Total genomic DNA was extracted from silica gel dried tissue following the CTAB protocol (Doyle and Doyle, 1987) with some modifications as reported by Bustamante et al. (2016) to avoid excess mucilage in the samples. DNA quality was tested with NanoDrop 2000. The DNA quality obtained with these modifications was good enough for Sanger sequencing. For each selected nuclear gene, the most promising pair of primers were selected according to previous cacti alignments (Table 1; primer ID “a”). To test the feasibility of primer amplification among Cactaceae species, we selected species from distant phylogenetic clades, namely: Opuntia pilifera, O. tehuacana, Grusonia invicta, Pilosocereus chrysacanthus, P. collinsii, Melocactus curvispinus, Mammillaria albilanata subsp. oaxacana, M. haageana subsp. Meissneri, and M. crucigera. We performed 15 μL PCRs with the commercial mix “Platinum Taq” (Invitrogen), and the reactions included 1.5 μL (1×) of 10× PCR buffer, 0.3 μL of dNTP mix, 0.3 μL of each primer (10 pmol/μL), 0.3 μL of BSA (0.4%), 0.6 μL of MgCl2 (1.5 μM), and 0.075 μL (0.375 units) of Taq DNA polymerase. Amplification tests were made using the touch-up PCR program (Table 2) as well as gradient PCR with two MgCl2 concentrations 1.5 and 2.5 μM (Table 3). To confirm the presence of PCR amplicons, these were run on 1% agarose gels. PCR cleaning and sequencing was performed at Laboratorio de Biología Molecular de la Biodiversidad y de la Salud, Instituto de Biología, UNAM, for sample sequencing the reactions included 0.4 μl of BigDye Terminator v.3.1 (Applied Biosystems), 2 μl of Buffer 5×, 4 μl of water, 1 μl of primer with a concentration of 10 μM and 3 μl of PCR product. Conditions of reaction sequencing were 30 cycles of 96°C for 10 s, 50°C for 5 s and 60°C for 4 min. After cycling, samples were purified with Centri-Sep (Thermo Fisher Scientific) plates following the manufacturer protocol. To each purified sample was added 25 μl of EDTA 0.5 mM and were run in a sequencer Applied Biosystems (Thermo Fisher Scientific) with polymer 7 (Thermo Fisher Scientific).

Touch-up PCR program for amplification tests and primer ID “a” in AT3G48380.

PCR program for gradient test and primer ID “a” in AT1G18270, using a concentration of 2.5 μM MgCl2.

Primer pairs amplifying a single band in representatives of Opuntia and Grusonia were further amplified for this work. Sequences were assembled in Sequencher v.5.4.6. Alignments were performed with the program Muscle v.3.8.31 (Edgar, 2004) and subsequently manually adjusted in PhyDE (Müller et al., 2005).

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