DNA extraction from leaves was performed using the cetyl trimethylammonium bromide (CTAB) protocol (Molecular Cloning, 3rd edition). Gene-specific primer pairs HOS1-1-target and HOS1-2-target (Supplementary Table S1 ) were used to amplify 381- and 372-bp long genomic regions containing the corresponding target sites, respectively. Obtained amplicons were ligated into a pJET (Thermo Fisher Scientific Inc., MA, USA) and sequenced using an ABI 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).
To genotype mutations by a heteroduplex mobility assay (HMA), polyacrylamide gel electrophoresis (PAGE) analysis of amplicons subjected to denaturation/renaturation cycle was performed following the procedure of Reference [45 (link)]. To screen for mutations using high-resolution melting (HRM) analysis, we performed polymerase chain reaction (PCR)-HRM reactions with the same primer sets and 2.5 x SYBR green PCR master mix (Evrogen, Moscow, Russia) using CFX96 thermocycler (Bio-Rad Laboratories). Melting curves were determined by incubating the reaction mixes from 60 °C to 95 °C with an increment 0.2 °C for 10 s with a plate reading. Precision Melt Analysis software (Bio-Rad Laboratories) was utilized to discriminate between native and mutant HOS1 alleles.
To genotype mutations by a heteroduplex mobility assay (HMA), polyacrylamide gel electrophoresis (PAGE) analysis of amplicons subjected to denaturation/renaturation cycle was performed following the procedure of Reference [45 (link)]. To screen for mutations using high-resolution melting (HRM) analysis, we performed polymerase chain reaction (PCR)-HRM reactions with the same primer sets and 2.5 x SYBR green PCR master mix (Evrogen, Moscow, Russia) using CFX96 thermocycler (Bio-Rad Laboratories). Melting curves were determined by incubating the reaction mixes from 60 °C to 95 °C with an increment 0.2 °C for 10 s with a plate reading. Precision Melt Analysis software (Bio-Rad Laboratories) was utilized to discriminate between native and mutant HOS1 alleles.
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