*3.3. Validation of the Developed SCAR Marker Using the Resistant and Susceptible Onion Lines*

The SCAR-OPAN1 marker was used to screen the resistant and susceptible lines. The 2-Kb-sized polymorphic band was amplified in the resistant lines S&P 7522, S&P 7521, S&P 7129, and S&P 7168 and was not amplified in the susceptible lines S&P 7483, S&P 7130, and S&P 7175 (Figure 3a). The developed SCAR-OPAN1 marker amplified the specific band only from the resistant lines, demonstrating its potential as a molecular marker for the selection of resistant lines. To validate the SCAR-OPAN1 marker, three resistant lines and four susceptible lines of 'Asia Seed' Co. were screened using the SCAR-OPAN1 marker (Figure 3b). Similar to the screening results of the 'Seeds & People' Co. onion lines, a product size of 2 Kb was amplified only in the resistant lines (Asia-12, Asia-42, and

Asia-53). These polymorphic bands between the resistant and susceptible lines allow the selection of gray mold-resistant onion lines for breeding programs.

**Figure 3.** Identification of resistant onion lines of 'Seeds & People' Co. and 'Asia Seed' Co. using the SCAR-OPAN1 marker. (**A**) The 2-Kb-sized polymorphic products were amplified in the resistant lines (S&P 7522, S&P 7521, S&P 7129, and S&P 7168) and were not amplified in the susceptible lines (S&P 7483, S&P 7130, and S&P 7175). M: 100-bp DNA ladder marker, Lane 1: S&P 7522, Lane 2: S&P 7521, Lane 3: S&P 7129, Lane 4: S&P 7168, Lane 5: S&P 7483, Lane 6: S&P 7130, and Lane 7: S&P 7175. (**B**) The 2-Kb products were amplified only in the resistant lines (Asia-12, Asia-42, and Asia-53) and were not amplified in the susceptible lines (Asia-30, Asia-35, Asia-45, and Asia-50). M: 100-bp DNA ladder marker, Lane 1: Asia-12, Lane 2: Asia-42, Lane 3: Asia-53, Lane 4: Asia-30, Lane 5: Asia-35, Lane 6: Asia-45, and Lane 7: Asia-50.
