2.3. Sequencing and Specific Primer Design

Genomic DNA was extracted from up to 100 mg of fresh tissues of donor plant leaves, calluses, and regenerants' leaves. Extraction was done with slight modifications for each development stage using the protocol: purification of total DNA from plant tissue (Mini Protocol, DNeasy Plant Mini Kit, Qiagen GmbH, Hilden, Germany). The final elution volume of DNA was 50 μL. The genomic DNA was quantified using a spectrophotometer (NanoDrop 1000, Thermo Scientific, Waltham, USA) to measure absorbance at 260 nm and the purity of DNA was checked. Stock DNA was diluted to make a working solution of 20 ng/μL for further PCR analysis.

PCR and sequencing primers were designed for amplification of the gene regions of Pme3 and Mlo (Table 1). Primers for conventional PCR and sequencing were developed utilizing PyroMark Assay Design software (Qiagen, Germany).

Sequence characteristics of primers developed in this study. PCR and sequencing primers were designed for amplification the gene regions of Pme3 and Mlo.

Amplification was carried out in a thermocycler (Veriti 96-Well Thermal Cycler, Applied Biosystems, Foster City, USA) using a Taq PCR Core Kit (Qiagen, Germany). Qiagen Taq DNA polymerase was used in 25 μL reactions with magnesium concentration adjusted to 2.0 mmol/L. The thermocycler was programmed for an initial denaturation of 5 min 94°C, followed by 35 cycles of denaturation for 1 min at 94°C, 1 min of annealing at 51°C, and 30 s of extension at 72°C. The final extension was carried out at 72°C for 10 min with a hold temperature of 4°C at the end. Negative control was included with each round of reactions. PCR products were cleaned-up by ExoSAP-IT (Affymetrix, USA) according to the manufacturer's protocol. Amplified PCR products were sequenced using an ABI 3130xl (Applied Biosystems, USA) capillary sequencer using BigDye Terminator v3.1 chemistry and for purification sequencing products BigDye XTerminator chemistry (Applied Biosystems, USA). Sequencing reactions used the primers designed for sequencing. All sequences were aligned using SeqScape version 2.5 (Applied Biosystems, USA) with default settings. The SV was expressed as the percentage of the differences in the nucleotide sequence in comparison with reference sequence of donor plant.