**6. Seed germination and collecting, pretreating and fixating root tips**

**1.** Wash seeds in 4% chlorine bleach for 5 min (Fig. 4A).

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‐

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

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

**1.** Prepare the nick translation mixture in centrifuge microtube surrounded by chopped

**2.** Vortex the mixture, spin down the volume and add the enzymatic solution rapidly (Fig.

**3.** Mix gently, spin down the volume and put the microtube either in a thermocycler or in a thermoblock at the recommended temperature (Fig. 3F). To find out if the reaction time recommended by the manufacturer was sufficient for obtaining fragments with 200-300 bp, the labeling process should be temporarily suspended by maintaining the tube in chopped ice. Meanwhile, an electrophoresis in 0.8% agarose gel should be per‐ formed with an aliquot of the reaction. If the DNA is sufficiently fragmented (Fig. 3H), add the stop buffer. Otherwise, the reaction must continue as long as necessary for cor‐

**4.** After adding the stop buffer, add 2 vol of cold absolute ethanol and 0.1 vol of 3 M so‐

**6.** Centrifuge for 20 min (14,000 rpm, room temperature), discard the supernatant and add

**7.** Centrifuge for 5 min (12,000 rpm, room temperature), discard the supernatant and dry

**8.** Re-suspend the pellet in 15-20 μL of 10 mM Tris-HCl (pH 8.0) and store at -20 °C.

tions could be impaired.

10 Plant Breeding from Laboratories to Fields

(Figs. 2C, 3G and 3H).

3D-E).

following the procedures below:

rect DNA fragmentation.

1 mL of 70% ethanol.

dium acetate, in order to precipitate DNA.

the pellet at room temperature or at 37 °C.

**5.** Mix gently by inverting and put in the freezer overnight.

ice without the enzymatic solution (Fig. 3B-C).


**Figure 4.** Washing and germinating seeds in (A) and (B); pretreatment and fixation of root tips in (C) and (D). (A) Seed washing in 4% chlorine bleach. (B) Roots with the length of 1 to 1.5 times the size of the seed, adequate for the collec‐ tion. (C) Cold pretreatment of root tips. (D) Fixation of root tips in absolute ethanol:acetic acid (3:1, v/v). A and C: Ana R. Oliveira & Ana C. Brasileiro-Vidal; B and D: Sandra P. Brammer.
