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

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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Use of fluorescence in situ hybridization for gross mapping of transgenes and screening for homozygous plants in transgenic barley (Hordeum vulgare L.)

Theoretical and Applied Genetics ◽

10.1007/s00122-002-0997-y ◽

2002 ◽

Vol 106 (1) ◽

pp. 92-100 ◽

Cited By ~ 15

Author(s):

H. Choi ◽

P. Lemaux ◽

M.-J. Cho

Keyword(s):

In Situ Hybridization ◽

Hordeum Vulgare ◽

Fluorescence In Situ Hybridization ◽

Hordeum Vulgare L ◽

Transgenic Barley

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Identification of all chromosome arms and their involvement in meiotic homoeologous associations at metaphase I in 2 Hordeum vulgare L. × Hordeum bulbosum L. hybrids

Genome ◽

10.1139/g05-071 ◽

2006 ◽

Vol 49 (1) ◽

pp. 73-78 ◽

Cited By ~ 12

Author(s):

R Pickering ◽

S Klatte ◽

R C Butler

Keyword(s):

In Situ Hybridization ◽

Hordeum Vulgare ◽

Fluorescence In Situ Hybridization ◽

Interspecific Hybrids ◽

Association Studies ◽

Hordeum Bulbosum ◽

Hordeum Vulgare L ◽

Chromosome Addition ◽

Chromosome Associations

We have identified all Hordeum bulbosum chromosomes in 2 diploid Hordeum vulgare × Hordeum bulbosum hybrids using suitable probes and fluorescence in situ hybridization. Using the parental idiograms allowed us to carry out a full analysis of chromosome associations among all chromosome arms in the hybrids. Association frequencies were generally lower for the short arms than for the long arms. There were also significant differences among the chromosome arms in association frequencies, partly correlated with the absolute length of the chromosome arm, as well as with the frequency of recombinant lines, which were recovered from partially fertile interspecific hybrids. The H. bulbosum idiogram will be useful for further chromosome association studies and will enable the identification of H. bulbosum chromosomes involved in chromosome addition or substitution lines.Key words: Hordeum vulgare, Hordeum bulbosum, interspecific hybrids, chromosome associations, meiosis, fluorescence in situ hybridization.

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Physical mapping of four sites of 5S rDNA sequences and one site of the α-amylase-2 gene in barley (Hordeum vulgare)

Genome ◽

10.1139/g93-071 ◽

1993 ◽

Vol 36 (3) ◽

pp. 517-523 ◽

Cited By ~ 101

Author(s):

I. J. Leitch ◽

J. S. Heslop-Harrison

Keyword(s):

In Situ Hybridization ◽

Hordeum Vulgare ◽

Physical Mapping ◽

5S Rdna ◽

Chromosome 1 ◽

Chromosome 2 ◽

Hordeum Vulgare L ◽

Rdna Sequences ◽

5S Rdna Sequence

The 5S rDNA sequences have been mapped on four pairs of barley (Hordeum vulgare L.) chromosomes using in situ hybridization and barley monotelotrisomic lines. The 5S rDNA sequences are located, genetically and physically, on the short arm of chromosome 1 (7I) and the long arms of chromosomes 2 (2I) and 3 (3I). The 5S rDNA sequence is also located on the physically long arm of chromosome 4 (4I). Only one site on chromosome 2(2I) has previously been reported. The characteristic locations of the 5S rDNA sequences make them useful as molecular markers to identify each barley chromosome. The physical position of the low-copy α-amylase-2 gene was determined using in situ hybridization; the location of this gene on the long arm of chromosome 1 (7I) was confirmed by reprobing the same preparation with the 5S rDNA probe. The results show that there is a discrepancy between the physical and genetic position of the α-amylase-2 gene.Key words: genetic mapping, physical mapping, barley, mapping, 5S DNA, α-amylase, in situ hybridization.

