*3.4. Validation of Di*ff*erential Gene Expression*

RNA-Seq results were validated using ten genes from di fferent response categories (increased, decreased, and non-di fferentially expressed genes upon treatment in both flag-leaf and panicle) for qRT-PCR (Figure S17A,B). Three selected genes from within the *qDTY1.1* region (LOC\_Os01g66120, LOC\_Os01g66820, and LOC\_Os01g67030) showed di fferential expression. LOC\_Os01g66120 (no apical meristem protein) was upregulated in both genotypes and both tissues under drought. LOC\_Os01g66820 (inactive receptor kinase At1g27190 precursor) was downregulated in DTY-IL flag-leaves under RDS. LOC\_Os01g67030 (auxin responsive protein) was upregulated under RDS in DTY-IL panicles and downregulated in Swarna panicles but not a ffected by RDS in flag-leaves. A high correlation between qRT-PCR results and RNA-Seq results was observed for flag-leaf ( *R*<sup>2</sup> = 0.88) and panicle ( *R*<sup>2</sup> = 0.91) tissues (Figure S17C,D), supporting RNA-Seq-based findings and interpretations. Targeted transcripts and respective primer sets used are shown in Table S14.

### *3.5. Colocalization of DEGs in the Introgression Fragments*

SNP genotyping revealed 16 N22-derived fragments in DTY-IL (Figure 10). The largest fragment was found on chromosome 1, encompassing *qDTY*1.1, followed by an introgression on chromosome 3, containing parts of *qDTY*3.2, which was also reported as N22-derived in an N22 by Swarna population [7]. Additional introgressions on chromosomes 4, 8, 9, and 10 did not overlap with major DTY QTL. Overlaying DEGs on the N22 introgressions identified 463 DEGs in the flag-leaf (Table S15-1) and 433 DEGs in the panicle (Table S15-2), of which 6 overlapped with the fine mapped region of *qDTY*1.1 [59], while 5 overlapped with the *qDTY*3.2 region.

**Figure 10.** Physical map of DTY-IL. Genome-wide physical position distribution of 1648 SNPs from the 7K genotyping assay across all rice chromosomes. Swarna SNP's alleles are represented in blue. N22 SNP alleles are represented in yellow. Names and ranges of N22-derived DTY QTLs (*qDTY*1.1 and *qDTY*3.2) are shown as red bars on the sides of the chromosome, more details are provided in Supplementary Table S15.

Of the 6 DEGs in the *qDTY*1.1 region (Table S16-2), LOC\_Os01g67030 was upregulated in the panicle (Log2 fold change = 3.1), was annotated as an auxin-responsive protein of 418 amino acids (AA) in Nipponbare. While LOC\_Os01g67030 was annotated in the *indica* reference MH63v2, it was missing in the N22v2 reference genome. FGENESH-based gene prediction in N22v2 revealed a putative homologue showing 91.4% identity with Nipponbare and 92.3% identity with MH63v2. Differences were found in the 5UTR, resulting in the loss of coding sequence for the first 37 AA in the MH63v2 and N22v2 alleles, as well as a number of nonsynonymous (NS) SNPs (Figure S18). NS SNPs unique to N22 corresponded to four AA changes (P52L, C60F, S80T, Q137P) and an AA deletion (G253del) (Figure S19). While the alignment of the 2 kb upstream *cis*-regulatory region of LOC\_Os01g67030 in Nipponbare and MH63v2 showed a 99.1% identity, N22v2 displayed a large deletion, including the 5UTR. With an identity of less than 15% to either Nipponbare or MH63v2, the N22v2 *cis*-regulatory region of LOC\_Os01g67030 was distinct with unique regulatory elements (Table S17) that could explain the observed expression differences.

Of the 5 DEG within *qDTY*3.2, LOC\_Os03g03510, downregulated in the panicle (Log2 fold change = −1.11617), was annotated as CAMK\_KIN1/SNF1/Nim1\_like.15-calcium/calmodulin-dependent protein kinase in Nipponbare, with corresponding annotations in MH63v2 and predictions in N22v2. LOC\_Os03g03510 contains a sucrose non-fermenting 1-related kinase 3 (SnRK3) domain and a CBL-interacting serine/threonine-protein kinase 3 (CIPK3) domain. While the coding sequences were largely conserved in multiple sequence alignment between Nipponbare, MH63v2, and N22v2 alleles, the N22v2 allele featured an altered stop codon resulting in a 35 AA C-terminal extension (Figure S20).
