*3.2. Development of a SCAR Marker for the Selection of Resistant Lines*

From the RAPD results, a SCAR marker was developed using a specific RAPD product showing polymorphisms between the resistant and susceptible lines (Figure S2). The 2-Kb bands were amplified in the resistant lines S&P 7522, S&P 7521, S&P 7129, and S&P 7168 by using the OPAN-1 primer. The amplified polymorphic bands were eluted, purified, and sequenced. From the sequencing results, 5 -ACT CCA CGT C-3 , which is a sequence of random primer OPAN-1, was identified at both ends of the fragment sequence (Figure 2). In addition, the sequences were analyzed using BLAST at the National Center for Biotechnology Information (NCBI), and insignificant results were obtained. Therefore, further studies are required to analyze this unknown DNA sequence. The SCAR marker primer was designed to include the 5 -ACT CCA CGT C-3 sequence of the OPAN-1 random primer. The forward primer was 5 -ACT CCA CGT CAT CGA TTC GAA-3 , and the reverse primer was 5 -ACT CCA CGT CCG AAC TAC AGA A-3 . The primer set for the SCAR marker was designed for considering the annealing temperature, GC content, the possibility of dimer formation, and hairpin loops. The developed SCAR marker was designated as SCAR-OPAN1.


**Figure 2.** The SCAR-OPAN-1 primer and sequence of a 2-Kb product amplified in four resistant lines by an OPAN-1 random primer. The SCAR-OPAN-1 primer contained the OPAN-1 random primer sequence. The sequence of the designed SCAR-OPAN-1 primer is shown in bold. The underlined sequence is the sequence of OPAN-1 random primer, 5 -ACT CCA CGT C-3 .
