**8. Genomic** *in situ* **hybridization**

#### **8.1. Treatment of slides**

Prior to the beginning of the GISH procedures, identify the slides and mark preparation area with a diamond-tipped pen (or similar), along the length of the blade (Fig. 6A). Avoid doing notes with permanent marker or using paper labels.

Slides stored for a long time must be dipped in Carnoy for 15 min, followed by an alcoholic series of 70% and 100% ethanol for 5 min each (Fig. 6B). The Carnoy helps to better fix the chromosome structure. However, this step is not necessary for newly prepared slides.

	- **•** Paraformaldehyde to be used at step 8 could be prepared during the steps 1-2.
	- **•** In general, for Triticeae species, the procedures from step 3 to 9 may be completely excluded without affecting the *in situ* hybridization.

#### **8.2.** *In situ* **hybridization according to Heslop-Harrison et al. [18] and Pedrosa et al. [19], with some modifications**

**8. Genomic** *in situ* **hybridization**

notes with permanent marker or using paper labels.

Prior to the beginning of the GISH procedures, identify the slides and mark preparation area with a diamond-tipped pen (or similar), along the length of the blade (Fig. 6A). Avoid doing

Slides stored for a long time must be dipped in Carnoy for 15 min, followed by an alcoholic series of 70% and 100% ethanol for 5 min each (Fig. 6B). The Carnoy helps to better fix the chromosome structure. However, this step is not necessary for newly prepared slides.

**1.** Let the slides to dry off for 30 min at 50-60 °C (Fig. 6C). This drying is important be‐ cause the grip of the chromosomes to the blade is improved during the process.

**•** Paraformaldehyde to be used at step 8 could be prepared during the steps 1-2.

**3.** Add 50 μL of 100 μg/mL RNase A [a 10 mg/mL RNase A solution diluted in 2×SSC (300 mM NaCl and 30 mM Na3C6H5O7.2H2O) at the proportion of 1:100], cover with a plastic coverslip (made of laboratory film or similar) and incubate in a moisture chamber for 1

**4.** Wash the slides three times in 2×SSC for 5 min each (Fig. 6G). After each wash cycle, the

**5.** Add 50 μL of 10 mM HCl, cover with plastic coverslip and maintain for 5 min (Fig. 6H).

**6.** Add 50 μL of 15 μg/mL pepsin (a 1 mg/mL pepsin solution diluted in 10 mM HCl at the proportion of 1.5:100), cover with plastic coverslip and incubate at 37 °C for 20 min (Fig. 6I-J). Before adding the pepsin solution, remove the excess of HCl with and absorbing

**8.** Fixate the chromosome preparation in 4% paraformaldehyde for 10 min (Fig. 6L). Be ex‐ tremely cautious when handling paraformaldehyde because it is highly toxic and carci‐

**•** In general, for Triticeae species, the procedures from step 3 to 9 may be completely

**10.** Dehydrate the slides in an alcoholic series of 70% and 100% ethanol for 3 min each (Fig.

**2.** Let the slides to cool for 5-10 min at room temperature (Fig. 6D).

used volume of 2×SSC must be replaced by a clean one.

**7.** Wash the slides three times in 2×SSC for 5 min each (Fig. 6K).

**9.** Wash the slides three times in 2×SSC for 5 min each (Fig. 6L).

excluded without affecting the *in situ* hybridization.

**11.** Let the slides to dry off for, at least, 1 h at room temperature (Fig. 6O).

**8.1. Treatment of slides**

14 Plant Breeding from Laboratories to Fields

h at 37 °C (Fig. 6E-F).

paper as illustrated in Fig. 6M.

nogenic.

6M-N).

The stringency value for the procedures below is 77%. The stringency value refers to the percentage of correct base pairing during the *in situ* hybridization process and is calculated according to formamide and salt (SSC) concentrations in the solution, as well as the reaction temperature [17]. Defrost all components and prepare the hybridization mixture in chopped ice (Fig. 7A).

**Figure 6.** Slide treatment for genomic *in situ* hybridization. (A) Marking the area of the slide that contains the chromo‐ some preparation. (B) Baths in absolute ethanol:acetic acid (3:1, v/v), 70% ethanol and 100% ethanol. (C) Drying slides in an incubation oven at 50-60 °C. (D) Slides in room temperature. (E) Digestion with RNase. (F) Incubation of the slides at 37 °C in a moisture chamber. (G) Washing the slides in 2×SSC to remove the RNase. (H) Addition of 10 mM HCl. (I) Digestion with pepsin. (J) Incubation of the slides at 37 °C in a moisture chamber. (K) Washing the slides in 2×SSC. (L) Treatment with 4% paraformaldehyde and posterior washes in 2×SSC. (M) Removal of the excess of 2×SSC with absorbing paper. (N) De‐ hydration in an alcoholic series (70% and 100%). (O) Drying the slides at room temperature. A, B, E, F, G, H, I, J, K, L, M and N: Sandra P. Brammer; C, D and O: Ana R. Oliveira & Ana C. Brasileiro-Vidal.

