www.sciencemag.org/cgi/content/full/337/6100/1336/DC1

Supplementary Material for

A Killer-Protector System Regulates both Hybrid Sterility and Segregation Distortion in Rice

Jiangyi Yang, Xiaobo Zhao, Ke Cheng, Hongyi Du, Yidan Ouyang, Jiongjiong Chen, Shuqing Qiu, Jianyan Huang, Yunhe Jiang, Liwen Jiang, Jihua Ding, Jia Wang,

Caiguo Xu, Xianghua Li, Qifa Zhang*

*To whom correspondence should be addressed. E-mail: qifazh@mail.hzau.edu.cn

Published 14 September 2012, Science 337, 1336 (2012)

DOI: 10.1126/science.1223702

This PDF file includes:

Materials and Methods

Figs. S1 to S10

Tables S1 to S7

References (33–41)

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Materials and methods

Rice materials

The genotypes of each NIL at S5 locus were determined using molecular markers

published previously (33) and ones developed in this study (table S5). The wild and

cultivated rice species used for haplotype survey were from collection maintained in our

own lab or provided by the International Rice Research Institute (Los Banos, the

Philippines), including 43 accessions of O. sativa, 16 accessions of O. rufipogon, 23

accessions of O. nivara, and one accession of O. glumaepatula (table S3).

Genetic transformation

The transgenic plants of BalillaORF5+ were prepared by Chen et al (11). For the ORF3

construct, the DNA fragment was isolated from the genomic DNA of Nanjing 11 by PCR

amplification, using primers SC1F and SC4R (table S6). The fragment was ligated into

the vector pCAMBIA1301 and transferred into Balilla by Agrobacterium-mediated

transformation. The genomic DNA fragment of ORF4 was cut from the Nipponbare BAC

clone a0014N19 (www.genome.arizona.edu/fpc/rice/) using enzyme Xba I and Kpn I

(TaKaRa, Japan). Transformation was conducted using the NIL of BL(NJ/NJ) as the

recipient. The genotypes of each transgenic plant and their progenies were examined by

GUS staining and PCR amplification using gene-specific primers (table S5).

Plant growth and fertility examination

NILs, transgenic plants and progenies from various crosses were grown in the

experimental field in normal rice growing seasons in Wuhan, China. Five panicles per

plant were harvested for examining spikelet fertility scored as the ratio of the number of

filled grains to the total spikelets. Rice seeds for haplotype survey were treated at 50-55ºC

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for five days and 4ºC for three days to break dormancy. The seeds were then germinated

in the 1/2 Murashige-Skoog medium to obtain seedlings.

Full-length cDNA cloning

Total RNA was extracted from leaves and developing young panicles using TRIzol

reagent (Invitrogen, CA, USA), and was subsequently reverse transcribed in a 20 μl

reaction by using DNase I and SuperScript III (Invitrogen, CA, USA) according to the

manufacturer’s instruction. The coding sequences of ORF3 from Balilla and Nanjing 11

were amplified by RT-PCR. The full-length cDNAs of ORF4 were obtained using the

SMART RACE cDNA Amplification Kit (Clontech, CA, USA) and RT-PCR

amplification. Primers were designed according to the sequences available in the public

databases (table S6).

Subcellular localization of proteins

To prepare the constructs for assaying subcellular localization, the full-length CDSs of

ORF3-, ORF3+, ORF4- and ORF4+ were amplified and inserted into pM999-GFP for

fusion with the reporter gene. Rice protoplast isolation, washing and transfection were

proceeded as described (34). Each time, 2 μg plasmid DNA was transfected. After

incubation at 26ºC for 12-16 h, protoplasts were observed for fluorescent signals by

confocal microscopy. Plasmids encoding the Golgi marker Man1-RFP (35) and the ER

marker RFP-HDEL (36) were as described previously.

DNA sequencing and sequence analysis

DNA extraction and sequencing followed Du et al. (32). The genomic DNA fragments of

ORF3 and ORF4 covering 2.9 kb and 2.3 kb, respectively, were amplified using total

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DNA as the template. For accessions showing double peaks at polymorphic sites (likely

heterozygous) in the chromatogram, PCR products were cloned using the pGEM-T Vector

System (Promega, WI, USA) to obtain multiple clones carrying different haplotypes. At

least ten clones per accession were sequenced, and the haplotype with the highest

frequency was taken as the genotype of the accession. The sequence data were assembled

and aligned using the Datacollection V3.6 software. Sequences were inspected using

Sequencher V4.5 program and manually edited using ContigExpress program in

Invitrogen Vector NTI advance V.10 package. Sequences from different accessions were

aligned using Clustal X version 1.83 (37) and adjusted manually with GeneDoc 2.7 (38).

Microarray analysis

Two biological replicated RNA samples used for microarray analysis were prepared from

ovaries at functional megaspore stage of BalillaORF5+ transgene-positive and negative

plants grown under normal field conditions. RNA isolation, purification and Affymetrix

microarray hybridization were carried out by the CapitalBio Corporation

(http://www.capitalbio.com, Beijing, China). R platform (http://www.R-project.org) and

Affy packages from bioconductor (http://www.bioconductor.org) were used to manage all

the CEL files. The Robust multiarray average (RMA) algorithm was employed for

background correction, normalization, gene expression calculation and log2

transformation of the probesets. The P-value for the differential expression gene between

two samples was obtained using the SAM (significance analysis of microarray) function

in the siggenes package from bioconductor.

Quantitative real-time PCR (qPCR) analysis

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qPCR reactions were performed using the RNA samples prepared from ovaries of

BalillaORF5+ transgene-positive, -negative and BalillaORF3+ORF5+ transgenic plants

at mature embryo-sac stage. The sequences of primers used for qPCR are listed in table

S7. TRIzol reagent (Invitrogen, CA, USA) was used to extract total RNA from the

samples. First-stand cDNA was synthesized from 5 μg total RNA using Superscript III

reverse transcriptase (Invitrogen, CA, USA) according to the manufacturer’s instructions.

qPCR was performed in a 25 μl reaction volume with 2 μl of first-strand cDNA, 12.5 μl

2×SYBR® Premix Ex Taq™ II (Perfect Real Time) (TaKaRa, Japan), 0.5 μl 50× ROX

Reference Dye II and 0.5 μl of each primer. The reactions were carried out using the

Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, CA, USA) with

the following profile: 95ºC for 30 s; 45 cycles of 95ºC for 5 s, 60ºC for 34 s. Rice profilin

(LOC_Os06g05880) (21) was used as the internal control. The relative expression levels

were analyzed using the 2−ΔΔCT method (39).

Detection of spliced OsbZIP50 mRNA

The first-stand cDNA synthesized for qPCR analysis was used for detection of the spliced

OsbZIP50 mRNA, with specific primers OsbZIP50-S (table S7) around the splice site.

PCR conditions were set as follows: 94ºC for 4 min; 38 cycles of 98ºC for 10 s, 55ºC for

30 s and 72ºC for 30 s (40). The PCR products were detected by electrophoresis using 4%

agarose.

