Aceto-carmine

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).

Flower buds and/or root tips of wheat, rye and barley were fixed for 45 min in ice-cold 4% (w/v) paraformaldehyde in 1xMTSB buffer (50 mM PIPES, 5 mM MgSO4, and 5 mM EGTA, pH 7.2). After washing in 1xMTSB, chromosome spreads were prepared by squashing. Young Ae. speltoides spikes were pretreated in ice-cold water for 24 h and then fixed in ethanol:acetic acid (3:1) for at least 4 days. Afterwards, the spikes were stained with aceto-carmine and chromosomes were prepared by squashing in 45% acetic acid.
Tissue sections of Ae. speltoides were prepared according to Steedman (1957 (link)) and Braszewska-Zalewska et al. (2013 (link)). Briefly, developing seeds were excised from spikelets and fixed in freshly prepared 3% (w/v) paraformaldehyde with 0.05% Triton X100 in 1 × PBS buffer on ice for 5 h. Then, dehydration was performed in an ethanol series (ethanol/1 × PBS buffer) from 30 to 96% for 30 min in each at room temperature and in 96% for 30 min at 37°C. Afterwards, the tissues were infiltrated with PEG1500-wax and embedded in a small casting mold (1–3 seeds per block). The blocks were cut into 10 μm slices using a Leica microtome (RM2265; knifes 35N from Feather company). The slices were transferred onto poly-L-lysine-coated slides using forceps and a brush and were stretched by adding a drop of 1 μl water over each slice. The rest of PEG-wax was removed from the dry slides by washing them in 90% ethanol.
Differentiated 2–16C leaf nuclei of A. thaliana were isolated and flow sorted according to their DNA content from differentiated rosette leaves after formaldehyde fixation using a FACS Aria (BD Biosciences) as described (Pecinka et al., 2004 (link)).
To evaluate the substructure of CENH3 containing chromatin, immunostaining was performed according to Jasencakova et al. (2000 (link)). CENH3 was detected with rabbit anti-grass CENH3 primary antibodies (Sanei et al., 2011 (link)) and goat anti-rabbit rhodamine (1:300; Jackson Immuno Research Laboratories) or goat anti-rabbit Alexa488 secondary antibodies (1:200; Molecular Probes). Spindle fibers were labeled with monoclonal mouse anti α-tubulin (1:200; clone DM 1A, Sigma) and anti-mouse Alexa488 (1:400; Molecular Probes) antibodies.
For FISH the 180-bp centromeric repeat sequence pAL of A. thaliana (Martinez-Zapater et al., 1986 (link)) was generated by PCR as described (Kawabe and Nasuda, 2005 (link)). The centromeric retrotransposon CRW2 of wheat was generated by PCR as described by Li et al. (2013 (link)). These probes as well as the subtelomeric repeat HvT01 (Schubert et al., 1998 (link)) and BAC7 containing centromere-specific repeats of barley (Hudakova et al., 2001 (link); Houben et al., 2007 (link)) were directly labeled by nick translation with TexasRed-dUTP, Alexa488-dUTP and Cy3-dUTP according to Ward (2002 ). FISH was performed according to Schubert et al. (2001 (link)).
For the colocalization of CENH3 immunosignals with centromeric FISH signals, immunostaining was performed first. The slides were treated with 10 mM citrate buffer (pH 6) in a microwave at 800 Watt for 60 s according to Chelysheva et al. (2010 (link)). Then the primary antibodies were applied and immunostaining was performed as described (Jasencakova et al., 2000 (link)). Prior FISH the slides were treated with ethanol:acetic acid (3:1) fixative for 10 min and freshly prepared 4% formaldehyde in 1 × PBS for 10 min, followed by three times washing for 5 min in 1 × PBS. These steps are important to stabilize the immunosignals during the following FISH procedure, which was performed as described (Ma et al., 2010 (link)). Nuclei and chromosomes were counterstained with DAPI (1 μg/ml) in Vectashield (Vector Laboratories).

Young spikes were collected from 7 to 10-week-old plants and carefully dissected to isolate anthers. For each dissected floret, one of the three developmentally equivalent anthers was squashed in aceto-carmine staining solution and meiocytes visualised using a ZEISS Optima microscope. When meiocytes at metaphase I were identified (for chiasma frequency analysis) or other defined stages (RNA analysis), the two remaining anthers were either fixed in 100% ethanol/acetic acid 3:1 (v/v) for 48 h and then subsequently transferred to 70% ethanol or snap frozen in liquid N₂ for later RNA-based analyses. Fixed anthers can eventually be stored at 4°C for a few months. For cytological analysis of meiocytes at metaphase I, pollen mother cells (PMCs) were released from the anther by crushing it on a slide in a drop of aceto-carmine staining solution. Anther debris was carefully removed, and a coverslip placed on the slide. The slides were then heated until separation of the chromosomes and aceto-carmine solution replaced by acetic acid 45%. Coverslips were then vertically pressed to spread out the chromosomes. Chromosome configurations of ~50 PMC per anther were analysed under a ZEISS Axio Observer Z1 inverted microscope. For each cell, the number of univalents, rod bivalents (pair of chromosomes linked by a unique chiasma), ring bivalents (pair of chromosomes linked by two chiasmata), trivalents (three chromosomes linked by two chiasmata) and quadrivalents (four chromosomes linked by three or four chiasmata) were counted. Frequency of chiasmata (the cytological manifestation of meiotic crossovers) was then calculated. Significant differences between mutant and corresponding wild-type control chiasma frequencies were assessed using Mann–Whitney tests adjusted for multiple comparisons.