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Mapping nonrecombining regions in barley using multicolor FISH

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Abstract

Fluorescence in situ hybridization (FISH) is a widely used method to localize DNA sequences on chromosomes. Out of the many uses, FISH facilitates construction of physical maps by ordering contigs of large-insert DNA clones, typically bacterial artificial chromosome (BAC) and establishing their orientation. This is important in genomic regions with low recombination frequency where genetic maps suffer from poor resolution. While BAC clones can be mapped directly by FISH in plants with small genomes, excess of repetitive DNA hampers this application in species with large genomes. Mapping single-copy sequences such as complementary DNA (cDNA) is an attractive alternative. Unfortunately, localization of single-copy sequences shorter than 10 kb remains a challenging task in plants. Here, we present a highly efficient FISH technique that enables unambiguous localization of single copy genes. We demonstrated its utility by mapping 13 out of 15 full-length cDNAs of variable length (2,127–3,400 bp), which were genetically defined to centromeric and pericentromeric regions of barley chromosome 7H. We showed that a region of 1.2 cM (0.7 %) on genetic map represented more than 40 % of the physical length of the chromosome. Surprisingly, all cDNA probes occasionally revealed hybridization signals on other chromosomes, indicating the presence of partially homologous sequences. We confirmed the order of 10 cDNA clones and suggested a different position for three cDNAs as compared to published genetic order. These results underline the need for alternative approaches such as FISH, which can resolve the order of markers in genomic regions where genetic mapping fails.

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Abbreviations

BAC-FISH:

Bacterial artificial chromosome-fluorescence in situ hybridization

Cy3, Cy5:

cyanine dyes

dUTP:

Deoxyuridine triphosphate

DEAC:

Diethylaminocoumarin

fl-cDNA:

Full-length complementary DNA

FISH:

Fluorescence in situ hybridization

FITC:

Fluorescein isothiocyanate

HU:

Hydroxyurea

rDNA:

Ribosomal DNA

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Acknowledgements

We appreciate technical advice from our colleague Marie Kubaláková. This work was supported jointly by the Czech Academy of Sciences and the German Academic Exchange Service–DAAD (grant award no. CZ07-DE12/2013-2014) by Internal Grant Agency of Palacky University in Olomouc (grant award no. IGA PrF/2012/001) and by the Ministry of Education, Youth and Sports of the Czech Republic and the European Regional Development Fund (Operational Programme Research and Development for Innovations No. ED0007/01/01).

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Authors and Affiliations

  1. Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany, Šlechtitelů 31, 783 71, Olomouc-Holice, Czech Republic

    M. Karafiátová, J. Bartoš, D. Kopecký & J. Doležel

  2. Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), 06466, Seeland, OT Gatersleben, Germany

    L. Ma, A. Houben & N. Stein

  3. Institute of Plant Science and Resources, Okayama University, Kurashiki, 710-0046, Japan

    K. Sato

  4. Institute of Experimental Botany, Šlechtitelů 31, 783 71, Olomouc-Holice, Czech Republic

    J. Doležel

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  1. M. Karafiátová
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  2. J. Bartoš
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  3. D. Kopecký
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  4. L. Ma
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  5. K. Sato
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  6. A. Houben
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  7. N. Stein
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  8. J. Doležel
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Correspondence to J. Doležel.

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Responsible Editor: Jiming Jiang

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Additional hybridization signals observed after FISH with probes for cDNA on complete metaphase spreads (DOC 327 kb)

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Karafiátová, M., Bartoš, J., Kopecký, D. et al. Mapping nonrecombining regions in barley using multicolor FISH. Chromosome Res 21, 739–751 (2013). https://doi.org/10.1007/s10577-013-9380-x

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