**Figure 7.** Genomic *in situ* hybridization. (A) Preparation of the hybridization mixture. (B) Denaturation of the probe at 75 °C. (C) Addition of the hybridization mixture in the chromosome preparation and covering with a glass coverslip. (D) Denaturation of the chromosomes in a metal plate inside a water bath at 73 °C. (E) Sealing the coverslip with rubber glue. (F) *In situ* hybridization at 37 °C. A, B, C, D and E: Ana R. Oliveira & Ana C. Brasi‐ leiro-Vidal; F: Sandra P. Brammer.

	- **•** For other plant groups, use 75 °C or above.


**Table 1.** Components of a 10 µL hybridization mixture.

#### **8.3. Post-hybridization baths and probe detection**

In this step, flasks with SSC solutions and the Coplin jar (with the solution of the first wash) must be previously in the water bath at 42 °C (Fig. 8A). Before the temperature of the water bath, the temperature inside the Coplin jar must also be checked. The same jar can be used for all baths by discarding the anterior SSC solution and adding the next one (Fig. 8B-C). Furthermore, the function of the wash procedures is to remove the excess of material from the *in situ* hybridization, mainly non-hybridized probes and incorrectly hybridized ones.


**Figure 7.** Genomic *in situ* hybridization. (A) Preparation of the hybridization mixture. (B) Denaturation of the probe at 75 °C. (C) Addition of the hybridization mixture in the chromosome preparation and covering with a glass coverslip. (D) Denaturation of the chromosomes in a metal plate inside a water bath at 73 °C. (E) Sealing the coverslip with rubber glue. (F) *In situ* hybridization at 37 °C. A, B, C, D and E: Ana R. Oliveira & Ana C. Brasi‐

**1.** Prepare the hybridization mixture (50% formamide, 10% dextran sulfate, 2×SSC, ca. 2.5-5 ng/μL of probe and 25-50 ng/μL of blocking) with a final volume of 10 μL per slide (Table 1). In case of using directly labeled probes, the mixture preparation and all the following steps must be done in partial darkness (avoid direct incidence of light). The formamide destabilizes the DNA molecule, helping in the denaturation. Thus, be care‐

**2.** For probe denaturing, incubate the mixture at 75 °C for 10 min in a water bath (Figs. 1D and 7B). Immediately put the mixture on ice for at least 5 min to keep the two DNA

**3.** Spin down the mixture and then add 10 μL per slide, cover with an 18×18 glass cover‐

**4.** Seal the coverslip with rubber glue (Fig. 7E) and incubate the slide in a moisture cham‐ ber at 37 °C overnight (ca. 16 h) or up to one day and a half (Figs. 1G and 7F).

100% formamide 5 μL 50% 50% dextran sulfate 2 μL 10% 20×SSC (saline-sodium citrate) 1 μL 2×

Ultrapure distilled water Q.s.p. 10 μL -

**Component Quantity Final concentration**

Probe 0.5-1 μL ca. 2.5-5 ng/μL Blocking DNA 0.5-1 μL ca. 25-50 ng/μL

slip and denature the slide at 73 °C for 10 min (Figs. 1E-F and 7C).

leiro-Vidal; F: Sandra P. Brammer.

16 Plant Breeding from Laboratories to Fields

strands open.

ful while handling it due to its high toxicity.

**•** For other plant groups, use 75 °C or above.

**Table 1.** Components of a 10 µL hybridization mixture.


During the washes of slides with directly labeled probes, the procedures must also be done in partial darkness due to the presence of fluorochromes. Moreover, the GISH procedures end after these washes when using this type of probes and the results may be already vi‐ sualized in an epifluorescence microscope after adding the DAPI-Vectashield, as better ex‐ plained below in the steps 11-12 (Fig. 1J).


**Figure 8.** Post-hybridization baths and probe detection. (A) Washing of the slides in a water bath at 42 °C. (B) Disposal of the saline-sodium citrate (SSC) solution. (C) Addition of the next SSC solution. (D) Addition of 4×SSC + 0.1% Tween 20 at room temperature. (E) Addition of 5% bovine serum albumin (BSA) for the blocking step. (F) Preparation of the antibody solution. (G) Addition of the antibody solution and covering with a plastic coverslip. (H) Incubation of the slides during the detection step at 37 °C. (I) Washing the Wash the slides three times for 10 min each in 4×SSC + 0.1% Tween 20 at 42 °C. These washes are needed for removing the excess of antibodies (Fig. 8I). Figures A, B, C, D, H and I: Sandra P. Brammer; E, F, G, J, K and L: Ana R. Oliveira & Ana C. Brasileiro-Vidal.

A metaphase cell of triticale (2*n* = 56) is shown with the 14 chromosomes from rye detected by GISH in Fig. 9. This cell was hybridized with blocking DNA of wheat and rye DNA probe labeled with digoxigenin and detected with FITC (fluorescein isothiocyanate). The chromosomes are counterstained with DAPI (Fig. 9A). Fig. 9B shows the image capture of the same chromosomes with the fluorescence filter for FITC; and, in Fig. 9C, is shown the superposition of both images.

**Figure 9.** Genomic *in situ* hybridization (GISH) in a triticale cell (2*n* = 56), using rye DNA as probe (green) and wheat DNA as blocking DNA. Chromosomes were counterstained with DAPI (blue). The same cell is represented in A (DAPI), B (FITC) and C (A + B image merging). The detail in A, B and C shows the 14th rye chromosome of the same cell. Ana C. Brasileiro-Vidal.
