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

Genotypisation of transfected protoplasts (folder “Transfected protoplasts”)

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 MIR160a, MIR160b and MIR390a genes, respectively. To determine types of mutations and polymorphism, PCR products were 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.

Genotypisation of transgenic lines (folder “Transgenic potato”)

Approximately 800 bp-long region surrounding coding sequence for each miRNA was amplified from genomic DNA isolated from CRISPR-edited MIR160a (cr-MIR160a), MIR160b (cr-MIR160b) and MIR390a (cr-MIR390a) 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 “PCR amplicons miRNA”). 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 “pJET_miRNA”).

Secondary structures of pre-miRNA precursors (folder “Secondary structures”)

Precursor sequences of wild-type potato MIR160a and MIR160b were obtained from miRBase (https://www.mirbase.org/; Accession No. MI0025955, MI0025956) and of wild-type potato MIR390a from the study of Križnik et al., 2017 (Križnik et al., 2017) (subfolder “WT pre-miRNAs“). The mutated pre-miRNAs of cr-MIR160a, cr-MIR160b and cr-MIR390a transgenic lines were extracted from Sanger sequencing results (subfolder “pJET_miRNA”). 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 et al., 2018) (subfolders “Desiree” and “Rywal”).