Identification of OsMTP1 as a possible element responsible for the sitl1 mutant
To identify the causal mutations in the sitl1 line, whole genome sequencing (WGS) was performed. A total of 116,584 SNPs and 13,196 Indels were identified as shared variants between WT (Oryza sativa , cv. Donganbyeo) and sitl1 based on rice reference genome (Oryza sativa , cv. Nipponbare) (Figure S7). In addition, a total of 435 SNPs and 195 Indels were not shared between sitl1 and WT genomes, indicating that these nonshared variants were caused by gamma-ray irradiation in the sitl1 (Figure 7a). To survey the effects of variants on the sitl1 genome, the variants were divided into the two groups, the genic region and the nongenic region according to their genome locations. Most variants of SNPs (87%) and Indels (78%) in the sitl1 were located in the nongenic region (Figure 7b). Fifty-seven out of 435 nonshared SNPs (13%) and 43 out of 195 Indels (22%) were located in the genic region (Figure 7b, left panel). Among them only 21 SNPs and 13 Indels were detected in the coding sequence (CDS) region of the genes (Figure 7b). The 21 SNPs located in the CDS regions included 12 missense, 8 synonymous, and 1 stop-gained variants (Figure 7b). The 13 Indels located in the CDS regions included 1 inframe insertion, 2 inframe deletion, 1 splice acceptor, 7 frameshift, and 2 stop-gained variants (Figure 7b).
To investigate the transcriptional changes driving the observed phenotypes due to the SNP and Indel variants in the sitl1 , Illumina-based RNA-seq was used to profile mRNA expression in root tissues. A set of 438 and 767 genes was identified whose mRNAs showed significantly increased or decreased transcript abundance respectively in the sitl1 (Figure S8 and Table S2-S3). The overrepresented gene functions of DEGs in the sitl1 were significantly associated with metal handling, stress, miscellaneous, protein, and transport functions (Figure S9). Analysis using metabolic pathways revealed that a large number of DEGs involved in a wide range of metabolisms including cell wall, light reactions, photosynthesis, and lipid metabolism were significantly downregulated in the sitl1 (Figure S10).
In an effort to evaluate whether the genes containing SNP and Indel variants in the sitl1 had altered gene expression levels, the mRNA expression profiles of 188 SNPs and 107 Indels variants were analyzed, which are commonly detected via WGS and RNA-seq. A total of 7 genes containing 4 SNP and 3 Indel variants showed significantly upregulated or downregulated expression levels in the sitl1compared to WT (Figure 7c,d). Of these, four SNP variants were located in the upstream of Os05g0227600, downstream of Os01g0195400 and Os09g036770, or the 3’ UTR region of Os08g0477100 (Figure 7c). In addition, two Indel variants were located in the upstream of Os08g0159500 and Os12g0226066 (Figure 7d). Lastly, one gene (Os01g0664100; MSU ID, Os04g47460, Mg2+ transporter, CorA-like ZnB domain) containing Indel variant in the CDS region has significantly downregulated mRNA abundance in the sitl1 (Figure 7d). This gene was selected and named (OsMTP1) as a putative causal gene for further functional analysis