*4.3. DNA Extraction and QTL Analysis*

DNA was extracted from leaf tissues using the extraction method, as described in Causse et al. (1994) [46]. Genotyping was conducted in 58 F3 and 17 F4 lines using 11 polymorphic SSR markers between CR2002 and Hwaseong. PCR was conducted, as described in Shim et al. (2019) [47]. PCR products were separated on 3% metaphor agar stained with StaySafe Nucleic Acid Gel Stain (RBC, Taiwan) or 4% polyacrylamide denaturing gel stained with Silver Staining Kit (Bioneer, Korea), respectively.

QTLs were determined by single-marker analysis (SMA). SMA was conducted to establish the effect of each marker on each trait. In SMA, a QTL was declared when the phenotype was associated with SSR genotype at *p* < 0.05 by one-way analysis of variance (ANOVA). The observed phenotypic variation was estimated using the coefficient of determination (R2). For Tukey's test, Minitab19 software was used. Student's *t*-test was conducted using Microsoft Excel. Boxplots were drawn using R version 3.5.3.
