*2.2. DNA Extraction, PCR Amplification, and Sequencing*

This procedure was performed with the 2% cetyl trimethylammonium bromide (CTAB) method according to Doyle and Doyle [27] with some modifications [28]. Genomic DNA was suspended in 100 μL of sterile HPLC water and quantified by spectrophotometry in a NanoDrop 2000c (Thermo Scientific, Waltham, MA, USA). To determine the DNA quality, absorbance values between 1.8 and 2.2 at A280/260 and A230/260 nm were considered acceptable. Finally, the DNA was diluted to 20 ng <sup>μ</sup>L−<sup>1</sup> and then stored at −<sup>20</sup> ◦C for PCR amplification.

Molecular identification of "PUE 4.0" strain was carried out based on the analysis of internal transcribed spacer (ITS) region sequences using primer pairs ITS5 (5 - GGAAGTAAAAGTCGTAACAAGG-3 )/ITS4(5 -TCCTCCGCTTATTGATATGC-3 ) [29]. The reaction mixture was prepared in a final volume of 15 μL with 1× Taq buffer DNA polymerase, 0.18 μM of each dNTP, 0.18 μL of each primer containing 10 pmol, 0.90 U of GoTaq DNA polymerase (Promega, Madison, WI, USA), and 40 ng μL−<sup>1</sup> DNA. PCR was performed in a Peltier PTC-200 DNA thermal cycler (Bio-Rad, Santa Rosa, CA, USA). The amplicons were verified by electrophoresis in a 1.5% agarose gel (Seakem, Invitrogen, Carlsbad, CA, USA) and stained with 10,000× GelRed (Biotium, Fremont, CA, USA). All PCR products were cleaned with ExoSAP-IT (Affymetrix, Santa Clara, CA, USA), and both strands were individually sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Carlsbad, CA, USA) in a 3130 Genetic Analyzer Sequencer (Applied Biosystems, Carlsbad, CA, USA) at Postgraduate College Facilities, Mexico, according to Juárez-Vázquez [30]. Sequences were assembled and edited using SeqMan (DNAStar, Madison, WI, USA) and compared to sequences established in GenBank™ using the Blast algorithm.
