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