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Precise karyotyping of carrot mitotic chromosomes using multicolour-FISH with repetitive DNA

Abstract

Carrot (Daucus carota L.) chromosomes are small and uniform in shape and length. Here, mitotic chromosomes were subjected to multicolour fluorescence in situ hybridization (mFISH) with probes derived from conserved plant repetitive DNA (18-25S and 5S rDNA, telomeres), a carrot-specific centromeric repeat (Cent-Dc), carrot-specific repetitive elements (DCREs), and miniature inverted-repeat transposable elements (MITEs). A set of major chromosomal landmarks comprising rDNA and telomeric and centromeric sequences in combination with chromosomal measurements enabled discrimination of carrot chromosomes. In addition, reproducible and unique FISH patterns generated by three carrot genome-specific repeats (DCRE22, DCRE16, and DCRE9) and two transposon families (DcSto and Krak) in combination with telomeric and centromeric reference probes allowed identification of chromosome pairs and construction of detailed carrot karyotypes. Hybridization patterns for DCREs were observed as pericentromeric and interstitial dotted tracks (DCRE22), signals in pericentromeric regions (DCRE16), or scattered signals (DCRE9) along chromosomes similar to those observed for both MITE families.

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Abbreviations

BAC:

bacterial artificial chromosome

DAPI:

4′,6-diamidino-2-phenylindole

DCRE:

Daucus carota repetitive element

FISH:

fluorescence in situ hybridization

FITC:

fluorescein isothiocyanate

mFISH:

multicolour fluorescence in situ hybridization

MITE:

miniature inverted-repeat transposable element

NOR:

nucleolar organizer region

SSC:

saline-sodium citrate

TE:

transposable element

TIR:

terminal inverted repeat

TRS:

telomeric repeat sequence

TSD:

target site duplications

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Correspondence to D. Grzebelus.

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Acknowledgments: This research was financed by the Ministry of Science and Higher Education of the Republic of Poland.

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Nowicka, A., Grzebelus, E. & Grzebelus, D. Precise karyotyping of carrot mitotic chromosomes using multicolour-FISH with repetitive DNA. Biol Plant 60, 25–36 (2016). https://doi.org/10.1007/s10535-015-0558-2

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Additional key words

  • Daucus carota repetitive elements
  • fluorescence in situ hybridization
  • miniature inverted-repeat transposable elements