TUNEL assays

Pistils at different developmental stages were fixed in a solution containing 50% ethanol,

5% glacial acetic acids, and 3.7% formaldehyde for 24 h at 4ºC. Vertical paraffin sections

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were used for in situ nick-end labeling of nuclear DNA fragmentation with a TUNEL

apoptosis detection kit (DeadEnd Fluorometric TUNEL system, Promega, WI, USA),

using procedures as described (41). The green fluorescence of fluorescein and red

fluorescence of propidium iodide were analyzed at 520±10 nm and 640±10 nm

respectively under a confocal microscope.

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Supplementary figures

Fig. S1. Sequence features of the transcripts of the three ORFs from Nanjing 11 (indica), Balilla (japonica), Dular (wide compatible variety, WCV) and 02428 (WCV). The UPRE-like sequence in the promoter of ORF3 was identical in all the varieties.

8

Ba MRAMAMAMARARAAASWLLLLVLLHMRIGASSSQLECKLEYTSLALLSCQETTPTTPTPS 60

02 MRAMAMAMVRARAAASWLLLLVLLHMRIGASSSQLECKLEYTSLALLSCQETTPTTPTPS 60

Du MRAMAMAMARARAAASWLLLLVLLHMRIGASSSQLECKLEYTSLALLSCQETTPTTPTPS 60

NJ MRAMAMAMARARAAASWLLLLVLLHMRIGASSSQLECKLEYTSLALLSCQETTPTTPTPS 60

********.*************************************************** Ba CCDALLYSLDIWPVNQREKGLCCLCVYVLARQPSFDLATTYITCRGSYAASVAQWTQQLI 120

02 CCDALLYSLDIWPVNQREKGLCCLCVYVLARQPSFDLATTYITCRGSYAASVAQWTQQLI 120

Du CCDALLYSLDIWPVNQREKGLCCLCVYVLARQPSFDLATTYITCRGFGRTTDATAYSRCS 120

NJ CCDALLYSLDIWPVNQREKGLCCLCVYVLARQPSFDLATTYITCRGFGRTMDATAYSRCS 120

********************************************** : * .: Ba RGVPPHDCNEPCGVDTGDHPPPLPSGKKNKTRRKKQKQQLGVGVIIAIVVCSLAAAGLLG 180

02 RGVPPHDCYEPCSVDTGDHPPPLPSGKKNKTRRKKQKQQLGVGVIIAIVVCSLAAAGLLG 180

Du SPRL-------------------------------------------------------- 124

NJ SPRLL------------------------------------------------------- 125

Ba YCLYHIFFSPAAKARRSPDPSSNNSSASSRQRRDGLEMSSGRLSSARFSTSALL 234

02 YCLYHIFFSPAAKARRSPDPSSNNSSASSRQRRDGLEMSSGRLSSARFSTSALL 234

Du ------------------------------------------------------

NJ ------------------------------------------------------

Fig. S2. Predicted sequences of the ORF4 proteins for each of the varieties used in genetic study. The residues in green and red represent the predicted signal peptide and the transmembrane domain respectively (by EBI InterProScan).

