials (e.g. individual chromosomes in metaphase spreads). This review article focuses on theapplications of FISH in genome research, including validation and correction of the genome assembly from thenext-generation sequencing (NGS) projects. DNA probes for specific DNA fragments of the assembly can beobtained from PCR amplicon or cloned products using different vector systems. Localization of these probeson their respective chromosomal regions can be visualized by FISH, providing useful information to crosscheckthe assembly data. Furthermore, the recent refinements in the FISH technology including using smartpooling scheme of differently colored DNA probes, together with consecutive FISH experiments (stripping andreprobing of the same slide) are described. These advances in multicolor FISH can provide crucial linkageinformation on association of linkage groups and assembly scaffolds, resulting in so-called cytogenetic maps.Integration of the cytogenetic maps and assembly sequences assists to resolve the chromosome-level genomeassembly and to reveal new insights in genome architecture and genome evolution. Especially, comparativechromosome painting with pooled DNA probes from one reference species can be used to investigate ancestralrelationships (chromosome homeology and rearrangements) among other not-yet-sequenced species. Inaddition, FISH using DNA probes for certain specific classes of repetitive DNA elements as well as for basicchromosome structures (e.g. centromere or telomere DNA repeats, ribosomal DNA loci) can be used to studythe genome organization and karyotype differentiation. We also discussed about limitations and futureperspectives of the FISH technology.

Use of fluorescence in situ hybridization to study the arrangement of DNA sequences in normal and disease-related chromosomes

1995 ◽  
Vol 53 ◽  
pp. 748-749
Author(s):  
Barbara J. F. Trask ◽  
Hillary Massa ◽  
Cynthia Friedman ◽  
Richard Esposito ◽  
Ger van den Engh ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Dna Sequences ◽  
Single Copy ◽  
Two Dimensions ◽  
Genetic Maps ◽  
Epifluorescence Microscopy ◽  
Metaphase Chromosomes ◽  
Interphase Nuclei

The sites of specific DNA sequences can be fluorescently tagged by fluorescence in situ hybridization (FISH). Different sequences can be labeled with different fluorochromes so that their arrangement can be studied using epifluorescence microscopy. The distances between points on the same or different chromosomes can be determined easily in a large number of interphase nuclei or metaphase chromosomes. A variety of probe types, ranging from single-copy sequences to highly repeated sequences can be employed. Our work has focussed on the analysis of hybridization patterns in two dimensions using conventional fluorescence microscopy.We have used FISH to study various aspects of genome organization that are difficult to study using other techniques. Examples of these applications will be presented.FISH is now the method of choice for determining the chromosomal location of DNA sequences. DNA sequences can be positioned in the genome with <1:1000 accuracy (to a 3-Mbp region within a 3000-Mbp genome). Through FISH, the cytogenetic, physical and genetic maps of chromosomes can be linked.

C-banding and fluorescence in situ hybridization studies of the wheat–alien hybrid 'Agrotana'

Genome ◽  
1994 ◽  
Vol 37 (3) ◽  
pp. 477-481 ◽  
Author(s):  
Jie Xu ◽  
R. L. Conner ◽  
A. Laroche
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Genomic Dna ◽  
Chromosome Engineering ◽  
C Banding ◽  
Chinese Spring Wheat ◽  
Mother Cells ◽  
Alien Chromatin ◽  
Chromosome Segments

'Agrotana', a wheat-alien hybrid (2n = 56), is a potential source of resistance to common root rot, stem rust, wheat streak mosaic virus, and the wheat curl mite. However, the origin of 'Agrotana', reported to be durum wheat × Agropyron trichophorum (pubescent wheatgrass), is uncertain. The objective of this investigation was to determine the chromosome constitution of 'Agrotana' using C-banding and fluorescence in situ hybridization techniques. The F1 hybrid of 'Agrotana' × 'Chinese Spring' wheat showed 7 I + 21 II in 14.9% of the pollen mother cells, evidence of the presence of the A, B, and D genomes in 'Agrotana'. The hybrid had 16 heavily C-banded chromosomes, namely 4A, and 1-7B of wheat, and a translocation that probably involved wheat chromosomes 2A and 2D. In situ hybridization using biotinylated genomic DNA of Ag. trichophorum cv. Greenleaf blocked with CS DNA failed to identify the alien chromosomes in 'Agrotana', indicating that the alien chromosomes were not likely derived from pubescent wheatgrass. In situ hybridization using labelled wheat genomic DNA blocked with 'Agrotana' DNA revealed that 'Agrotana' had 40 wheat, 14 alien, and 2 (a pair) wheat–alien translocated chromosomes. There was no homology between wheat and the alien chromosomes or chromosome segments involved in the wheat–alien recombinant. Two of the seven pairs of alien chromosomes were homoeologous to each other. The ability to identify alien chromatin in wheat using labelled wheat DNA instead of labelled alien DNA will be particularly useful in chromosome engineering of wheat germplasms having alien chromatin of unknown origin.Key words: wheat–alien hybrid, C-banding, fluorescence in situ hybridization, labelled wheat DNA as probe.

Fluorescence in situ hybridization on plant extended chromatin DNA fibers for single-copy and repetitive DNA sequences

2011 ◽  
Vol 30 (9) ◽  
pp. 1779-1786 ◽  
Author(s):  
Kun Yang ◽  
Hecui Zhang ◽  
Richard Converse ◽  
Yong Wang ◽  
Xiaoying Rong ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Repetitive Dna ◽  
Dna Sequences ◽  
Single Copy ◽  
Repetitive Dna Sequences

scholarly journals Extending the Useful Life Span of DNA Probes for Fluorescence In Situ Hybridization

BioTechniques ◽  
1999 ◽  
Vol 26 (6) ◽  
pp. 1068-1072
Author(s):  
Allen T. Christian ◽  
Holly E. Garcia ◽  
James D. Tucker
Keyword(s):  
In Situ Hybridization ◽  
Life Span ◽  
Fluorescence In Situ Hybridization ◽  
Dna Probes ◽  
Useful Life

Identification of the entire chromosome complement of bread wheat by two-colour FISH

Genome ◽  
1997 ◽  
Vol 40 (5) ◽  
pp. 589-593 ◽  
Author(s):  
C. Pedersen ◽  
P. Langridge
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Dna Sequences ◽  
Banding Pattern ◽  
Aegilops Tauschii ◽  
Chromosome Complement ◽  
Chromosome Identification ◽  
Satellite Sequence ◽  
Entire Chromosome

Using the Aegilops tauschii clone pAs1 together with the barley clone pHvG38 for two-colour fluorescence in situ hybridization (FISH) the entire chromosome complement of hexaploid wheat was identified. The combination of the two probes allowed easy discrimination of the three genomes of wheat. The banding pattern obtained with the pHvG38 probe containing the GAA-satellite sequence was identical to the N-banding pattern of wheat. A detailed idiogram was constructed, including 73 GAA bands and 48 pAs1 bands. Identification of the wheat chromosomes by FISH will be particularly useful in connection with the physical mapping of other DNA sequences to chromosomes, or for chromosome identification in general, as an alternative to C-banding.Key words: Triticum aestivum, chromosome identification, fluorescence in situ hybridization, repetitive DNA sequences.

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