*3.1. qCC2 is Associated with Chlorophyll Content and Leaf Senescence*

Chlorophyll content, one of the most important traits due to its role in photosynthesis, is regulated by polygenic loci. Several studies have identified a number of QTLs across various genetic resources and environments [3–6]. A number of studies reported QTLs on the short arm of chromosome 2. Four QTLs have been mapped for stay-green traits on chromosome 2 between RM145 and RM322 using 190 doubled haploid lines from a cross between 'Zhenshan 97 and 'Wuyujing 2- [2]. Jiang et al. (2012) reported QTL for chlorophyll content from heading to the maturity stage (*qCHM2*) located on the short arm of chromosome 2 between RM145 and RM29 [5]. In this study, three QTLs for chlorophyll content were detected using progeny from an interspecific cross between *O. sativa* and *O. grandiglumis*. *qCC2* was located on chromosome 2 between RM3390 and RM7288, which were approximately 1.4 Mbp apart. This QTL was detected in F3 and F4 generations and at two different developmental stages, respectively, and confirmed by substitution mapping (Figure S3). *qCC2* shared a similar location with QTLs reported in previous studies [2,5]. Although allelism tests are needed to clarify the relationships among these QTLs, these studies indicated the presence of genes associated with chlorophyll content and leaf senescence on the short arm region of chromosome 2.

When Hwaseong and CR2002 were tested for dark-induced senescence, CR2002 showed higher chlorophyll content with delayed leaf senescence than Hwaseong (Figure 2a,b). *Fv*/*Fm* values were also significantly different at 4 days after incubation (Figure 2c). Endogenous expression levels of SAGs and CDGs were severely down-regulated in CR2002 than Hwaseong, and these results indicated that *qCC2* was associated with leaf senescence in CR2002.

#### *3.2. GW2 Positively Regulates Leaf Senescence*

*GW2* encodes a RING-type E3 ubiquitin ligase and resides in the *qCC2* region (1.4 Mbp). The *O. grandiglumis GW2* allele in CR2002 has the 1-bp deletion in 4th exon, which causes a premature stop and truncated protein (Figure S4) [21]. Many studies have suggested ubiquitination as a candidate pathway for the regulation of senescence. *SAUL1* encodes E3 ubiquitin ligase, which is required for suppression of premature senescence in *Arabidopsis* [26]. Also, *spl11* encodes E3 ubiquitin ligase, which regulates cell death in rice [27]. E3 ubiquitin ligase *Big Brother* (*BB*) gene and ubiquitin receptor *DA1* gene negatively regulate leaf size and promote senescence [19]. Mutations in *DA1* and *BB* enhance leaf growth, an effect that is synergistically increased in the double mutant. A *da1-1*/*eod1-2* double mutant especially exhibits a longer lifespan than wild type or the single mutants [19]. In addition, DA1 physically interacts with DA2, an ortholog of *GW2*, and *DA2* acts synergistically with *DA1* to regulate seed size in *Arabidopsis* [18]. Based on these previous results, *GW2* was chosen as a candidate gene for the delayed senescence phenotype in CR2002. The transcript level of *GW2* was down-regulated at 2 DAI and showed a similar expression level at 0 DAI and at 4 and 6 DAI in the dark (Figure 4e and Figure S5). This expression pattern was similar to the *NYC4* gene, which functions in the degradation of chlorophyll and chlorophyll-protein complexes during DIS [28]. The effect of *gw2-ko* was examined in DIS (Figure 5c). *gw2-ko* showed delayed senescence and down-regulated expression of SAGs and CDGs. Compared to Hwaseong and Dongjin, most of the SAGs and CDGs genes were consistently down-regulated in CR2002 and *gw2-ko* during DIS. However, transcript levels of *RCCR1* (*red chlorophyll catabolite reductase 1*), *PAO* (*Pheophorbide a oxygenase*), and *OsI57* (*putative 3-ketoacyl-CoA thiolase*) did not show significant differences between Dongjin and *gw2-ko* or Hwaseong and CR2002 in DIS. These results suggested that *RCCR1* and *PAO* might function down-stream in the chlorophyll degradation pathway [8]. Because the transcript levels of *RCCR1* and *PAO* in *gw2-ko* were not significantly different from wild-type Dongjin, *GW2* might affect genes up-stream in the chlorophyll degradation pathway.
