In situ DNA sequence mapping with surface-spread mouse pachytene chromosomes

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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High resolution mapping of in situ hybridized biotinylated DNA to surface-spread Drosophila polytene chromosomes

Chromosoma ◽

10.1007/bf00293158 ◽

1985 ◽

Vol 93 (2) ◽

pp. 113-122 ◽

Cited By ~ 25

Author(s):

Horst Kress ◽

Elliot M. Meyerowitz ◽

Norman Davidson

Keyword(s):

High Resolution ◽

Polytene Chromosomes ◽

High Resolution Mapping ◽

Resolution Mapping ◽

Surface Spread

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The use of double fluorescence in situ hybridization to physically map the positions of 5S rDNA genes in relation to the chromosomal location of 18S–5.8S–26S rDNA and a C genome specific DNA sequence in the genus Avena

Genome ◽

10.1139/g96-068 ◽

1996 ◽

Vol 39 (3) ◽

pp. 535-542 ◽

Cited By ~ 63

Author(s):

Concha Linares ◽

Juan González ◽

Esther Ferrer ◽

Araceli Fominaya

Keyword(s):

In Situ Hybridization ◽

Dna Sequence ◽

Diploid Species ◽

5S Rdna ◽

Rdna Genes ◽

26S Rdna ◽

A Genome ◽

Rdna Loci ◽

C Genome

A physical map of the locations of the 5S rDNA genes and their relative positions with respect to 18S–5.8S–26S rDNA genes and a C genome specific repetitive DNA sequence was produced for the chromosomes of diploid, tetraploid, and hexaploid oat species using in situ hybridization. The A genome diploid species showed two pairs of rDNA loci and two pairs of 5S loci located on both arms of one pair of satellited chromosomes. The C genome diploid species showed two major pairs and one minor pair of rDNA loci. One pair of subtelocentric chromosomes carried rDNA and 5S loci physically separated on the long arm. The tetraploid species (AACC genomes) arising from these diploid ancestors showed two pairs of rDNA loci and three pairs of 5S loci. Two pairs of rDNA loci and 2 pairs of 5S loci were arranged as in the A genome diploid species. The third pair of 5S loci was located on one pair of A–C translocated chromosomes using simultaneous in situ hybridization with 5S rDNA genes and a C genome specific repetitive DNA sequence. The hexaploid species (AACCDD genomes) showed three pairs of rDNA loci and six pairs of 5S loci. One pair of 5S loci was located on each of two pairs of C–A/D translocated chromosomes. Comparative studies of the physical arrangement of rDNA and 5S loci in polyploid oats and the putative A and C genome progenitor species suggests that A genome diploid species could be the donor of both A and D genomes of polyploid oats. Key words : oats, 5S rDNA genes, 18S–5.8S–26S rDNA genes, C genome specific repetitive DNA sequence, in situ hybridization, genome evolution.

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A unified DNA sequence and non‐ DNA sequence mapping model of complex traits

The Plant Journal ◽

10.1111/tpj.14354 ◽

2019 ◽

Vol 99 (4) ◽

pp. 784-795

Author(s):

Yanru Zeng ◽

Xuli Zhu ◽

Chixiang Chen ◽

Kalins Banerjee ◽

Lidan Sun ◽

...

Keyword(s):

Dna Sequence ◽

Complex Traits ◽

Sequence Mapping ◽

Mapping Model

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Organization and distribution of a Sau3A tandem repeated DNA sequence in Picea (Pinaceae) species

Genome ◽

10.1139/g98-054 ◽

1998 ◽

Vol 41 (4) ◽

pp. 560-565 ◽

Cited By ~ 12

Author(s):

Garth R Brown ◽

Craig H Newton ◽

John E Carlson

Keyword(s):

In Situ Hybridization ◽

Dna Sequence ◽

Picea Glauca ◽

Tandem Repeats ◽

Picea Sitchensis ◽

Repeated Dna ◽

Metaphase Chromosomes ◽

Genes Encoding ◽

Repeated Dna Sequence

Repeated DNA families contribute to the large genomes of coniferous trees but are poorly characterized. We report the analysis of a 142 bp tandem repeated DNA sequence identified by the restriction enzyme Sau3A and found in approximately 20 000 copies in Picea glauca. Southern hybridization indicated that the repeated DNA family is specific to the genus, was amplified early in its evolution, and has undergone little structural alteration over evolutionary time. Fluorescence in situ hybridization localized arrays of the Sau3A repeating element to the centromeric regions of different subsets of the metaphase chromosomes of P. glauca and the closely related Picea sitchensis, suggesting that mechanisms leading to the intragenomic movement of arrays may be more active than those leading to mutation of the repeating elements themselves. Unambiguous identification of P. glauca and P. sitchensis chromosomes was made possible by co-localizing the Sau3A tandem repeats and the genes encoding the 5S and 18S-5.8S-26S ribosomal RNAs.Key words: Picea, repeated DNA, in situ hybridization, centromere.

