**2. DNA isolation (probe and blocking DNA)**

ic *in situ* hybridization (GISH). On the other hand, the genome of the second parent (unlabeled) is used as blocking DNA, aiming to avoid non-specific hybridizations due to the similarity of the two parental genomes. Thus, both parental genomes (the probe and the blocking DNA) must be used together in the same hybridization mixture. The proportion probe:blocking DNA

**Figure 1.** Main steps of the genomic *in situ* hybridization (GISH). (A) Direct and indirect probe labeling. (B) Fragmenta‐ tion of the blocking DNA. (C) Slide preparation. (D) Probe and blocking DNA denaturation in a hybridization mixture. (E) Addition of the hybridization mixture with the probe and the blocking DNA. (F) Denaturation of the chromosome DNA. (G) *In situ* hybridization of probe and blocking DNA in the target sequence of the chromosome. (H) Detection of the probe in the chromosome DNA of one parent, in an indirect labeling. (I) Chromosome DNA molecule of the sec‐ ond parent associated to the unlabeled blocking DNA. (J) Visualization of hybridization signals associated to a probe (green) in a fluorescence microscope. Unmarked chromosomes are visualized with a counter-staining (blue). When the probe labeling is direct, the detection step of the GISH can be excluded. The fluorochromes are the signaling mol‐ ecules and can be directly visualized in a fluorescence microscope with the appropriate filter. Santelmo Vasconcelos &

Ana C. Brasileiro-Vidal.

should be adequate to avoid the detection of the second parent (see Fig. 1).

4 Plant Breeding from Laboratories to Fields

In a GISH, isolating the genomic DNA of the plant materials is the first step for producing the probe and the blocking DNA, being a critical procedure due to the necessity of obtaining DNA as intact as possible and free of contaminants (such as polysaccharides). For plant ma‐ terials, the DNA isolation can be affected by several factors, such as the procedures for col‐ lecting and storing the plant tissue as well as the method for DNA isolation itself. The leaf tissue is the most common to be used for DNA extraction. However, tissues from other parts, as seeds, roots and cultivated cells in suspension, can also be employed.

#### **2.1. CTAB method from Embrapa Wheat, according to Bonato [15]**


#### **2.2. Selective precipitation of polysaccharides, according to Michaels et al. [16]**


#### **3. DNA quantification**

**6.** Add 700 μL of CIAA (chloroform:isoamyl alcohol, 24:1, v/v). Mix gently for 10 min.

**8.** Transfer the supernatant to new centrifuge tubes and add again 700 μL of CIAA. Mix

**10.** Transfer the supernatant to new centrifuge tubes and add 500 μL of cold (-20 °C) iso‐ propanol, mix gently to precipitate the DNA and incubate for, at least, 30 min at -20 °C.

**14.** Wash the pellet with 600 μL of cold 96% ethanol. Discard the 96% ethanol and dry the

**15.** Re-suspend the pellet in 100 μL of 10 mM Tris-HCl (pH 8.0) or ultrapure distilled water.

**17.** Store samples at -20 °C or -80 °C. For long term conservation, the best results are ob‐

**1.** Add 500 μL of the precipitation solution [10 mM Tris-HCl (pH 8.0) and 250 mM NaCl].

**2.** Dissolve the pellet by vortexing. The complete dissolution of the pellet is important to not lose DNA. Samples with much polysaccharide contamination tend to dissolve more

**3.** Add 180 μL of cold absolute ethanol. Mix the solution by vortexing and put immediate‐

**6.** Transfer the aqueous phase to a new tube. In this step, the DNA is in the aqueous phase

**7.** Add 700 μL of isopropanol and mix gently, inverting the tubes approximately 50 times.

**7.** Centrifuge for 7 min (10,000 rpm, room temperature).

**9.** Centrifuge for 7 min (10,000 rpm, room temperature).

**11.** Centrifuge for 5 min (10,000 rpm, room temperature).

**12.** Discard the supernatant carefully in order to not lose the pellet.

**16.** Add 3 μL of 10 mg/mL RNase A, mix and incubate for 1 h at 37 °C.

tained when pelleted materials are stored in 70% ethanol.

**13.** Wash the pellet with 600 μL of cold 70% ethanol. Discard the 70% ethanol.

**2.2. Selective precipitation of polysaccharides, according to Michaels et al. [16]**

**4.** Put in the refrigerator (10 °C) for 20 min or in the freezer (-20 °C) overnight.

gently for 10 min.

6 Plant Breeding from Laboratories to Fields

pellet at room temperature.

slowly.

ly in chopped ice.

