Innuprep doublepure kit

The Polymerase Chain Reaction (PCR) to amplify TcMPX was performed using 200 ng of DNA, and specific primers were designed using the software Primer1 (http://primer1.soton.ac.uk/primer1.html): TcMPX-Fw (5′-ATGTTTCGTCGTATGGCCG-3′) and TcMPX-Rv (5′-TCATGCGTTTTTCTCAAAATATTCA-3′). The conditions employed were as follows: 5 U Platinum™ Pfx enzyme (Invitrogen, Massachusetts, USA), 10 mM of each primer, and 4 mM MgCl₂. One initial step of 94°C for 3 min, 35 cycles (30 s at 94°C, 30 s at 60°C, and 45 s at 68°C), and a final elongation step (5 min at 68°C). The amplicons obtained (330-bp fragment) were purified using the innuPREP DOUBLEpure Kit (Analytik Jena, Jena, Germany). They were sequenced in the Laboratorio de Secuenciación Genómica de la Biodiversidad y de la Salud (UNAM) with the Sanger technique, using a 3500xL genetic analyzer (Applied Biosystems, Massachusetts, USA). The obtained sequences for Qro and Ninoa were analyzed using the software Chromas. The consensus sequence was determined using the software Bioedit. Finally, the two sequences were uploaded to the GenBank as QroMPX (accession number QKE53461.1) and NinoaMPX (QKE53460.1).

PCR products were purified either using the innuPREP DOUBLEpure Kit (Analytikjena) or the QIAquick PCR Purification Kit Protocol (QIAGEN) following the manufacturer’s instructions. Purified PCRs were sent for sequencing to Eurofins Genomics (Ebersberg, Germany), the allelic distribution profiles were determined using the online toolkit TIDE with standard parameters [8 (link)].

The SMART RACE cDNA Amplification Kit (Clontech) was used. First-strand cDNA was synthesized from 5 μg RNA, primed with the adaptor-linked oligo(dT) primer 3′-CDS, as described in the instructions for the synthesis of 3′-RACE-ready cDNA. In case of 5′-RACE, the reverse GSPs used to synthesize the first-strand 5′-RACE-ready cDNA of the respective genes were CYP81-5RACE1 and CPR-5RACE1 (Supplementary Table 7). Touchdown PCR was employed for the subsequent amplification step. The 25 μl reaction mixture contained 1 μl each of the cDNA samples in 1 × reaction buffer Y, 0.4 mM dNTPs, 0.4 μM primers and 1.25 U peqGOLD Taq-DNA-Polymerase (Peqlab). The PCR conditions were as follows. Initial denaturation at 94 °C for 2 min; 10 cycles with 94 °C for 30 s, annealing at the T_m of the respective GSPs for 45 s with ΔT −0.5 °C per cycle, 72 °C for 90 s; 30 cycles with 94 °C for 30 s, annealing at the T_m of the respective GSPs −5 °C for 45 s, 72 °C for 2 min; and final extension at 72 °C for 15 min. The RACE products were run on an agarose gel stained with Midori Green (Nippon Genetics) and the bands at the expected size were excised and purified using the innuPREP DOUBLEpure Kit (Analytic Jena). The purified products were cloned into the pGEM-T Easy vector (Promega) and sequenced. The primers used for the RACE reactions are listed in Supplementary Table 7.