*2.1. QTL Analysis for Chlorophyll Content*

CR2002 exhibited a stay-green phenotype and a higher SPAD value (a parameter of leaf greenness) than Hwaseong (Figure 1b). CR2002, which had four *O. grandiglumis* introgressions, also showed differences in several agronomic traits, including grain weight (Table 1, Figure 1a, and Figure S1). To identify loci associated with chlorophyll content and the stay-green phenotype, QTL analysis was conducted using F3 and F4 populations derived from a cross between Hwaseong and CR2002. Chlorophyll contents were measured at the heading stage and one month after heading. A total of three significant QTLs located on chromosomes 1, 2, and 6 were detected in the F3 and F4 populations (Table 2 and Figure S2). A QTL for chlorophyll content (*qCC2*) was located on chromosome 2 between RM12813 and RM12983. This QTL was repeatedly detected in F3 and F4 generations at both stages, heading (Chlorophyll I) and one month after heading (Chlorophyll II). *qCC2* explained 24.6% of the phenotypic variation, and the *O. grandiglumis* allele contributed to the increased chlorophyll content, indicating that *qCC2* was a major QTL responsible for leaf greenness at heading and ripening stage.

**Figure 1.** CR2002 showed a higher SPAD value and delayed senescence than Hwaseong in the field condition. Comparison of (**a**) plant morphology and (**b**) SPAD value (*n* = 30) of two parents. Error bars indicate the standard error. DAT: days after transplanting.



\* Data are presented as mean ± standard deviation. The student's *t*-test was conducted for *p*-value. Chlorophyll content I and II were measured at the heading stage and 1 month after heading, respectively.


**Table 2.** QTLs (quantitative trait loci) for chlorophyll content based on one-way ANOVA in the F3 and F4.

<sup>1</sup> Chlorophyll content I and II were measured at heading stage and heading after 1 month, respectively. <sup>2</sup> H/H and G/G indicate the mean chlorophyll contents of homozygous for Hwaseong and *O. grandiglumis* genotypes, respectively.

The *qCC2* region was confirmed and fine-mapped by substitution mapping (Figure S3). SPAD values were measured four times, from 95 to 125 days after transplanting (DAT), for four substitution lines and two parental lines. CR7036 and CR7039 showed significantly higher SPAD values at 125 DAT than CR7038 and CR7058, suggesting that *qCC2* was located in the marker interval between RM3390 and RM7288 (about 1.4 Mbp).
