Multi-Genome In Situ Hybridization of Wheat Progenitors

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).

Free full text: Click here

Othmeni M., Grewal S., Walker J., Yang C.Y., King I.P, & King J. (2022). Assessing the Potential of Using the Langdon 5D(5B) Substitution Line for the Introgression of Aegilops tauschii Into Durum Wheat. Frontiers in Plant Science, 13, 927728.

Publication 2022

DEAC-dUTP

Alexa 594 Chromosome Ctab Donor Dutp Genome In situ hybridization Metaphase Microscope Wheat

Corresponding Organization : University of Nottingham

Top 5 similar protocols

Variable analysis

independent variables

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

dependent variables

Metaphase chromosome spreads

control variables

Preparation of metaphase chromosome spreads as described in Kato et al. (2004) and King et al. (2017)
DNA extraction from young leaves using the CTAB method (Zhang et al., 2013)
DNA labeling using the nick translation procedure (Luchniak et al., 2002)
Probe mix ratio of 1:2:2 for T. urartu, Ae. speltoides, and Ae. tauschii
Counterstaining with DAPI
Microscopy and image acquisition using the Zeiss Axio Imager.Z2 upright epifluorescence microscope and ISIS software

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!