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Ba MGRRRLLLSDLLLLLLIAIAVAGAAAAIAIPRAAPAFGVETGWPPEHCLRCFAPPDAPFV 60

Du MGRRRLLLSDLLLLLLIAIAVAGAAAAIAIPRAAPAFGVETGWPPEHCLRCFAPPDAPFV 60

02 MGRRRLLLSDLLLLLLIAIAVAGAAAAIAIPRAAPAFGVETGWPPEHCLRCFAPPDAPFV 60

NJ MGRRRLLLSDLLLLLLIAIAVAGAAAAIAIPRAAPAFGVETGWPPEHCLRCFAPPDAPFV 60

************************************************************

Ba LGAAAAIHLGNTNSCIAGYDDDDAPLGAKRSYYQFCIPSWVALAHDNGTVISGEAAMNRA 120

Du LGAAAAIHLGNTNSCIAGYDDDDAPLGAKRSYYQFCIPSWVALAHDNGTVISGEAAMNRA 120

02 LGAAAAIHLGNTNSCIAGYDDDDAPLGAKRSYYQFCIPSWVALAHDNGTVISGEAAMNRA 120

NJ LGAAAAIHLGNTNSCIAGYDDDDAPLGAKRSYYQFCIPSWVALAHDNGTVISGEAAMNRA 120

************************************************************

Ba ALSPSTAVSAFMRLLHRRVEDDVVKREIELVPYKFTKMLGWVSVQLDTDAEFSVDHLAGI 180

Du ALSPSTAVSAFMRLLHRRVEDDVVKREIELVPYKFTKMLGWVSVQLDTDAEFSVDHLAGI 180

02 ALSPSTAVSAFMRLLHRRVEDDVVKREIELVPYKFTKMLGWVSVQLDTDAEFSVDHLAGI 180

NJ ALSPSTAVSAFMRLLHRRVEDDVVKREIELVPYKFTKMLGWVSVQLDTDAEFSVDHLAGI 180

************************************************************

Ba LISHLKHTAEAHLGRHINNAVITLPSRLSYSADGRQVLSSAAKEYSGFRAVKVVDEHIAA 240

Du LISHLKHTAEAHLGRHINNAVITLPSRLSYSADGRQVLSSAAKEYSGFRAVKVVDEHIAA 240

02 LISHLKHTAEAHLGRHINNAVITLPSRLSYSADGRQVLSSAAKEYSGFRAVKVVDEHIAA 240

NJ LISHLKHTAEAHLGRHINNAVITLPSRLSYSADGRQVLSSAAKEYSGFRAVKVVDEHIAA 240

************************************************************

Ba AAAYGHHTKQGDRKAILVFHLGGRTSHATIFKFVDGTARLIATRAHHFLGGDDFTARIVD 300

Du AAAYGHHTKQGDRKAILVFHLGGRTSHATIFKFVDGTARLIATRAHHFLGGDDFTARIVD 300

02 AAAYGHHTKQGDRKAILVFHLGGRTSHATIFKFVDGTARLIATRAHHFLGGDDFTARIVD 300

NJ AAAYGHHTKQGDRKAILVFHLGGRTSHATIFKFVDGTARLIATRAHHFLGGDDFTARIVD 300

************************************************************

Ba HMVEHIKEQHGRDVRQEEKAMVRLRVACEHAKKALSEQQETLVQMDSLLDDGAVFSATLT 360

Du HMVEHIKEQHGRDVRQEEKAMVRLRVACEHAKKALSEQQETLVQMDSLLDDGAVFSATLT 360

02 HMVEHIKEQHGRDVRQEEKAMVRLRVACEHAKKALSEQQETLVQMDSLLDDGAVFSATLT 360

NJ HMVEHIKEQHGRDVRQEEKAMVRLRVACEHAKKALSEQQETLVQMDSLLDDGAVFSATLT 360

************************************************************

Ba RAKFEELNHDLLDRAMALVKEVVVTTGGVEVVDEVLVVGGSARIPKVRQLVKDYFNGNGN 420

Du RAKFEELNHDLLDRAMALVKEVVVTTGGVEVVDEVLVVGGSARIPKVRQLVKDYFNGNGN 420

02 RAKFEELNHDLLDRAMALVKEVVVTTGGVKVVDEVLVVGGSARIPKVRQLVKDYFNGNGN 420

NJ RAKFEELNHDLLDRAMALVKEVVVTTGGVEVVDEVLVVGGSARIPKVRQLVKDYFNGNGN 420

*****************************:******************************

Ba GTHPNSRGCKGPVDVEPEDAVLHGAALLSRPLPVAEGTAAARSIGSVGGI--------- 470

Du GTHPNSRGCKGPVDVEPEDAVLHGAALLSRPLPVAEGTAAARSIGSVGGI--------- 470

02 GTHPNSRGCKGPVDVEPEDAVLHGAALLSRPLPVAEGTAAARSIDFDHWFRGRHLISLV 479

NJ GTHPNSRGCKGPVDVEPEDAVLHGAALLSRPLPVAEGTAAARSIDFDHWFRGRHLISLV 479

********************************************. :

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Fig. S3. Predicted sequences of the ORF3 proteins for each of the varieties used in genetic study. The residues in green represent the predicted signal peptide and those in red indicate the heat shock protein Hsp70 homology region (by EBI InterProScan).

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Fig. S4. Expression profiles of ORFs 3, 4 and 5 in various tissues covering the entire life cycle of the rice plants. Tissues: 1, Callus, 15 days after induction; 2, Callus, 15 days after induction at 31ºC; 3, Callus, 15 days after subculture; 4, Callus, 5 days after regeneration; 5, Callus, screening stage; 6, Germinating seed, 72 h after soaking in water; 7, Plumule, 48 h after germination; 8, Radicle, 48 h after germination; 9, Seedling, 3 days after sowing; 10, Seedling, trefoil stage; 11, Shoot, seedling with 2 tillers; 12, Root, seedling with 2 tillers; 13, Sheath, young panicle (less than 1 mm) at secondary branch primordium differentiation stage; 14, Sheath, young panicle (40-50 mm) at meiosis stage; 15, Leaf, young panicle (less than 1 mm) at secondary branch primordium differentiation stage; 16, Leaf, young panicle (40-50 mm) at meiosis stage; 17, Flag leaf, 5 days before heading; 18, Flag leaf, 14 days after flowering; 19, Panicle, young panicle (less than 1 mm) at secondary branch primordium differentiation stage; 20, Panicle, young panicle (3-5 mm) at pistil/stamen primordium differentiation stage; 21, Panicle, young panicle (10-15 mm) at pollen-mother cell formation stage; 22, Panicle, young panicle (40-50 mm) at meiosis stage; 23, Panicle, heading stage; 24, Stem, 5 days before heading; 25, Stamen, one day before flowering; 26, Stem, heading stage; 27, Palea/lemma, one day before flowering; 28, Spikelet, 3 days after flowering; 29, Endosperm, 7 days after pollination (embryo is excluded); 30, Endosperm, 14 days after pollination (embryo is excluded); 31, Endosperm, 21 days after pollination (embryo is excluded).

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Fig. S5. Subcellular localizations of ORF3 and ORF4 proteins in rice protoplasts. (A-D) A protoplast cell co-expressing ORF3+-GFP (A) and an ER marker RFP-HDEL (B), a merged image (C), and a bright-field image (D). (E-H) A protoplast cell co-expressing ORF3--GFP (E) and an ER marker RFP-HDEL (F), a merged image (G), and a bright-field image (H). (I-L) A protoplast cell co-expressing ORF4--GFP (I) and an ER marker RFP-HDEL (J), a merged image (K), and a bright-field image (L). (M-P) A protoplast cell co-expressing ORF4--GFP (M) and a Golgi marker Man1-RFP (N), a merged image (O), and a bright-field image (P). (Q-T) A protoplast cell co-expressing ORF4+-GFP(Q) and a Golgi marker Man1-RFP (R), a merged image (S), and a bright-field image (T). Bars=10 μm.

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Fig. S6. qPCR verification for expression of ER stress-responsive (A) and PCD related genes (B) in BalillaORF5+ transgene-negative (3-4+5-) and transgene-positive (3-4+5+) plants. Ovary tissues at mature embryo-sac stage were used for the analysis. Y-axis represents relative expression level. Means ± SEM are based on three biological replicates.

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Fig. S7. Detection of spliced OsbZIP50 mRNA in BalillaORF5+ transgene-positive plants. The bands of unspliced (U) and spliced (S) OsbZIP50 mRNA are amplified using OsbZIP50-S primers. M represents the 20 bp DNA ladder marker. Lane 1, 2 and 3 represent the BalillaORF5+ transgene-positive (3-4+5+), transgene-negative (3-4+5-) and wild type (Balilla: 3-4+5-) plants respectively.

15

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Fig. S8. TUNEL assay of embryo-sac during megaspore formation in Balilla and BalillaORF5+. The red fluorescence indicates nuclei stained with propidium iodide, and yellow fluorescence is TUNEL-positive signal arising from the red fluorescence of background staining merged with the green fluorescence of TUNEL-positive nuclei staining. (A-B) Before megasporocyte meiosis. No TUNEL-positive signal is detected in either Balilla (A) or BalillaORF5+ (B). (C-D) Megasporocyte undergoing meiosis. Weak TUNEL signal is detected in nucellus cells surrounding megasporocyte in BalillaORF5+ (D), but not in Balilla (C). (E-F) Degeneration of three magaspores. Strong TUNEL-positive signal is observed in degraded megaspores in both Balilla (E) and BalillaORF5+ (F). In addition, TUNEL-positive signal is detected in the un-degraded megaspore in BalillaORF5+ (F). (G-H) Developing embryo-sac. Strong TUNEL-positive signal is detected in megaspore and nucellus cells surrounding embryo sac in BalillaORF5+ (H), but not in Balilla (G). (I-J) Mature embryo sac stage. Embryo sac is matured in Balilla (I) but aborted in BalillaORF5+ (J). TUNEL-positive signal is detected in nucellus cells surrounding antipodals in Balilla (I). Strong TUNEL-positive signal is detected in aborted embryo-sac and nucellus cells in BalillaORF5+ (J). Bars = 50 μm.

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Fig. S9. qPCR verification for expression of ER stress-responsive and PCD related genes in BalillaORF5+ transgene-negative (3-4+5-), transgene-positive (3-4+5+) and BalillaORF3+ORF5+ transgene-positive (3+4+5+) plants. Ovary tissues at mature embryo-sac stage were used for the analysis. Y-axis represents relative expression level. Means ± SEM are based on two biological replicates.

