Cytogenetic analyses for the perennial wheat entries included chromosome counting of the root tip cells, meiotic behaviors of chromosomes, and characterization of chromosome constitutions by genomic in situ hybridization (GISH) and Oligonucleotide in situ hybridization. Seeds were germinated at 23°C for 24 h, 4°C for 48 h, and 23°C for 27.5 h. Root tips were incubated in ice water at 0–4°C for 24 h and fixed in Carnoy’s solution (ethanol: glacial acetic acid = 3:1) for 24 h. They were stained in 1% aceto-carmine for at least 5 h prior to squashing in 45% acetic acid. Chromosome numbers were counted under a light microscope (Leica DM LS2, Mannheim, Germany). Inflorescences were sampled at 8:00 to 9:00 am or 15:00 to 16:00 pm, fixed in Carnoy’s solution for 5–10 h, stored in 75% ethanol, and anthers at appropriate stages were stained in 1% aceto-carmine. Chromosome behaviors were observed under a light microscope.
DNA extraction from fresh leaves was performed using the CTAB method (Doyle and Doyle, 1987 (link)). The St-genome DNA as probe can be used for GISH analysis of Th. intermedium chromosome (Chen et al., 1998 (link); Chen, 2005 (link)). The probe was prepared by labeling 1 μg (2 μl) Ps. strigose and common wheat “Chinese Spring” genomic DNA in 4 μl of DIG-Nick-Translation mix (Roche, Mannheim, Germany) and 14 μL of ddH2O at 15°C for 90 min. The reaction was terminated by 1 μL 0.5 mol EDTA (pH 8.0) at 65°C for 10 min. DNA of Chinese Spring wheat and Th. intermedium were sheared to be used as a blocker. Anti-digoxin Rhodamine and DAPI (Roche, Mannheim, Germany) were added prior to incubation in the dark. A Leica DM6000B fluorescence microscope (Leica, Mannheim, Germany) was used for observing the hybridization signals, and images were captured with a Leica digital camera (Model DFC480).
An oligonucleotide (oligo hereafter) multiplex containing oligos pAs1-1, pAs1-3, AFA-4 (GAA) 10, and pSc119.2-1 was used in oligonucleotide in situ hybridization to discriminate wheat chromosomes. The synthetic oligo pAs1-1, pAs1-3, and AFA-4 were 5′ end-labeled with 6-carboxytetramethyl-rhodamine (TAMRA) for red signals. The synthetic oligo pSc119.2-1 and (GAA) 10 were 5′ end-labeled with 6-carboxyfluorescein (6-FAM) for yellow-green signals. Genomic DNA from Ps. strigose was labeled with fluorescein-12-dUTP by the nick translation method as described above and used as a probe for bright green signals. The protocol of GISH/FISH using the synthesized probes was previously described by Wang et al. (2017) (link).
DNA extraction from fresh leaves was performed using the CTAB method (Doyle and Doyle, 1987 (link)). The St-genome DNA as probe can be used for GISH analysis of Th. intermedium chromosome (Chen et al., 1998 (link); Chen, 2005 (link)). The probe was prepared by labeling 1 μg (2 μl) Ps. strigose and common wheat “Chinese Spring” genomic DNA in 4 μl of DIG-Nick-Translation mix (Roche, Mannheim, Germany) and 14 μL of ddH2O at 15°C for 90 min. The reaction was terminated by 1 μL 0.5 mol EDTA (pH 8.0) at 65°C for 10 min. DNA of Chinese Spring wheat and Th. intermedium were sheared to be used as a blocker. Anti-digoxin Rhodamine and DAPI (Roche, Mannheim, Germany) were added prior to incubation in the dark. A Leica DM6000B fluorescence microscope (Leica, Mannheim, Germany) was used for observing the hybridization signals, and images were captured with a Leica digital camera (Model DFC480).
An oligonucleotide (oligo hereafter) multiplex containing oligos pAs1-1, pAs1-3, AFA-4 (GAA) 10, and pSc119.2-1 was used in oligonucleotide in situ hybridization to discriminate wheat chromosomes. The synthetic oligo pAs1-1, pAs1-3, and AFA-4 were 5′ end-labeled with 6-carboxytetramethyl-rhodamine (TAMRA) for red signals. The synthetic oligo pSc119.2-1 and (GAA) 10 were 5′ end-labeled with 6-carboxyfluorescein (6-FAM) for yellow-green signals. Genomic DNA from Ps. strigose was labeled with fluorescein-12-dUTP by the nick translation method as described above and used as a probe for bright green signals. The protocol of GISH/FISH using the synthesized probes was previously described by Wang et al. (2017) (link).
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