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High-density fluorescence in situ hybridization signal detection on barley (Hordeum vulgare L.) chromosomes with improved probe screening and reprobing procedures

Genome ◽

10.1139/g10-098 ◽

2011 ◽

Vol 54 (2) ◽

pp. 151-159 ◽

Cited By ~ 14

Author(s):

Akio Kato

Keyword(s):

In Situ Hybridization ◽

Hordeum Vulgare ◽

Fluorescence In Situ Hybridization ◽

Gel Electrophoresis ◽

Agarose Gel ◽

Agarose Gel Electrophoresis ◽

Hordeum Vulgare L ◽

Pcr Products ◽

Break Points

The barley ( Hordeum vulgare L.) genome was screened to identify sequences that could be used for fluorescence in situ hybridization (FISH). From 2000 transformed bacterium colonies carrying barley clones, 56 colonies were selected on the basis of the patterns that their PCR products produced when subjected to agarose gel electrophoresis. Among them, 42 (75%) exhibited fluorescent signals on barley chromosomes after in situ hybridization using the directly labeled PCR products. Sequencing revealed seven clones, pHv-365, pHv-177, pHv-1112, pHv-689, pHv-1476, pHv-1889, and pHv-1972, to be newly identified FISH-positive sequences. The remainder possess previously described sequences such as 5S, GAA microsatellite, centromere repeats, HVT01, and pHvMWG2315 (324 bp repeat). It is shown here that a combination of five probes, which produce strong signals on barley chromosomes, pHv-38 (5S), pHv-365, pHv-961 (HVT01), GAA, and TAG microsatellites, offer unequivocal recognition of each chromosome. The combination of three probes, i.e., pHv-1123 (barley 324 bp repeat), GAA, and TAG, decorated entire chromosomes with fine dotted signals and was useful for detecting the break points of aberrant chromosomes. The signals’ distributions of pHv-177, pHv-1112, and TAG were highly polymorphic. An improved reprobing procedure and its usefulness are also discussed.

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Repeated DNA sequences isolated by microdissection. I. Karyotyping of barley (Hordeum vulgare L.)

Genome ◽

10.1139/g95-144 ◽

1995 ◽

Vol 38 (6) ◽

pp. 1082-1090 ◽

Cited By ~ 24

Author(s):

Winfried Busch ◽

Regina Martin ◽

Reinhold G. Herrmann ◽

Uwe Hohmann

Keyword(s):

In Situ Hybridization ◽

Hordeum Vulgare ◽

Fluorescence In Situ Hybridization ◽

Dna Sequences ◽

Fish Analysis ◽

Hordeum Vulgare L ◽

Agropyron Elongatum ◽

D Genome ◽

In Situ Experiments

We report on microdissection, cloning and sequence, and Southern and fluorescence in situ hybridization (FISH) analysis of one moderately and one highly amplified repetitive DNA element, pHvMWG2314 and pHvMWG2315, respectively, isolated from barley (Hordeum vulgare L.) chromosome arm 3HL. The pHvMWG2315 sequence hybridizes to all 14 telomeric or subtelomeric regions of the barley chromosomes as determined by FISH. The 50 different hybridization sites that include intercalary signals allow the discrimination of all 14 chromosome arms and the construction of a karyotype of barley. The tandemly repeated subtelomeric element of 331 bp exists in all Triticeae species tested (H. vulgare, Agropyron elongatum, Secale cereale, Triticum tauschii, T. turgidum, and T. aestivum). It is AT rich (66%), exhibits 84% sequence homology to subfragments of the D genome "specific" 1-kb element pAsl of T. tauschii and 75% homology to the interspersed genome-specific DNA sequence pHcKB6 from H. chilense. The repetitive sequence pHvMWG2314 is moderately amplified in barley and highly amplified in hexaploid wheat. The in situ experiments revealed no distinct signals on barley chromosomes, indicating a dispersed character for the sequence. The significance of the results for the identification of chromosomes and chromosome aberrations in FISH experiments are discussed.Key words: karyotype, fluorescence in situ hybridization, FISH, DNA sequencing.

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