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Fig. S10. Schematic drawing of polymorphisms in the coding sequences of ORF3, ORF4 and ORF5. Boxes indicate the exons and lines represent the introns. Other non-coding regions are indicated by dashed lines. Single nucleotide polymorphisms (SNPs) are indicated by lines in the gene model with solid circles. Insertion/deletions (InDels) are indicated by lines in the gene model with triangles.

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Supplementary tables Table S1. Differential expression of some known ER stress-responsive genes in ORF5+ microarray. Function Gene Loci of RGAP Fold P value Gene annotation ER stress-responsive CRT2-1 Os01g67054 1.78 0.0093 calreticulin precursor protein, putative, expressed ER stress-responsive OsBiP1 Os02g02410 4.1 0.0061 DnaK family protein, putative, expressed ER stress-responsive OsJ2 Os03g57340 0.33 0.032 chaperone protein dnaJ, putative, expressed ER stress-responsive CRT2-2 Os03g61670 3.87 0.0238 calreticulin precursor, putative, expressed ER stress-responsive Calnexin Os04g32950 1.72 0.036 calreticulin precursor protein, putative, expressed ER stress-responsive ERdj3B-like Os05g06440 1.98 0.0204 dnaJ homolog subfamily B member 11 precursor, putative, expressed ER stress-responsive OsBiP4 Os05g35400 2.96 0.0125 DnaK family protein, putative, expressed ER stress-responsive ORF3 Os06g10990 3.06 0.0006 DnaK family protein, putative, expressed ER stress-responsive DGL1-1 Os07g10830 1.96 0.0159 dolichyl-diphosphooligosaccharide-protein glycosyltransferase 48 kD subunit ER stress-responsive CRT1 Os07g14270 2.21 0.0243 calreticulin precursor protein, putative, expressed ER stress-responsive SDF2-like Os08g17680 9.65 0.0003 stromal cell-derived factor 2-like protein precursor, putative, expressed ER stress-responsive Fes1-like Os09g33780 5.89 0.0024 expressed protein ER stress-responsive PDIL1-1 Os11g09280 2.61 0.0034 OsPDIL1-1 protein disulfide isomerase PDIL1-1, expressed ER stress-responsive OsbZIP60 Os05g34050 0.49 0.0294 bZIP transcription factor domain containing protein, expressed ER stress-responsive OsbZIP50 Os06g41770 5.58 0.0038 bZIP transcription factor domain containing protein, expressed ER stress-responsive OsIRE1 Os07g28820 0.61 0.0136 ribonuclease 2-5A family protein, expressed ER stress-responsive OsbZIP39 Os07g44950 0.27 0.0174 bZIP transcription factor domain containing protein, expressed

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Table S2. Functional classification of differentially expressed genes in ORF5+ microarray (fold≥2 or ≤0.5 with P<0.05).

Loci of RGAP Fold Gene annotation

Lipid transport and metabolism Os02g40440 13.14 GDSL-like lipase/acylhydrolase, putative, expressed Os09g04624 6.32 GDSL-like lipase/acylhydrolase, putative, expressed Os06g24404 4.84 GDSL-like lipase/acylhydrolase, putative, expressed Os06g47910 3.87 GDSL-like lipase/acylhydrolase, putative, expressed Os09g04624 3.40 GDSL-like lipase/acylhydrolase, putative, expressed Os08g02094 2.96 GDSL-like lipase/acylhydrolase, putative, expressed Os01g61570 2.61 GDSL-like lipase/acylhydrolase, putative, expressed Os06g34120 2.41 GDSL-like lipase/acylhydrolase, putative, expressed Os01g22640 2.37 GDSL-like lipase/acylhydrolase, putative, expressed Os05g44200 2.27 GDSL-like lipase/acylhydrolase, putative, expressed Os07g39750 2.20 GDSL-like lipase/acylhydrolase, putative, expressed Os01g22660 2.19 GDSL-like lipase/acylhydrolase, putative, expressed Os09g04710 2.19 GDSL-like lipase/acylhydrolase, putative, expressed Os02g09620 2.15 GDSL-like lipase/acylhydrolase, putative, expressed Os02g57110 2.13 GDSL-like lipase/acylhydrolase, putative, expressed Os06g06520 2.12 GDSL-like lipase/acylhydrolase, putative, expressed Os09g39430 2.10 GDSL-like lipase/acylhydrolase, putative, expressed Os05g06720 2.07 GDSL-like lipase/acylhydrolase, putative, expressed Os06g05550 2.06 GDSL-like lipase/acylhydrolase, putative, expressed Os01g11710 1.97 GDSL-like lipase/acylhydrolase, putative, expressed Os02g01140 1.96 GDSL-like lipase/acylhydrolase, putative, expressed Os01g46080 0.41 GDSL-like lipase/acylhydrolase, putative, expressed Os01g46169 0.36 GDSL-like lipase/acylhydrolase, putative, expressed Os07g39740 0.23 GDSL-like lipase/acylhydrolase, putative, expressed Os06g06290 0.12 GDSL-like lipase/acylhydrolase, putative, expressed Os05g11910 0.08 GDSL-like lipase/acylhydrolase, putative, expressed Os01g11650 0.08 GDSL-like lipase/acylhydrolase, putative, expressed Os11g24070 0.28 LTPL10 - Protease inhibitor/seed storage/LTP family protein precursor, expressed Os01g62980 0.45 LTPL101 - Protease inhibitor/seed storage/LTP family protein precursor, expressed Os04g33920 0.14 LTPL102 - Protease inhibitor/seed storage/LTP family protein precursor, expressed Os03g50960 5.20 LTPL118 - Protease inhibitor/seed storage/LTP family protein precursor, expressed Os12g02320 0.32 LTPL12 - Protease inhibitor/seed storage/LTP family protein precursor, expressed Os04g52260 2.60 LTPL124 - Protease inhibitor/seed storage/LTP family protein precursor, expressed Os10g40614 7.13 LTPL147 - Protease inhibitor/seed storage/LTP family protein precursor, expressed Os01g49650 3.59 LTPL150 - Protease inhibitor/seed storage/LTP family protein precursor, expressed Os03g02050 0.12 LTPL151 - Protease inhibitor/seed storage/LTP family protein precursor, expressed Os05g47730 0.20 LTPL153 - Protease inhibitor/seed storage/LTP family protein precursor, expressed Os01g12020 3.11 LTPL18 - Protease inhibitor/seed storage/LTP family protein precursor, expressed Os07g46210 4.87 LTPL2 - Protease inhibitor/seed storage/LTP family protein precursor, expressed Os08g43290 6.37 LTPL44 - Protease inhibitor/seed storage/LTP family protein precursor, expressed Os09g35700 16.93 LTPL45 - Protease inhibitor/seed storage/LTP family protein precursor, expressed Os04g09520 2.59 LTPL59 - Protease inhibitor/seed storage/LTP family protein precursor, putative Os01g59870 3.83 LTPL65 - Protease inhibitor/seed storage/LTP family protein precursor, expressed

