*4.1. Plant Materials*

CR2002 was derived from a cross between *O. sativa* Korean elite line 'Hwaseong' as a recurrent parent and wild species *O. grandiglumis* (2n = 48, CCDD, Acc. No. 101154) [41]. Four *O. grandiglumis* chromosome segments were found in CR2002 on chromosomes 1, 2, 6, and 10. CR2002 was backcrossed with Hwaseong to produce an F2 population. A total of 705 F2 plants were genotyped using SSR markers from the introgression regions. Fifty-eight F3 plants with different combinations of *O. grandiglumis* homozygous segments were selected and advanced to the F3. Among the 58 F3 lines, 17 that have different recombination break-points within the *qCC2* region were selected and advanced to the F4 generation. QTL analysis was performed using two replications of the 58 F3 lines and 17 F4 lines, which were grown in the Chungnam National University paddy field during 2015 and 2016, respectively. For substitution mapping, four F4 recombinant lines in the *qCC2* region were investigated in 2017. To determine whether *OsGW2* is associated with leaf senescence, a *gw2*-knockout (*gw2-ko*) mutant (PFG\_1B-10017.R) in the *japonica* rice 'Dongjin' background was used for evaluating leaf senescence [42,43]. An F2 population (*n* = 107) derived from a cross between Dongjin and *gw2-ko* was generated and used for DIS assay to confirm whether the T-DNA insertion in *OsGW2* locus was responsible for delayed senescence.

#### *4.2. Phenotypic Evaluation*

A total of 10 traits, including chlorophyll content and three-grain morphology traits (Table 1), were evaluated in the parental lines (*n*= 10) using the methods, as described in Yoon et al. (2006) [41].

To evaluate chlorophyll content, three methods were used. First, fluorescence ratio F735/F700 was measured in F3 and F4 generation plants, and the relative chlorophyll content (mg/m2) was evaluated using CCM-300 (OPTI-SCIENCE, Hudson, NH, USA). This data was used for QTL analysis. Second, SPAD values were measured to compare parental lines (Hwaseong, CR2002, Dongjin, and *gw2-ko*) using a SPAD-502Plus (KONICA MINOLTA, Tokyo, Japan). Third, total chlorophyll was extracted from detached leaves and used for dark-induced senescence using pre-chilled 80% acetone [7]. The absorbance of supernatants was measured at 645 and 663 nm using a UV/VIS spectrophotometer (Hanson Tech., Seoul, Korea) [44]. For the dark-induced senescence (DIS) assay, fully expanded flag leaves were detached and placed in 3 mM MES buffer (pH 5.8) and incubated at 27 ◦C under complete darkness [45]. *Fv*/*Fm* ratio was measured using a Hansatech PEA MK2 (Hansatech, Norfolk, UK). The middle part of leaf samples from DIS was adapted in the dark for 30 min to complete the oxidation of QA, and then the *Fv*/*Fm* value was measured. A student's *t*-test was performed for trait comparisons.
