Seeds were germinated on moist filter paper in a Petri dish at room temperature for 2–3 days. Growing roots were cut from seedlings and treated in nitrous oxide with 15 bars of pressure for approximately 2 h. The roots were subsequently fixed in 90% acetic acid for 5 min and then stored in 70% v/v ethanol at -20°C. Chromosome spread preparation was performed as previously described (Kato et al., 2004 (link)).
Oligo-pSc119.2-1 combined with Oligo-pTa535-1 was used to distinguish the whole set of 42 wheat chromosomes (Tang et al., 2014 (link)). Oligo-pSc119.2-1 (10 ng/µl) and Oligo-pTa535-1 (10 ng/µl) were 5’ end-labeled with 6-carboxyfluorescein (6-FAM) and 6-carboxytetramethylrhodamine (6-Tamra) (InvitrogenTM, Shanghai, China), respectively. Genomic DNA was isolated from the leaves of Th. intermedium accession PI 440001, T. urartu accession TMU38, Ae. speltoides accession AE739, Ae. tauschii accession TQ27, and CS using the cetyltrimethylammonium bromide (CTAB) method (Murray and Thompson, 1980 (link)). The green or red probes with a concentration of 100 ng/µl were prepared according to the nick translation method (Kato et al., 2011 (link)). The genomic DNA of Th. intermedium, T. urartu and the plasmid of St2-80 reported by Wang et al. (2017) (link) were labeled with Alexa Fluor-488-5-2'-deoxyuridine 5'-triphosphate (dUTP) (InvitrogenTM, Shanghai, China). The genomic DNA of A. tauschii and the centromeric retrotransposon of wheat (CRW) clone 6C6 was labeled with Texas-red-5-dCTP (InvitrogenTM, Shanghai, China). The genomic DNA of CS and A. speltoides in a concentration of 3,000 ng/µl was used for blocking in multicolor-GISH (mc-GISH). For each slide, FISH was performed in 10 µl reaction volumes, in which 0.2 µl Oligo-pSc119.2-1, 0.2 µl Oligo-pTa535-1, and 0.3 µl 6C6, 0.5 µl St2-80 were used and the 2x SSC, 1x TE buffer was used to adjust the volume. For Th. Intermedium chromatin detection, 10 µl reaction volumes for each slide contain 0.5 µl labeled genomic DNA of PI 440001 and 2.5 µl genomic DNA of CS. For the mc-GISH on wheat, the 10 µl reaction volumes for each slide contain the 2 µl labeled genomic DNA of TMU38, 2 µl genomic DNA of AE739, and 1 µl labeled genomic DNA of TQ27. All chromosomes were counterstained with 4, 6-diamidino-2-phenylindole (DAPI) (Vectashield, Vector Laboratories, Burlingame, CA, USA). Chromosomes on microscope slides were examined using a BX61 fluorescence microscope (Olympus, Tokyo, Japan) equipped with a U-CMAD3 camera (Olympus, Tokyo, Japan) and appropriate filter sets. The signal capture and picture processing were performed using MetaMorph software (Molecular Devices, LLC., San Jose, CA, USA). The final image adjustment was done in Adobe Photoshop CS5 (Adobe Systems Incorporated, San Jose, CA, USA).
Oligo-pSc119.2-1 combined with Oligo-pTa535-1 was used to distinguish the whole set of 42 wheat chromosomes (Tang et al., 2014 (link)). Oligo-pSc119.2-1 (10 ng/µl) and Oligo-pTa535-1 (10 ng/µl) were 5’ end-labeled with 6-carboxyfluorescein (6-FAM) and 6-carboxytetramethylrhodamine (6-Tamra) (InvitrogenTM, Shanghai, China), respectively. Genomic DNA was isolated from the leaves of Th. intermedium accession PI 440001, T. urartu accession TMU38, Ae. speltoides accession AE739, Ae. tauschii accession TQ27, and CS using the cetyltrimethylammonium bromide (CTAB) method (Murray and Thompson, 1980 (link)). The green or red probes with a concentration of 100 ng/µl were prepared according to the nick translation method (Kato et al., 2011 (link)). The genomic DNA of Th. intermedium, T. urartu and the plasmid of St2-80 reported by Wang et al. (2017) (link) were labeled with Alexa Fluor-488-5-2'-deoxyuridine 5'-triphosphate (dUTP) (InvitrogenTM, Shanghai, China). The genomic DNA of A. tauschii and the centromeric retrotransposon of wheat (CRW) clone 6C6 was labeled with Texas-red-5-dCTP (InvitrogenTM, Shanghai, China). The genomic DNA of CS and A. speltoides in a concentration of 3,000 ng/µl was used for blocking in multicolor-GISH (mc-GISH). For each slide, FISH was performed in 10 µl reaction volumes, in which 0.2 µl Oligo-pSc119.2-1, 0.2 µl Oligo-pTa535-1, and 0.3 µl 6C6, 0.5 µl St2-80 were used and the 2x SSC, 1x TE buffer was used to adjust the volume. For Th. Intermedium chromatin detection, 10 µl reaction volumes for each slide contain 0.5 µl labeled genomic DNA of PI 440001 and 2.5 µl genomic DNA of CS. For the mc-GISH on wheat, the 10 µl reaction volumes for each slide contain the 2 µl labeled genomic DNA of TMU38, 2 µl genomic DNA of AE739, and 1 µl labeled genomic DNA of TQ27. All chromosomes were counterstained with 4, 6-diamidino-2-phenylindole (DAPI) (Vectashield, Vector Laboratories, Burlingame, CA, USA). Chromosomes on microscope slides were examined using a BX61 fluorescence microscope (Olympus, Tokyo, Japan) equipped with a U-CMAD3 camera (Olympus, Tokyo, Japan) and appropriate filter sets. The signal capture and picture processing were performed using MetaMorph software (Molecular Devices, LLC., San Jose, CA, USA). The final image adjustment was done in Adobe Photoshop CS5 (Adobe Systems Incorporated, San Jose, CA, USA).
Free full text: Click here