21

Os03g57970 2.28 LTPL73 - Protease inhibitor/seed storage/LTP family protein precursor, expressed Os12g43363 3.15 fatty acid hydroxylase, putative, expressed Os03g01820 0.46 fatty acid hydroxylase, putative, expressed Os04g48880 0.15 fatty acid hydroxylase, putative, expressed Os08g41780 0.46 triacylglycerol lipase precursor, putative, expressed Os03g22670 0.36 triacylglycerol Lipase, putative, expressed Os06g11090 0.50 CXE carboxylesterase, putative, expressed Os07g34280 0.02 CXE carboxylesterase, putative, expressed Os03g09250 0.37 inositol-3-phosphate synthase, putative, expressed Os09g24710 2.20 alpha/beta hydrolase fold, putative, expressed Os05g33940 0.37 alpha/beta hydrolase fold, putative, expressed

Secondary metabolites biosynthesis, transport and catabolism Os03g04340 1.98 C-methyltransferase, putative, expressed Os12g13730 2.89 pleiotropic drug resistance protein 2, putative, expressed Os08g29570 0.22 pleiotropic drug resistance protein 3, putative, expressed Os09g16330 0.36 pleiotropic drug resistance protein, putative, expressed Os06g46500 5.11 monocopper oxidase, putative, expressed Os06g01490 2.68 monocopper oxidase, putative, expressed Os08g05820 0.42 monocopper oxidase, putative, expressed Os09g03939 3.61 white-brown complex homolog protein 11, putative, expressed Os12g22284 0.31 white-brown complex homolog protein 11, putative, expressed Os05g02890 0.30 white-brown complex homolog protein 16, putative, expressed Os11g07930 0.47 oxidoreductase, short chain dehydrogenase/reductase family domain containing Os08g37130 0.25 oxidoreductase, short chain dehydrogenase/reductase family domain containing Os03g18740 0.15 oxidoreductase, short chain dehydrogenase/reductase family domain containing Os02g09250 0.48 cytochrome P450 71D10, putative, expressed Os05g29750 0.48 cytochrome P450 71E1, putative, expressed Os01g43844 2.28 cytochrome P450 72A1, putative, expressed Os01g29150 2.27 cytochrome P450 72A1, putative, expressed Os04g01140 3.44 cytochrome P450 93A2, putative, expressed Os07g33480 2.81 cytochrome P450 domain containing protein, expressed Os03g04650 2.94 cytochrome P450 protein, putative, expressed Os06g30640 0.38 cytochrome P450, putative Os03g07250 5.80 cytochrome P450, putative, expressed Os11g29720 4.92 cytochrome P450, putative, expressed Os11g27730 4.15 cytochrome P450, putative, expressed Os03g07250 3.75 cytochrome P450, putative, expressed Os10g16974 2.44 cytochrome P450, putative, expressed Os05g41440 2.07 cytochrome P450, putative, expressed Os05g01120 0.53 cytochrome P450, putative, expressed Os06g43430 0.52 cytochrome P450, putative, expressed Os01g10040 0.49 cytochrome P450, putative, expressed Os10g05490 0.45 cytochrome P450, putative, expressed Os02g02000 0.41 cytochrome P450, putative, expressed Os03g12500 0.45 cytochrome P450, putative, expressed Os02g02000 0.41 cytochrome P450, putative, expressed Os01g63930 0.38 cytochrome P450, putative, expressed Os08g36310 0.32 cytochrome P450, putative, expressed Os02g12690 0.30 cytochrome P450, putative, expressed

22

Os02g30100 0.26 cytochrome P450, putative, expressed Os08g43390 0.20 cytochrome P450, putative, expressed

Carbohydrate transport and metabolism Os05g23924 0.17 glycosyl hydrolase family 10 protein, putative, expressed Os02g51620 2.54 glycosyl hydrolase family 3 protein, putative, expressed Os07g43820 0.54 glycosyl hydrolase, putative, expressed Os01g19750 0.49 glycosyl hydrolase, putative, expressed Os10g28080 0.44 glycosyl hydrolase, putative, expressed Os11g47600 0.23 glycosyl hydrolase, putative, expressed Os01g64110 0.20 glycosyl hydrolase, putative, expressed Os11g47500 0.17 glycosyl hydrolase, putative, expressed Os11g47580 0.14 glycosyl hydrolase, putative, expressed Os11g47530 0.11 glycosyl hydrolase, putative, expressed Os08g40680 0.09 glycosyl hydrolase, putative, expressed Os05g15770 0.04 glycosyl hydrolase, putative, expressed Os08g40690 0.03 glycosyl hydrolase, putative, expressed Os05g15770 0.03 glycosyl hydrolase, putative, expressed Os11g33270 4.37 glycosyl hydrolases family 16, putative, expressed Os07g34580 3.40 glycosyl hydrolases family 16, putative, expressed Os10g39840 3.30 glycosyl hydrolases family 16, putative, expressed Os07g29750 3.11 glycosyl hydrolases family 16, putative, expressed Os06g48200 0.35 glycosyl hydrolases family 16, putative, expressed Os06g48180 0.07 glycosyl hydrolases family 16, putative, expressed Os03g25790 0.23 glycosyl hydrolases family 17 protein, expressed Os05g37130 3.43 glycosyl hydrolases family 17, putative, expressed Os06g34020 3.26 glycosyl hydrolases family 17, putative, expressed Os05g37130 3.17 glycosyl hydrolases family 17, putative, expressed Os01g71474 2.82 glycosyl hydrolases family 17, putative, expressed Os01g71380 0.15 glycosyl hydrolases family 17, putative, expressed Os01g71350 0.05 glycosyl hydrolases family 17, putative, expressed Os01g71670 0.04 glycosyl hydrolases family 17, putative, expressed Os05g31140 0.03 glycosyl hydrolases family 17, putative, expressed Os04g33740 5.76 glycosyl hydrolases, putative, expressed Os04g33720 0.43 glycosyl hydrolases, putative, expressed Os02g01590 0.11 glycosyl hydrolases, putative, expressed Os06g12280 2.36 glycosyl transferase 8 domain containing protein, putative, expressed Os03g30000 2.10 glycosyl transferase 8 domain containing protein, putative, expressed Os01g52710 2.01 glycosyl transferase 8 domain containing protein, putative, expressed Os02g41520 0.52 glycosyl transferase 8 domain containing protein, putative, expressed Os03g20120 0.51 glycosyl transferase 8 domain containing protein, putative, expressed Os02g50600 0.24 glycosyl transferase 8 domain containing protein, putative, expressed Os06g49810 2.03 glycosyl transferase family 8, putative, expressed Os01g04920 2.12 glycosyl transferase, group 1 domain containing protein, expressed Os03g08600 3.44 glycosyl transferase, putative, expressed Os05g35200 3.25 glycosyl transferase, putative, expressed Os07g49370 5.86 glycosyltransferase family 43 protein, putative, expressed Os10g13810 0.38 glycosyltransferase family 43 protein, putative, expressed Os06g27560 0.21 glycosyltransferase protein, putative, expressed Os01g02920 0.08 glycosyltransferase protein, putative, expressed