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In situ localization of two repetitive DNA sequences to surface-spread pachytene chromosomes of rye

Genome ◽

10.1139/g92-082 ◽

1992 ◽

Vol 35 (4) ◽

pp. 551-559 ◽

Cited By ~ 25

Author(s):

S. M. Albini ◽

T. Schwarzacher

Keyword(s):

In Situ Hybridization ◽

Synaptonemal Complex ◽

Dna Sequences ◽

Meiotic Prophase ◽

Hybridization Signal ◽

Metaphase Chromosomes ◽

Somatic Metaphase ◽

Rdna Signal ◽

Surface Spread

Surface-spread pollen mother cells at meiotic prophase from Secale cereale (rye) were used for fluorescent DNA:DNA in situ localization of two tandemly repeated DNA sequences: pTa71, a wheat rDNA clone, and pSc119.2, a cloned 120-bp repeat from rye heterochromatin. The fluorescent hybridization signal, consisting of many yellow-green dots, was closely associated with the bivalent axes, corresponding to the synaptonemal complex, and located in the surrounding chromatin. The rDNA signal was associated with one bivalent, the smallest of the seven, at a distance about 13% of the bivalent length from the telomere. This corresponded to the position of the nucleolar organizing region of silver-stained synaptonemal complexes analyzed under the electron microscope and published data for somatic metaphase chromosomes. The relative length of the axis covered with the rDNA signal is less than expected from somatic metaphases, but it corresponds more closely to the proportion of the sequences in the genome. The hybridization signal with the 120-bp repeat was located mainly at the telomeric regions of several bivalents that showed thickenings of the axis after DAPI staining, probably corresponding to somatic C-bands. These major and some minor intercalary sites agree with the distribution of the 120-bp repeat in somatic metaphase. Fluorescent in situ hybridization to plant surface-spread pachytene chromosomes, which can be obtained in large numbers, has great potential for studying meiotic prophase, high-resolution mapping of DNA sequences, and investigating the relationship of DNA sequences to the synaptonemal complex.Key words: in situ hybridization, cereals, pachytene, meiosis, synaptonemal complex, physical mapping.

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

Genome ◽

10.1139/g11-034 ◽

2011 ◽

Vol 54 (9) ◽

pp. 718-726 ◽

Cited By ~ 9

Author(s):

Mateus Mondin ◽

Margarida L.R. Aguiar-Perecin

Keyword(s):

Dna Sequence ◽

Ribosomal Dna ◽

Karyotype Evolution ◽

5S Rdna ◽

Rdna Locus ◽

Variable Number ◽

Chromosome 1 ◽

Distamycin A ◽

Rdna Sites

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 A3 (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.

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Current status and the future of fluorescence in situ hybridization (FISH) in plant genome research

Genome ◽

10.1139/g06-076 ◽

2006 ◽

Vol 49 (9) ◽

pp. 1057-1068 ◽

Cited By ~ 224

Author(s):

Jiming Jiang ◽

Bikram S. Gill

Keyword(s):

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Dna Sequence ◽

Dna Sequences ◽

Molecular Cytogenetics ◽

Plant Genome ◽

Current Status ◽

Sequence Information ◽

Resolution Mapping

Fluorescence in situ hybridization (FISH), which allows direct mapping of DNA sequences on chromosomes, has become the most important technique in plant molecular cytogenetics research. Repetitive DNA sequence can generate unique FISH patterns on individual chromosomes for karyotyping and phylogenetic analysis. FISH on meiotic pachytene chromosomes coupled with digital imaging systems has become an efficient method to develop physical maps in plant species. FISH on extended DNA fibers provides a high-resolution mapping approach to analyze large DNA molecules and to characterize large genomic loci. FISH-based physical mapping provides a valuable complementary approach in genome sequencing and map-based cloning research. We expect that FISH will continue to play an important role in relating DNA sequence information to chromosome biology. FISH coupled with immunoassays will be increasingly used to study features of chromatin at the cytological level that control expression and regulation of genes.

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Chromosome preparations from protoplasts: in situ hybridization banding pattern of a dispersed DNA sequence in rye (Secale cereale L.)

Genome ◽

10.1139/g91-080 ◽

1991 ◽

Vol 34 (4) ◽

pp. 524-527 ◽

Cited By ~ 2

Author(s):

N. Jouve ◽

C. L. McIntyre ◽

J. P. Gustafson

Keyword(s):

In Situ Hybridization ◽

Dna Sequence ◽

Dna Sequences ◽

Banding Pattern ◽

Banding Technique ◽

Differential Distribution ◽

Cellular Debris ◽

Biotin Labeling ◽

Secale Cereale L

The utilization of genome-specific DNA sequences coupled with in situ hybridization for chromosome karyotyping in wheat, rye, and triticale has been of limited value because of the presence of cellular and cytoplasmic debris. The use of protoplasts, thus eliminating cellular debris, has been shown to improve the level of detection of low-copy and unique DNA sequences in cereals. Therefore, the use of protoplasts could represent an appropriate tool to improve the results of karyotyping cereal chromosomes with genome-specific DNA sequences. This paper describes the results on the comparative application of protoplasts and squash preparations in the analysis of physical mapping of a dispersed DNA sequence (pSc119.1) to rye chromosomes by in situ hybridization. Individual chromosomes of rye were not distinguishable by their hybridization patterns to pSc119.1 when squash preparations were used. These showed an undefined distribution of the DNA probe that covered apparently the entire length of each rye chromosome. However, considerable improvement was observed for the differential distribution of the pSc119.1 DNA sequence in protoplast preparations. The karyotypic banding pattern of pSc119.1 showed a better banding pattern than can be observed using the C-banding technique. Therefore, the use of protoplasts hybridized with dispersed DNA markers could be of more value in monitoring chromosome karyotypes than existing cytological techniques.Key words: biotin labeling, dispersed sequences, rye.

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