**2.2 PCR amplification**

14 Agricultural Science

Molecular markers analyses occurred in the Plant Biotechnology Laboratory - UFRPE. The optimization of the DNA extraction protocol was performed using fresh young leaves samples of heliconia, harvested in the earliest stage of development and treated under three conditions: harvested, packed in a polystyrene box containing liquid nitrogen and taken to the Laboratory for immediate DNA extraction; harvested and frozen at -20°C for 1 day

In the DNA extraction, Doyle and Doyle (1990) protocol were used with modifications, which was prepared at a 2x CTAB (hexadecyltrimethylammonium bromide) buffer solution. It was added 700 microliter extraction buffer to 200 mg of macerated leaves in test tubes and taken to bath at 65°C. The tubes, after cooled at room temperature, were centrifuged and the supernatant transferred to new tubes. Supernatant was added to 700 microliter (L) CIA

The supernatant was added to 700 microliter (L) CIA (Chloroform-Isoamyl Alcohol) and then centrifuged. After this process, supernatant was added to 500 L of cold isopropanol and stored for 24 hours in a freezer at -20ºC. Subsequently, it was washed twice with 70% ethanol and with 95% ethanol. The precipitate was dried at room temperature for 20 minutes and then resuspended with 300 L TE containing RNAse, incubated at 37°C for 30 minutes, then 5M NaCl and 300 L of cooled isopropanol were added, in which the DNA was precipitated. Solution was incubated at 4°C throughout the night and the pellet

Primers Sequence Number of

A set of 12 primers (Table 2) and 10 combinations of this primers were selected and tested for the ITS analysis: ITS1-ITS4; ITS5-ITS4; ITS1-ITS2; ITS5-ITS2; ITS3-ITS4 based on White *et* 

ITS 1 5'- TCCGTAGGTGAACCTGCGG -3' 19 ITS 2 5'-GCTGCGTTCTTCATCGATGC-3' 20 ITS 3 5'-GCATCGATGAAGAACGCAGC -3' 20 ITS 4 5'-TCCTCCGCTTATTGATATGC-3' 20 ITS 5 5´-GGAAGTAAAAGTCGTAACAAGG -3´ 22 EF11 5-GTGGGGCATTTACCCCGCC-3' 19 EF22 5´-AGGAACCCTTACCGAGCTC-3´ 19 *trnL* 5´-GGTTCAAGTCCCTCTATCCC -3´ 20 *trnF* 5´-ATTTGAACTGGTGACACGAG-3´ 20 *trnS* 5´-TACCGAGGGTTCGAATC -3´ 17 *rps5'* 5´-ATGTCCCGTTATCGAGGACCT -3´ 21 *rps3'* 5' –ATATTCTACAACTAACAACTC – 3' 21 Table 2. Sequence of primers used in amplification reactions in genotypes of the UFRPE

Basis

before extraction; harvest and preserved in silica gel for 5 days before extraction.

(Chloroform-Isoamyl Alcohol) and then centrifuged was performed.

resuspended in 300 L TE. The DNA was quantified in 0.8% agarose gel.

Heliconia Germplasm Collection used in this study

The DNA amplification using PCR was performed to a final volume of 25 L containing 1 L template DNA, 0.3 L Taq-DNA polymerase (Invitrogen), 2.51 L Tris-HCl (pH 8, 0), and 0.75 L MgCl2, 2 L of each dNTPs, 1 L primer, 1 L oligonucleotide 1 and 2; and 15.45 L milli-Q water to complete the reaction.

Amplifications were performed in a thermocycler MJ Reseach, Inc., PTC100 under the following conditions: step 1 - following a denaturation step of 95°C for 3 minutes; step 2 – 94°C for 1 minute; step 3 – 58°C for 1 minute for annealing temperature; step 4 – 72°C for 1 min (repeat steps 2/3/4 for 29 cycles) followed by a final extension at 72°C for 10 minutes and 10°C for 24h. The PCR product visualization was performed in 1.5% agarose gel stained with SYBER Gold (Invitrogen), visualized under ultraviolet light and recorded on a digital Vilber Lourmat photographer.