23

Os04g44740 0.17 glycosyltransferase sugar-binding region containing DXD motif, putative, expressed Os03g37010 0.53 glycosyltransferase, putative, expressed Os04g12010 0.44 glycosyltransferase, putative, expressed Os07g46380 0.39 glycosyltransferase, putative, expressed Os03g19310 0.25 glycosyltransferase, putative, expressed Os01g02900 0.09 glycosyltransferase, putative, expressed Os01g72610 0.03 glycosyltransferase, putative, expressed Os07g26630 5.66 aquaporin protein, putative, expressed Os01g13120 4.28 aquaporin protein, putative, expressed Os02g13870 1.99 aquaporin protein, putative, expressed Os04g44060 0.46 aquaporin protein, putative, expressed Os02g41860 0.42 aquaporin protein, putative, expressed Os01g74450 0.41 aquaporin protein, putative, expressed Os02g41860 0.22 aquaporin protein, putative, expressed Os10g35050 0.08 aquaporin protein, putative, expressed Os05g09500 2.78 hexokinase, putative, expressed Os05g01380 0.53 polygalacturonase inhibitor precursor, putative, expressed Os05g01444 0.51 polygalacturonase inhibitor precursor, putative, expressed Os03g59330 6.61 polygalacturonase, putative, expressed Os03g03350 2.51 polygalacturonase, putative, expressed Os02g54030 2.08 polygalacturonase, putative, expressed Os02g54030 2.01 polygalacturonase, putative, expressed Os05g20020 0.13 polygalacturonase, putative, expressed Os05g02310 4.46 soluble inorganic pyrophosphatase, putative, expressed Os01g64670 0.45 soluble inorganic pyrophosphatase, putative, expressed Os03g24870 8.62 transporter family protein, putative, expressed Os06g05980 3.05 transporter family protein, putative, expressed Os05g49260 0.52 transporter family protein, putative, expressed Os04g37980 0.51 transporter family protein, putative, expressed Os09g24924 0.40 transporter family protein, putative, expressed Os07g05640 0.38 transporter family protein, putative, expressed Os12g32760 0.36 transporter family protein, putative, expressed Os04g41460 0.31 transporter family protein, putative, expressed Os04g38026 0.20 transporter family protein, putative, expressed Os07g01560 0.19 transporter family protein, putative, expressed Os03g11900 0.15 transporter family protein, putative, expressed Os03g43720 0.06 transporter family protein, putative, expressed Os12g29220 0.29 nodulin MtN3 family protein, putative, expressed Os04g34490 2.91 nodulin, putative, expressed Os06g01840 2.45 nodulin, putative, expressed Os04g31924 2.25 nodulin, putative, expressed Os03g04770 0.29 beta-amylase, putative, expressed Os10g32810 0.18 beta-amylase, putative, expressed Os11g07020 0.51 fructose-bisphospate aldolase isozyme, putative, expressed Os11g07020 0.47 fructose-bisphospate aldolase isozyme, putative, expressed Os06g40640 0.43 fructose-bisphospate aldolase isozyme, putative, expressed Os06g40640 0.40 fructose-bisphospate aldolase isozyme, putative, expressed Os08g02700 0.05 fructose-bisphospate aldolase isozyme, putative, expressed Os02g41780 9.85 transporter-related, putative, expressed

24

Os06g30950 3.54 transporter-related, putative, expressed Os06g03540 2.57 oligopeptide transporter, putative, expressed Os06g03700 2.51 oligopeptide transporter, putative, expressed Os02g46850 0.53 oligopeptide transporter, putative, expressed Os11g03230 2.91 nucleoside-triphosphatase, putative, expressed Os12g02980 2.85 nucleoside-triphosphatase, putative, expressed Os11g03230 2.12 nucleoside-triphosphatase, putative, expressed Os12g38750 7.84 nucleotide pyrophosphatase/phosphodiesterase, putative, expressed Os08g41880 2.92 nucleotide pyrophosphatase/phosphodiesterase, putative, expressed Os12g38760 2.44 nucleotide pyrophosphatase/phosphodiesterase, putative, expressed Os02g17920 2.12 glyoxalase family protein, putative, expressed Os02g07160 0.23 glyoxalase family protein, putative, expressed

Transcription Os01g74020 10.14 MYB family transcription factor, putative, expressed Os05g50340 9.41 MYB family transcription factor, putative, expressed Os05g04820 7.59 MYB family transcription factor, putative, expressed Os04g50770 6.18 MYB family transcription factor, putative, expressed Os01g50720 3.76 MYB family transcription factor, putative, expressed Os01g18240 3.40 MYB family transcription factor, putative, expressed Os02g49986 2.98 MYB family transcription factor, putative, expressed Os09g36730 2.50 MYB family transcription factor, putative, expressed Os08g33660 2.38 MYB family transcription factor, putative, expressed Os12g13570 2.32 MYB family transcription factor, putative, expressed Os08g06370 2.06 MYB family transcription factor, putative, expressed Os09g01960 0.53 MYB family transcription factor, putative, expressed Os07g37210 0.49 MYB family transcription factor, putative, expressed Os05g48010 0.27 MYB family transcription factor, putative, expressed Os02g46030 0.23 MYB family transcription factor, putative, expressed Os12g37690 0.22 MYB family transcription factor, putative, expressed Os02g42850 0.19 MYB family transcription factor, putative, expressed Os11g45740 0.11 MYB family transcription factor, putative, expressed Os07g48870 0.09 MYB family transcription factor, putative, expressed Os08g06110 0.02 MYB family transcription factor, putative, expressed Os06g51260 0.01 MYB family transcription factor, putative, expressed Os06g43090 0.47 MYB transcription factor TaMYB1, putative, expressed Os07g48596 7.90 Myb transcription factor, putative, expressed Os05g51160 2.33 Myb transcription factor, putative, expressed Os01g09640 0.45 Myb transcription factor, putative, expressed Os06g10820 0.17 helix-loop-helix DNA-binding domain containing protein Os02g51320 3.27 helix-loop-helix DNA-binding domain containing protein, expressed Os01g18290 0.38 helix-loop-helix DNA-binding domain containing protein, expressed Os12g41650 0.30 helix-loop-helix DNA-binding domain containing protein, expressed Os01g50940 0.25 helix-loop-helix DNA-binding domain containing protein, expressed Os03g43810 0.18 helix-loop-helix DNA-binding domain containing protein, expressed Os02g45170 3.76 helix-loop-helix DNA-binding protein, putative, expressed Os08g37730 3.03 helix-loop-helix DNA-binding protein, putative, expressed Os03g22680 0.42 RING finger and CHY zinc finger domain-containing protein 1, putative, expressed Os01g52110 0.16 RING finger and CHY zinc finger domain-containing protein 1, putative, expressed Os01g56070 0.50 RING finger protein, putative, expressed

