Preparation of the root metaphase chromosome spreads, the protocol for the mcGISH and the image capture was as described in King et al. (2017 ). All slides were probed with labelled genomic DNA of the three putative diploid progenitors of bread wheat, that is T. urartu (A genome), Ae. speltoides (B genome) and Ae. tauschii (D genome). Additionally, introgression lines with segments from Th. bessarabicum and Am. muticum were probed with the respective wild relative's labelled genomic DNA. The genomic DNA of (i) T. urartu was labelled by nick translation with ChromaTide™ Alexa Fluor™ 488‐5‐dUTP (Invitrogen; C11397; coloured green), (ii) Ae. speltoides was labelled by nick translation with DEAC‐dUTP (Jena Bioscience; NU‐803‐DEAC; coloured blueish purple), (iii) Ae. tauschii was labelled with ChromaTide™ Alexa Fluor™ 594‐5‐dUTP (Invitrogen; C11400; coloured red), and (iv) Th. bessarabicum and Am. muticum were labelled by nick translation with ChromaTide™ Alexa Fluor™ 546‐14‐dUTP (Invitrogen; C11401; coloured yellow).
Deac dutp
Manufactured by Jena Biosciences
DEAC‐dUTP is a fluorescently labeled nucleotide analog that can be incorporated into DNA during synthesis. It is designed for labeling and detection applications in molecular biology and biochemistry.
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2 protocols using deac dutp
1
Multicolor Genomic In Situ Hybridization
2
Multi-Genome In Situ Hybridization of Wheat Progenitors
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Slides of metaphase chromosome spreads were prepared as described in Kato et al. (2004 (link)) and King et al. (2017 (link)). Multi-color genomic in situ hybridization (Mc-GISH) was conducted using the labeled total genomic DNA of the three potential wheat progenitor species: the A-genome donor T. urartu, the B-genome donor Ae. speltoides, and the D-genome donor Ae. tauschii. DNA was extracted from young leaves using the CTAB method (Zhang et al., 2013 (link)) and labeled using the nick translation procedure (Luchniak et al., 2002 (link)): T. urartu was labeled with ChromaTideTM Alexa FluorTM 488-5-dUTP (Invitrogen; C11397; green), Ae. tauschii with ChromaTideTM Alexa FluorTM 594-5-dUTP (Invitrogen; C11400; red), and Ae. speltoides with DEAC-dUTP (Jena Bioscience; NU-803-DEAC; blue). Slides were hybridized with a probe mix containing 1.5 μL Ae. urartu, 3 μL Ae. speltoides, and 3 μL Ae. tauschii (a ratio of 1:2:2). Slides were counterstained with 4'-6-diamidino-2-phenylindole (DAPI) before being analyzed with the fully automated Zeiss Axio Imager.Z2 upright epifluorescence microscope (Carl Zeiss Ltd., Oberkochen, Germany). Images were taken using a MetaSystems Coolcube 1 m CCD camera and processed with ISIS (image processing) software (Metasystems GmbH, Altlussheim, Germany).
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