Geldoc go

Excised mouse toes were added to 100 μL tissue digestion solution (bimake) and digested for 15 min at 55 °C, then incubated for 5 min at 95 °C to inactivate enzymes. Tubes were centrifuged at 12,000 rpm for 5 min, with supernatant used as the PCR template (1000 ng). Primer sequences are listed in Table S6. Next, PCR amplification was performed, followed by agarose gel electrophoresis and imaging analysis using a gel imaging system (BIO-RAD, GelDoc Go).

Type I. Classic PCR was performed using the primers shown in Table 3, in the Tercyc gene cycler (DNA Technology, Moscow, Russia). Primers were selected on the basis of cranberry genome fragment (JOTO01169953.1). The 40 µL reaction mixture contained DreamTaq Green PCR Master Mix (2X) buffer (Thermo Fisher, Waltham, MA, USA), 100 ng DNA, and 10 µM primers. The following program was used for PCR, 5 min at 93 °C; 40 cycles of 17 s at 93 °C, 30 s at X °C and 90 s at 72 °C; then 5 min at 72 °C, where X was the annealing temperature, depending on the primers used.
Type II. Real-time PCR from colonies was performed for additional control of early threshold values to search for clones containing the target insert. Real-time PCR was carried out in ANK-32 cycler (Sintol, Moscow, Russia). Primers for plasmid pJET1.2 from the CloneJET PCR Cloning Kit (Thermo Fisher, Waltham, MA, USA) were used in the reaction. The 20 μL of the reaction mixture contained SsoAdvanced Universal SYBR Green Supermix buffer (Bio-Rad, Hercules, CA, USA), 10 μM primers, and a suspension of E. coli bacterial cells taken from a colony of transformants. The following program was used for real-time PCR: 60 s at 50 °C, 40 cycles of 5 min at 95 °C, 30 s at 58 °C, 60 s 72 °C, and 18 s at 95 °C.
PCR products were separated on agarose gel in 1x TBE buffer and visualized using GelDoc Go (BioRad, Hercules, CA, USA).

Genomic DNA was extracted by the method of Dorokhov and Klocke [65 ]. The DNA stock solution was adjusted to a concentration of 100–150 ng/mL with nuclease-free sterile water as the working concentration for the polymerase chain reaction (PCR) and stored at −20 °C. PCRs were performed using a thermocycler (C1000, BioRad, Hercules, CA, USA) in 20 mL of a PCR mixture containing 100–150 ng of genomic DNA, 2 units of Taq DNA polymerase, 1X PCR buffer (10 mM Tris HCL), 2.5 mM of MgCl₂, 100 mM of each dNTP and 10 mM of each primer. The recommended PCR protocol was used in amplifications. PCR products were separated on 1.5 to 3% agarose gels (depending on gene product size) and visualized under UV light using the digital gel imaging system (GelDocGo, BioRad, Hercules, CA, USA).
All cultivars were evaluated with molecular markers (Table 3) linked to 11 known genes, Yr2, Yr5, Yr7, Yr9, Yr10, Yr15, Yr17, Yr18, Yr24, Yr25 and Yr60.