*2.3. Development of the SCAR Marker*

To develop the SCAR marker, the 2-Kb polymorphic bands obtained between four resistant and three susceptible lines by using the OPAN-1 primer (5 -ACT CCA CGT C-3 ) were sequenced and identified. The amplified polymorphic products were eluted from a 1% agarose gel and purified using the NucleoSpin® Gel and PCR Clean-up Kit (Macherey-Nagel, Duren, Germany). The eluted products were ligated into the pGEM-T easy vector (Promega, Madison, WI, USA) for sequencing. The plasmid was purified using the Fast DNA-spinTM Plasmid DNA Purification Kit (iNtRON Biotechnology, Seongnam, Korea), and the fragments were sequenced by Macrogen® (Seoul, Korea). The SCAR primer set was designed from the sequence of the 2-Kb polymorphic band between the resistant and susceptible lines. PCR using the SCAR primer set was performed under the following conditions: initial denaturation for 10 min at 95 ◦C, 35 cycles of 30 s at 95 ◦C, 30 s at 61 ◦C, 1 min at 72 ◦C, and a final extension for 10 min at 72 ◦C. Thereafter, the amplified products of the resistant onion lines were confirmed by electrophoresis in a 1% agarose gel. In addition, to confirm the versatility of the SCAR marker, three gray mold-resistant and four susceptible onion lines from the 'Asia Seed' Co. were also analyzed.