**5.** Centrifuge for 20 min (10,400 rpm, 4 °C).

**8.** Centrifuge for 20 min (10,400 rpm, 4 °C).

Leave the tubes for 15 min at room temperature.

**9.** Discard the supernatant and dry the pellet at room temperature.

**10.** Add 500 μL of 70% cold ethanol and invert the tube approximately 20 times.

and the pellet may be discarded.

After the isolation procedures, the resultant DNA must be quantified prior to probe labeling and preparation of the blocking DNA. Thus, an electrophoresis in agarose gel (0.8%) with an aliquot of each isolated DNA should be performed, using λ-DNA as reference with different amounts (e.g. 50 ng, 100 ng and 150 ng). After the electrophoretic run, a comparison be‐ tween reference bands and bands of the isolated DNA can be made. In the sample of the rye DNA (Fig. 2A, sample 2), for instance, it is suggested that the band of the sample presents the same fluorescence intensity of the 100 ng reference λ-DNA. Thus, as 1 μL of the rye DNA was loaded in the gel, then concentration of the isolated rye DNA is 100 ng/μL. For the wheat DNA (Fig. 2A sample 1), the band is also similar to the 100 ng reference λ-DNA. However, in this case, only 0.5 μL of the sample where loaded in the gel. Thus, the concen‐ tration of the isolated wheat DNA is 200 ng/μL.

**Figure 2.** Analysis of genomic DNA by electrophorese in 0.8% agarose. (A) Quantification of genomic DNA of wheat (sample 1; 0.5 μL of DNA) and rye (B; sample 2; 1 μL of DNA). The two first bands are the weight markers with 50 ng and 100 ng (1 μL). (B) Verification of the fragmentation of wheat DNA, which will be used as blocking DNA, by auto‐ claving and (C) rye DNA after labeling by nick translation. The 100 bp DNA ladder was used as marker. Sandra P. Brammer.

#### **4. Fragmentation of the blocking DNA**

In general, the species involved in the production of hybrids are closely related. Therefore, when a GISH is performed with the genomic DNA of one parental species, a non-specific hybridization often happens in chromosomes derived from the second parental species, mainly due to the presence of repetitive DNA that are common between the two parents. In order to avoid this non-specific hybridization, the unlabeled genomic DNA of the second parent should be used in the *in situ* hybridization. As the probe, the blocking DNA should be approximately 300 bp long, or even shorter (50-300 bp) (Fig. 2B, sample 2). In the specific case of the blocking DNA, the exact amount of DNA of the sample has to be known. Consid‐ ering the total value of the wheat DNA sample as 100 μL, 99.5 μL still remain after the quan‐ tification (100 ng/μL). It is important to remember that the DNA should be quantified (total amount in ng or μg) prior to its fragmentation in autoclave (as explained below), in boiling water, in sonicator or by nick translation (without the marked nucleotides). The DNA frag‐ mentation in autoclave can be made as follows:

	- **•** Using a 1.5 mL centrifuge microtube of good quality is recommended to avoid break‐ age of the tube. To avoid evaporation of the sample, the microtubes should be sealed.
	- **•** For GISH in wheat × rye hybrids, the blocking DNA (wheat DNA) must be at the concentration of 500 ng/μL due to the concentration of the probe of 50 ng/μL (propor‐ tion 1:10, probe:blocking DNA). However, for hybrids between other species, the concentration of the blocking DNA may be higher, if the proportion probe:blocking DNA is different. For instance, if the proportion to be used is 1:20, the blocking DNA should be at 1 μg/μL.

#### **5. Nick translation**

mainly due to the presence of repetitive DNA that are common between the two parents. In order to avoid this non-specific hybridization, the unlabeled genomic DNA of the second parent should be used in the *in situ* hybridization. As the probe, the blocking DNA should be approximately 300 bp long, or even shorter (50-300 bp) (Fig. 2B, sample 2). In the specific case of the blocking DNA, the exact amount of DNA of the sample has to be known. Consid‐ ering the total value of the wheat DNA sample as 100 μL, 99.5 μL still remain after the quan‐ tification (100 ng/μL). It is important to remember that the DNA should be quantified (total amount in ng or μg) prior to its fragmentation in autoclave (as explained below), in boiling water, in sonicator or by nick translation (without the marked nucleotides). The DNA frag‐

**1.** Prepare an aliquot containing 5-50 μg of the previously quantified DNA in 100 μL (di‐ luted in ultrapure distilled water). The sample 1 of the Fig. 2A, for instance, was at 200

