Sequences and secondary structures of mutated pre-miRNAs from CRISPR/Cas9-edited MIR160a, MIR160b and MIR390a genes of potato

<strong>Genotypisation of transfected protoplasts</strong> (folder<strong> “Transfected protoplasts”</strong>) Genomic DNA isolated from transfected and non-transfected protoplast of cv. Desiree was used as a template to amplify approximately 800 bp-long region surrounding coding sequence for miR160a-5p, miR160b-5p and miR390a-5p, encoded by <em>MIR160a</em>, <em>MIR160b</em> and <em>MIR390a</em> genes, respectively. To determine types of mutations and polymorphism, PCR products were cloned into pJET, transformed into <em>E. coli</em>, isolated from 9 or 10 colonies per PCR product and Sanger sequenced with primers specific for amplicons from both directions. <strong>Genotypisation of transgenic lines</strong> (folder<strong> “Transgenic potato”</strong>) Approximately 800 bp-long region surrounding coding sequence for each miRNA was amplified from genomic DNA isolated from CRISPR-edited <em>MIR160a</em> (cr-<em>MIR160a</em>), <em>MIR160b </em>(cr-<em>MIR160b</em>) and <em>MIR390a</em> (cr-<em>MIR390a</em>) transgenic and non-transgenic plants (NT; cv. Rywal and cv. Desiree). For the screening of transgenic lines with desired mutations, PCR products were Sanger sequenced (subfolder <strong>“PCR amplicons miRNA”</strong>). To determine types of mutations and polymorphism, PCR products were for the selected transgenic lines and NT plants cloned into pJET, transformed into E. coli, isolated from 9 or 10 colonies per PCR product and Sanger sequenced with primers specific for amplicons from both directions (subfolder <strong>“pJET_miRNA”</strong>). <strong>Secondary structures of pre-miRNA precursors</strong> (folder <strong>“Secondary structures”</strong>) Precursor sequences of wild-type potato <em>MIR160a</em> and <em>MIR160b</em> were obtained from miRBase (https://www.mirbase.org/; Accession No. MI0025955, MI0025956) and of wild-type potato <em>MIR390a</em> from the study of Križnik <em>et al.</em>, 2017 (Križnik <em>et al.</em>, 2017) (subfolder <strong>“</strong><strong>WT pre-miRNAs</strong><strong>“</strong>). The mutated pre-miRNAs of cr-<em>MIR160a</em>, cr-<em>MIR160b</em> and cr-<em>MIR390a</em> transgenic lines were extracted from Sanger sequencing results (subfolder <strong>“pJET_miRNA”</strong>). The secondary structures were drawn and miRNA/miRNA* duplex regions were highlighted (orange – 5p miRNA coding region, blue – 3p miRNA coding region) using RNA Folding/Annotation tool of The Small RNA Workbench v4.5 (Stocks <em>et al.</em>, 2018) (subfolders <strong>“Desiree” </strong>and<strong> “Rywal”</strong>).