25

Os02g50290 0.54 RING zinc finger protein, putative, expressed Os03g24590 0.16 mTERF domain containing protein, expressed Os07g13170 3.76 AP2 domain containing protein, expressed Os03g60430 3.27 AP2 domain containing protein, expressed Os04g55560 2.92 AP2 domain containing protein, expressed Os03g08500 0.55 AP2 domain containing protein, expressed Os05g25260 0.54 AP2 domain containing protein, expressed Os02g51300 0.54 AP2 domain containing protein, expressed Os05g03040 0.53 AP2 domain containing protein, expressed Os08g42550 0.53 AP2 domain containing protein, expressed Os01g07120 0.48 AP2 domain containing protein, expressed Os05g29810 0.43 AP2 domain containing protein, expressed Os08g42550 0.41 AP2 domain containing protein, expressed Os01g07120 0.37 AP2 domain containing protein, expressed Os03g08490 0.37 AP2 domain containing protein, expressed Os06g07030 0.33 AP2 domain containing protein, expressed Os04g46440 0.28 AP2 domain containing protein, expressed Os07g42510 0.07 AP2 domain containing protein, expressed Os03g64260 0.04 AP2 domain containing protein, expressed Os09g35760 0.53 homeobox and START domains containing protein, putative, expressed Os10g23090 2.37 homeobox associated leucine zipper, putative, expressed Os09g35910 0.41 homeobox associated leucine zipper, putative, expressed Os02g35770 0.19 homeobox associated leucine zipper, putative, expressed Os02g35770 0.18 homeobox associated leucine zipper, putative, expressed Os02g43330 0.17 homeobox associated leucine zipper, putative, expressed Os04g45810 0.16 homeobox associated leucine zipper, putative, expressed Os09g21180 0.01 homeobox associated leucine zipper, putative, expressed Os02g13310 2.12 homeobox domain containing protein, expressed Os03g55990 0.54 homeobox domain containing protein, expressed Os07g48560 0.40 homeobox domain containing protein, expressed Os07g39320 0.36 homeobox domain containing protein, expressed Os03g10210 0.02 homeobox domain containing protein, expressed Os03g56110 2.48 homeobox protein knotted-1, putative, expressed Os03g51710 2.40 homeobox protein knotted-1, putative, expressed Os01g53220 5.78 HSF-type DNA-binding domain containing protein, expressed Os01g39020 0.52 HSF-type DNA-binding domain containing protein, expressed Os06g35960 0.32 HSF-type DNA-binding domain containing protein, expressed Os02g13800 0.29 HSF-type DNA-binding domain containing protein, expressed Os03g20310 0.46 transcription factor HBP-1b, putative, expressed Os01g06560 0.22 transcription factor HBP-1b, putative, expressed Os04g45940 0.45 transcription factor like protein, putative, expressed Os03g03070 2.33 transcription factor, putative, expressed Os06g15480 2.20 transcription factor, putative, expressed Os09g10840 0.45 transcription factor, putative, expressed Os07g48820 0.22 transcription factor, putative, expressed Os07g39800 2.08 transcription repressor HOTR, putative, expressed Os10g18370 0.08 transcriptional regulator, putative, expressed Os01g03840 3.02 ZOS1-02 - C2H2 zinc finger protein, expressed Os01g35040 0.40 ZOS1-09 - C2H2 zinc finger protein, expressed

26

Os11g38330 2.53 ZOS11-06 - C2H2 zinc finger protein, expressed Os01g63980 0.24 ZOS1-17 - C2H2 zinc finger protein, expressed Os12g07280 0.47 ZOS12-02 - C2H2 zinc finger protein, expressed Os12g38960 2.12 ZOS12-07 - C2H2 zinc finger protein, expressed Os12g39400 0.12 ZOS12-09 - C2H2 zinc finger protein, expressed Os02g47920 0.27 ZOS2-16 - C2H2 zinc finger protein Os02g57790 0.37 ZOS2-19 - C2H2 zinc finger protein Os03g41390 0.21 ZOS3-15 - C2H2 zinc finger protein, expressed Os03g60560 0.34 ZOS3-21 - C2H2 zinc finger protein, expressed Os04g59380 3.95 ZOS4-14 - C2H2 zinc finger protein, expressed Os07g39960 0.20 ZOS7-07 - C2H2 zinc finger protein Os07g40300 0.08 ZOS7-10 - C2H2 zinc finger protein, expressed

Cell wall/ membrane/ envelope biogenesis Os05g02770 0.32 glycine-rich cell wall structural protein precursor, putative, expressed Os01g06310 0.31 glycine-rich cell wall structural protein precursor, putative, expressed Os04g56030 0.05 glycine-rich cell wall structural protein precursor, putative, expressed Os02g37480 0.05 glycine-rich cell wall structural protein precursor, putative, expressed Os04g05050 5.38 pectate lyase precursor, putative, expressed Os01g66840 2.89 pectinacetylesterase domain containing protein Os10g40290 4.53 pectinacetylesterase domain containing protein, expressed Os04g51340 3.06 pectinacetylesterase domain containing protein, expressed Os01g21630 0.20 pectinacetylesterase domain containing protein, expressed Os11g36240 2.05 pectinesterase, putative Os02g54190 7.69 pectinesterase, putative, expressed Os01g13320 3.22 pectinesterase, putative, expressed Os07g47830 2.46 pectinesterase, putative, expressed Os05g29790 0.28 pectinesterase, putative, expressed Os04g52320 3.39 QRT3, putative, expressed Os03g28330 8.94 sucrose synthase, putative, expressed Os06g09450 2.06 sucrose synthase, putative, expressed Os02g58480 2.04 sucrose synthase, putative, expressed Os03g22120 0.18 sucrose synthase, putative, expressed Os03g07480 0.16 sucrose transporter, putativ, expressed Os08g34150 0.45 OsTIL-2 Temperature-induced lipocalin-2, expressed Os03g16980 2.37 NAD dependent epimerase/dehydratase family domain containing protein, expressed Os06g44180 2.04 NAD dependent epimerase/dehydratase family protein, putative, expressed Os06g25439 0.50 NAD dependent epimerase/dehydratase family protein, putative, expressed Os03g32170 0.49 NAD dependent epimerase/dehydratase family protein, putative, expressed Os05g51670 0.23 NAD dependent epimerase/dehydratase family protein, putative, expressed

Protein turnover, chaperones Os04g03050 3.30 OsSub34 - Putative Subtilisin homologue, expressed Os07g39020 0.04 OsSub53 - Putative Subtilisin homologue, expressed Os10g25450 3.98 OsSub60 - Putative Subtilisin homologue, expressed Os01g58290 0.24 OsSub9 - Putative Subtilisin homologue, expressed Os06g02780 3.06 aspartic protease, putative, expressed Os03g08790 4.86 aspartic proteinase nepenthesin precursor, putative, expressed Os09g30414 0.47 aspartic proteinase nepenthesin-2 precursor, putative, expressed Os02g48860 0.19 aspartic proteinase nepenthesin-2 precursor, putative, expressed Os01g44130 2.26 aspartic proteinase oryzasin-1 precursor, putative, expressed

