Supplementary information
Divergent DNA methylation patterns associated with gene
expression in rice cultivars with contrasting drought and salinity
stress response
Rohini Garg1*, VVS Narayana Chevala1, Rama Shankar1 & Mukesh Jain1*
1
Functional and Applied Genomics Laboratory, National Institute of Plant Genome Research
(NIPGR), Aruna Asaf Ali Marg, New Delhi - 110067, India
Supplementary Table S1 Details of bisulphite sequencing libraries generated in the study.
IR64
N22
Pokkali
Total read pairs
82237670
79798554
106014605
High-quality read pairs
82237654
79798533
106014601
Uniquely mapped read pairs
52632006
49374576
65592598
Mapping efficiency (%)
64.00
61.87
61.87
Genome coverage (%)
87.24
89.08
88.28
Genome Cs
163078985
163078985
163078985
Cs covered
131729131
136104482
134552064
C coverage
Chloroplast DNA
conversion rate (%)
Error rate
80.78%
83.46%
82.51%
99.55007
99.5742318
99.4819746
0.0045
0.01
0.00755
Total methylated Cs
14906181
18210399
16819406
11.32%
12.30%
11.50%
Total methylated C's (%)
Only the reads mapped uniquely to the rice nuclear genome were included in the analysis to identify the
methylated cytosines.
IR64, N22 and PK represent IR64, Nagina 22 and Pokkali rice cultivars, respectively.
Supplementary Table S2 Summary of sequencing data generated for RNA-seq and mapping
on the rice genome.
IR64
N22
PK
Raw reads
58565758
96289178
62150476
High quality reads
54230024
81129568
54962644
Total mapped reads
50117149
73756916
50848127
IR64, N22 and PK represent IR64, Nagina 22 and Pokkali rice cultivars, respectively.
Supplementary Table S3 List of differentially expressed genes between the rice cultivars.
Available as separate MS Excel file
Supplementary Table S4 List of genes with methylation level correlated with differential
gene expression between the rice cultivars.
Available as separate MS Excel file
Supplementary Table S5 Summary of small RNA sequencing data generated and preprocessing.
Total raw reads
Unique reads after QC
Reads uniquely mapped to rice
genome
IR64
N22
Pokkali
25959467
16051585
37240914
1927142
1376538
2088005
1536225
1090668
1699184
(79.72%)
(79.23%)
(81.38%)
885816
(45.97%)
653002
(47.44%)
Small RNA reads (21-24nt)
IR64, N22 and PK represent IR64, Nagina 22 and Pokkali rice cultivars, respectively.
962624
(46.10%)
Supplementary Table S6 List of primers used for real-time PCR analysis in this study.
Gene ID
Primer sequence
LOC_Os09g07510 CAGAGATGCTGCTGCAAACAA
ATCGCCCATTCTGGACCAA
LOC_Os11g31640 TCCAGGGAGTGCCTTAGACAA
AGGTGTGGCAGGGAATGC
LOC_Os02g48560 CATCCCGCCTCACTGCTT
CATGGACCACGTAGGAGAAGGA
LOC_Os03g22720 TTCTTGGAGGCGTTGTCATG
AGGTTTTCACCTGGAGATTTGTG
LOC_Os10g39130 CAACAAGATATCCAGCAAGTCAAAG
TGGACTCATCAAGAGCTTCAAGTTT
LOC_Os01g14610 TCGTGGCGGAGGATGAGA
GGCCTCCATGCGAATGTT
LOC_Os02g42810 GCCGAGAAAAGGGTTGCA
TGCATATCTCGAGCCCAAGTC
LOC_Os11g20160 TGGTGACGCGTTCCAGTGTA
GCAGAACAAGCTTGAGCATGACT
LOC_Os08g01794 AGGGAATGATCGACACAAAAATG
TTTGCGGCGTGTAACTTGAC
LOC_Os03g48970 GAGAAAGCCGAACCAAACAAAG
GGAGGGATCCGGCGATAC
LOC_Os11g01154 CTCAGGATGCTCTGCGACTTC
TAGCGCCATTCTGGACAAGA
LOC_Os06g43860 GGCTCACTCTAAATGGCCATACC
AACCCTGTTTCCTGCACCAA
LOC_Os04g16722 GGAGCACCTAACAACGCATCTT
CCCCAGAATGAAAGGGTGATC
LOC_Os01g72530 GGCTCCGACCAGGACATC
TGAGATCGAACCTGCAGATCA
LOC_Os01g72370 CCTACTACTACTGGTCTGGCTTGGT
CCCGCTTCCGTCTCAAAAG
LOC_Os01g53220 TCCTCCTCCCCTCCTACTTCA
GCGGAATCCGTAGGTGTTGA
Supplementary Figure S1 DNA methylation level in each sequence context (CG, CHG and
CHH) in three rice cultivars. The fraction of mCs with different levels of methylation are
shown in the line graph. The average value of methylation level in each sequence context is
also shown.
Fraction of total methylcytosines (%)
70
60
70
CG
CHG
60
CG
70
CHG
60
CHH
CHH
50
50
50
40
40
40
30
30
40.7
66.5
66.6
67.6
10
0
0
87.9
20
10
10
CHH
42.6
87.9
20
20
CHG
30
41.0
86.7
CG
0
10 20 30 40 50 60 70 80 90 100
10 20 30 40 50 60 70 80 90 100
10 20 30 40 50 60 70 80 90 100
Methylation level of cytosine (%)
Methylation level of cytosine (%)
Methylation level of cytosine (%)
IR64
N22
Pokkali
Supplementary Figure S2 Global methylome maps of rice in different rice cultivars.