**2.** Put the microtube in a closed flask to avoid both the opening of the microtube and the direct contact of the sample with the autoclave steam. Put the flask in the autoclave. **3.** Turn on the autoclave and when the temperature reaches 121 °C, mark 5 min and then

**4.** After removing the microtube from autoclave, expect the microtube to cool and spin down the volume. Run an electrophoresis with the autoclaved DNA and a 100 bp lad‐ der (as reference) in a 0.8% agarose gel (Fig. 2B). The fragmented DNA must be be‐

**•** For GISH in wheat × rye hybrids, the blocking DNA (wheat DNA) must be at the concentration of 500 ng/μL due to the concentration of the probe of 50 ng/μL (propor‐ tion 1:10, probe:blocking DNA). However, for hybrids between other species, the concentration of the blocking DNA may be higher, if the proportion probe:blocking DNA is different. For instance, if the proportion to be used is 1:20, the blocking DNA

**5.** Add 2 volumes (vol) of cold absolute ethanol and 0.1 vol of 3 M sodium acetate (or 0.05

**11.** Re-suspend the pellet in 10 mM Tris-HCl (pH 8.0) or in ultrapure distilled water, in or‐ der to reach the required concentration (in this case, 500 ng/μL). Take into account that there are losses in the total quantity of DNA during the steps of precipitation and re-

**•** Using a 1.5 mL centrifuge microtube of good quality is recommended to avoid break‐ age of the tube. To avoid evaporation of the sample, the microtubes should be sealed.

mentation in autoclave can be made as follows:

8 Plant Breeding from Laboratories to Fields

turn it off.

tween 100-300 bp.

suspension.

should be at 1 μg/μL.

**8.** Wash the pellet with 1 mL of 70% ethanol.

**10.** Dry the pellet at room temperature or at 37 °C.

vol of 7.5 M sodium acetate) to precipitate the DNA. **6.** Mix gently by inverting and store overnight at -20 °C. **7.** Centrifuge for 20 min (14,000 rpm, room temperature).

**9.** Centrifuge for 5 min (14,000 rpm, room temperature).

ng/μL. Thus, in 100 μL of sample there are 19.9 μg of DNA.

The procedures of probe labeling by nick translation are performed by using 1 μg of DNA. The components that are needed for the labeling reaction are: unmarked nucleotides (dATPs, dCTPs, dGTPs and, in a minor concentration, dTTPs), marked nucleotide (dUTPs) and an enzyme solution with DNase I and DNA polymerase I (Fig. 3A-C). The enzyme DNase I hydrolyzes the DNA by generating random nicks in the double-stranded DNA.

**Figure 3.** Nick translation reaction. (A) Total genomic DNA to be labeled. (B) Components of the reaction in chopped ice. (C) Preparation of the reaction mixture without the enzymatic solution. (D) The reaction mixture in a vortex. (E) Fast centrifugation of the mixture and addition of the enzymes. (F) Nick translation in a thermoblock at 15-16 °C, ac‐ cording to the manufacturer's recommendation. (G) Fragmented and labeled DNA. (H) Agarose gel showing a frag‐ mented DNA with approximately 200-300 bp. Santelmo Vasconcelos & Ana C. Brasileiro-Vidal.

**•** A low number of nicks may lead to an inefficient insertion of marked nucleotides, thus generating larger probes. On the other hand, excessive nicks result in very short probes.

The DNA polymerase I has three different activities: 1) an exonuclease function that re‐ moves nucleotides from the breakage site in the sense 5' 3'; 2) a polymerase function that inserts new nucleotides in the 3' end, by using the opposite strand as template; and 3) a re‐ pair function in the sense 3' 5'. Thus, marked and unmarked nucleotides are incorporated by the new synthesized DNA (see Fig. 3F; [17]). Additionally, only part of the thymines may be replaced by marked uracils. If all thymines are changed, the *in situ* hybridization reac‐ tions could be impaired.

During the nick translation reaction, the DNA structure becomes extremely fragile, resulting in the breakage of the double-stranded DNA. Besides the incorporation of marked nucleoti‐ des, the nick translation also fragments the DNA. Therefore, the longer the reaction lasts, smaller the fragments will be. The ideal size for the probe is around 200-300 bp because if it is above 500 bp, the *in situ* hybridization will not work properly; if the probe is much short‐ er, it could be washed away during the post-hybridization baths. Thus, the size of the frag‐ ments must be checked through electrophoresis in agarose gel before stopping the reaction (Figs. 2C, 3G and 3H).

Nick translation reactions are generally performed with commercial labeling kits, which should be performed according to the manufacturer's recommendations, although always following the procedures below:

