*4.5. RNA-Seq Analysis*

The 30-day-old Hwaseong and CR2002 plants were dark-treated to induce senescence for three days. From plants each before (day 0) and 3 days after dark-treatment (day 3), the 5th leaves were collected with three replicates, and total RNA was extracted, as described above. A total of 12 transcriptome libraries were constructed using TruSeq RNA Sample Prep Kit v2 (Illumina, San Diego, CA, USA), according to the manufacturer's instructions. The library construction was conducted by GnC Bio Inc. (Daejeon, Korea). The constructed libraries were sequenced on a HiSeq 4000 instrument with 150 bp of paired-end reads. The sequence data generated from this study have been deposited at the National Center for Biotechnology Information Sequence Read Archive (SRA) under accession number PRJNA602373. Sequencing results were checked using FastQC (version 0.11.7). Raw sequences were processed to obtain clean reads by removing adapters and low-quality reads, using SCYTHE (https://github.com/vsbuffalo/scythe) and SICKLE (https://github.com/najoshi/sickle), respectively. High-quality reads were mapped on rice reference genome IRGSP-1.0, and transcript abundances were quantified using Salmon version 0.8.2 [49]. Differentially expressed genes (DEGs) were determined with more than 2-fold change with significance at Benjamini-Hochberg false discovery rate adjusted *p* < 0.05 using Limma-Voom package [50,51]. GO enrichment was carried out using agriGO v2.0 singular enrichment analysis [52].

#### **5. Conclusions**

A mapping population derived from an interspecific cross between *O. sativa* and *O. grandiglumis* facilitated the discovery of *qCC2*, a major QTL for chlorophyll content. The *O. grandiglumis* allele at *qCC2* increased chlorophyll content and delayed senescence. Building on several previous studies, *GW2*, which encodes E3 ubiquitin ligase, was selected as the most probable candidate gene for this phenotype, and this was confirmed through genetic analysis of a knockout mutant. The *gw2-ko* mutant showed delayed leaf senescence under dark conditions with down-regulation of SAGs and CDGs, and the delayed senescence trait completely segregated with the *gw2-ko* gene. On the basis of this work, the RING-type ubiquitin ligase *GW2* appeared to function as a positive regulator of leaf senescence in rice. This study also represented the first investigation of global gene expression in rice leaves subjected

to complete darkness. The results of this whole-genome transcriptome analysis of Hwaseong and CR2002 highlighted the processes and genes that are likely to play a role in the mechanisms underlying leaf senescence in rice (e.g., carbohydrate metabolism, pyrophosphate-dependent energy conservation, and ethylene signaling). The QTL analysis and the genotype-specific regulation suggested that senescence was regulated at the transcriptional level, although the possibility of post-translational regulation by GW2 requires further investigation.

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/5/1704/ s1.

**Author Contributions:** K.-C.S. and S.H.K. performed experiments and analyzed data. K.-C.S. and S.-N.A. conceived the study, designed, and supervised the study. Y.-A.J., H.-S.L., J.-W.K., and C.A. investigated the traits. H.-J.K. and T.H.T. assisted in analyzing the RNA-seq data. K.-C.S., T.H.T., and S.-N.A. wrote and edited the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was carried out with the support of "Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01321401)", Rural Development Administration and the Basic Science Research Program through the National Research Foundation (NRF) of Korea funded by the Ministry of Education (NRF-2017R1A2B2007554).

**Acknowledgments:** The *gw2*-knockout (*gw2-ko*) mutant seeds were provided by G. An, Kyung Hee University. We thank Cheol Seong Jang and Ju Hee Kim, Kangwon National University, for their critical comments on this work and Young-Sook Kim for her technical assistance.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