Density plots of methylcytosines in different sequence contexts (I, CG; II, CHG and III,
CHH), genes (IV), transposable elements (V) and small RNAs (VI) are shown on both DNA
strands (outside, sense strand and inside, antisense strand). Chromosome name and scale are
indicated on the outer rim.
I
II
III
IV
V
VI
IR64
I
II
III
IV
V
VI
N22
I
II
III
IV
V
VI
Pokkali
Supplementary Figure S3 Box-plot showing distribution of DNA methylation level on both
DNA strands in each sequence context in three rice cultivars.
Sense-strand
Antisense-strand
100
Methylation level (%)
80
60
40
20
0
CG
CHG
IR64
CHH
CG
CHG
N22
CHH
CG
CHG
Pokkali
CHH
Supplementary Figure S4 Fraction of mCs identified in different genomic features in each
sequence context in three rice cultivars.
Fraction of total methylcytosines (%)
25
20
15
10
5
0
20
15
10
5
0
20
15
10
5
0
CG
Promoter
Gene body
5' -UTR
Exon
Intron
CDS
3' -UTR
Downstream
CHG
CHH
IR64
N22
Pokkali
Supplementary Figure S5 DNA methylation patterns in the gene body and flanking
sequences in different rice cultivars. Average methylation level of mCs within gene body and
flanking (upstream and downstream) sequences in each sequence context in different
cultivars. TSS, transcriptional start site and TTS, transcriptional termination site.
1.00
0.90
Methylation level (%)
0.80
0.70
0.60
0.50
0.40
0.30
0
Gene body
Upstream
TSS
IR64 CG
IR64 CHG
IR64 CHH
Downstream
TTS
N22 CG
N22 CHG
N22 CHH
Pokkali CG
Pokkali CHG
Pokkali CHH
Supplementary Figure S6 Fraction of differentially methylated regions (DMRs) identified
within the gene body or flanking (2 kb upstream and downstream) sequences among rice
cultivars.
Gene body
Flanking
70
Number of DMRs (%)
60
50
40
30
20
10
0
Hyper
Hypo
N22 vs IR64
Hyper
Hypo
Pokkali vs IR64
0
post-embryonic development
reproduction
multicellular organismal development
PK/IR64 Hyper
regulation of gene expression, epigenetic
protein modification process
translation
biosynthetic process
transport
N22/IR64 Hypo
signal transduction
cellular homeostasis
response to biotic stimulus
response to abiotic stimulus
response to stress
N22/IR64 Hyper
DNA metabolic process
secondary metabolic process
carbohydrate metabolic process
protein metabolic process
lipid metabolic process
0.35
nucleotide and nucleic acid metabolic process
metabolic process
Fraction of DMR-associated genes
Supplementary Figure S7 Gene ontology (GO) categories represented in the DMRassociated genes for N22/IR64 and PK/IR64.
PK/IR64 Hypo
0.3
0.25
0.2
0.15
0.1
0.05
Supplementary Figure S8 DMRs associated with on-off gene expression state. The scatter
plots show correlation of DMRs in N22/IR64 (a) and PK/IR64 (b) with switch on-off
expression state of nearby genes (within 2 kb). The plots show that hypermethylation leads to
switch-off state of gene expression and hypomethylation leads to switch-on state of gene
expression. (c) GO enrichment analysis of DMR-associated genes showing on-off gene
expression state. The significantly enriched GO terms are highlighted in different colours as
per scale (P-value) given at the bottom.
Supplementary Figure S9 Validation of DNA methylation results. Scatter plot showing
correlation of methylation levels predicted by whole genome bisulphite sequencing (WGBS)
and locus-specific bisulphite sequencing (LSBS) in IR64 (a), Nagina 22 (b) and Pokkali (c)
rice cultivars. The Pearson correlation (r) obtained for each sequence context has been
indicated.
(a)
IR64
100
Methylation level (LSBS)
100
100
r = 0.83
r= 0.77
CG
CHG
r = 0.71
80
80
80
60
60
60
40
40
40
20
20
20
0
0
0
0
20
40
60
80
Methylation level (WGBS)
100
CHH
0
20
40
60
80
Methylation level (WGBS)
0
100
20
40
60
80
Methylation level (WGBS)
100
(b)
N22
100
Methylation level (LSBS)
r = 0.78
r = 0.73
r = 0.71
CHG
80
80
80
60
60
60
40
40
40
20
20
20
0
0
(c)
40
60
80
Methylation level (WGBS)
100
CHH
0
0
20
0
20
40
60
80
Methylation level (WGBS)
100
0
20
40
60
80
Methylation level (WGBS)
100
Pokkali
100
100
r = 0.79
Methylation level (LSBS)
100
100
CG
r = 0.74
CG
100
CHG
80
80
80
60
60
60
40
40
40
20
20
20
0
0
0
20
40
60
80
Methylation level (WGBS)
100
r = 0.70
CHH
0
0
20
40
60
80
Methylation level (WGBS)
100
0
20
40
60
80
Methylation level (WGBS)
100
Supplementary Figure S10 Validation of differential gene expression results. Scatter plot
showing correlation of transcript levels (log2 fold change) predicted by RNA-seq and qRTPCR. The Pearson correlation (r) has been indicated.