27

Os05g51220 2.29 aspartic proteinase, putative, expressed Os09g31031 30.45 ubiquitin family protein, putative, expressed Os09g31031 8.73 ubiquitin family protein, putative, expressed Os02g38410 3.65 ubiquitin family protein, putative, expressed Os02g38410 2.37 ubiquitin family protein, putative, expressed Os03g15370 2.05 ubiquitin fusion protein, putative, expressed Os04g58800 3.43 ubiquitin-conjugating enzyme, putative, expressed Os06g30970 0.19 ubiquitin-conjugating enzyme, putative, expressed Os09g38920 0.07 cysteine protease Os02g57410 0.31 cysteine protease family protein, putative, expressed Os01g27140 0.23 OsGrx_C7 - glutaredoxin subgroup III, expressed Os12g10670 2.89 AAA-type ATPase family protein, putative, expressed Os04g40290 2.03 AAA-type ATPase family protein, putative, expressed Os03g02330 0.38 AAA-type ATPase family protein, putative, expressed Os05g44340 0.28 heat shock protein 101, putative, expressed Os04g57880 7.75 heat shock protein DnaJ, putative, expressed Os06g13060 2.06 heat shock protein DnaJ, putative, expressed Os11g10990 0.50 heat shock protein DnaJ, putative, expressed Os03g56540 0.33 heat shock protein DnaJ, putative, expressed Os05g33010 0.30 heat shock protein DnaJ, putative, expressed Os06g44160 0.28 heat shock protein DnaJ, putative, expressed Os05g33010 0.24 heat shock protein DnaJ, putative, expressed Os05g42120 4.31 heat shock protein, putative, expressed Os04g01740 0.37 heat shock protein, putative, expressed Os05g51360 20.58 DnaK family protein, putative, expressed Os02g02410 4.22 DnaK family protein, putative, expressed Os06g10990 3.05 DnaK family protein, putative, expressed Os02g48110 2.39 DnaK family protein, putative, expressed Os05g35400 2.37 DnaK family protein, putative, expressed Os03g16920 0.24 DnaK family protein, putative, expressed Os03g16860 0.12 DnaK family protein, putative, expressed Os01g62290 0.10 DnaK family protein, putative, expressed Os05g38530 0.07 DnaK family protein, putative, expressed Os05g45350 0.26 dnaJ domain containing protein, expressed Os05g45350 0.15 dnaJ domain containing protein, expressed Os05g48810 0.11 dnaJ domain containing protein, expressed Os05g06440 2.05 dnaJ homolog subfamily B member 11 precursor, putative, expressed Os10g07210 2.94 hsp20/alpha crystallin family protein, putative, expressed Os02g03570 2.19 hsp20/alpha crystallin family protein, putative, expressed Os04g36750 0.44 hsp20/alpha crystallin family protein, putative, expressed Os02g54140 0.03 hsp20/alpha crystallin family protein, putative, expressed Os02g54140 0.02 hsp20/alpha crystallin family protein, putative, expressed

Signal transduction mechanisms Os02g41580 0.43 CAMK_CAMK_like.14 - CAMK includes calcium/calmodulin depedent protein Os03g03660 1.95 CAMK_CAMK_like.17 - CAMK includes calcium/calmodulin depedent protein Os03g27280 0.29 CAMK_CAMK_like.19 - CAMK includes calcium/calmodulin depedent protein Os03g41460 2.49 CAMK_CAMK_like.20 - CAMK includes calcium/calmodulin depedent protein Os11g07040 2.60 CAMK_CAMK_like.43 - CAMK includes calcium/calmodulin depedent protein Os12g39630 3.17 CAMK_CAMK_like.49 - CAMK includes calcium/calmodulin depedent protein

28

Os05g41090 3.05 CAMK_CAMK_like_CG18020d.1 - CAMK includes calcium/calmodulin depedent Os03g22050 8.81 CAMK_KIN1/SNF1/Nim1_like.16 - CAMK includes calcium/calmodulin depedent Os09g25090 0.29 CAMK_KIN1/SNF1/Nim1_like.34 - CAMK includes calcium/calmodulin depedent Os10g42190 0.43 leucine rich repeat containing protein, expressed Os04g51580 0.29 leucine rich repeat containing protein, expressed Os04g08390 2.09 Leucine Rich Repeat family protein, expressed Os07g28570 2.25 leucine rich repeat protein, putative, expressed Os07g31840 2.93 leucine-rich repeat family protein, putative, expressed Os01g52880 2.58 leucine-rich repeat family protein, putative, expressed Os07g05190 2.28 leucine-rich repeat family protein, putative, expressed Os11g35450 2.64 leucine-rich repeat receptor protein kinase EXS precursor, putative, expressed Os03g03570 2.09 leucine-rich repeat transmembrane protein kinase, putative, expressed Os02g44990 0.06 OsFBDUF13 - F-box and DUF domain containing protein, expressed Os02g51350 0.36 OsFBK10 - F-box domain and kelch repeat containing protein, expressed Os04g52830 2.10 OsFBK15 - F-box domain and kelch repeat containing protein, expressed Os06g39370 0.10 OsFBK16 - F-box domain and kelch repeat containing protein, expressed Os05g35110 0.50 OsFBL22 - F-box domain and LRR containing protein, expressed Os06g07000 0.36 OsFBL28 - F-box domain and LRR containing protein, expressed Os06g40360 0.28 OsFBL30 - F-box domain and LRR containing protein, expressed Os08g09450 2.21 OsFBL41 - F-box domain and LRR containing protein, expressed Os12g33210 2.05 OsFBL59 - F-box domain and LRR containing protein, expressed Os02g06520 0.48 OsFBLD1 - F-box, LRR and FBD domain containing protein, expressed Os07g18560 3.19 OsFBLD6 - F-box, LRR and FBD domain containing protein, expressed Os11g34460 16.81 OsFBO10 - F-box and other domain containing protein, expressed Os02g41910 0.54 OsFBO12 - F-box and other domain containing protein, expressed Os03g12940 0.50 OsFBO13 - F-box and other domain containing protein, expressed Os02g47640 2.31 OsFBT4 - F-box and tubby domain containing protein, expressed Os04g59130 2.42 OsFBT6 - F-box and tubby domain containing protein, expressed Os04g59130 2.06 OsFBT6 - F-box and tubby domain containing protein, expressed Os04g33820 0.15 OsFBX132 - F-box domain containing protein, expressed Os04g39080 0.31 OsFBX140 - F-box domain containing protein, expressed Os04g40910 0.52 OsFBX146 - F-box domain containing protein, expressed Os01g52980 0.54 OsFBX23 - F-box domain containing protein, expressed Os01g52980 0.45 OsFBX23 - F-box domain containing protein, expressed Os01g59180 0.54 OsFBX27 - F-box domain containing protein, expressed Os09g28120 2.44 OsFBX330 - F-box domain containing protein, expressed Os10g03850 0.54 OsFBX352 - F-box domain containing protein, expressed Os10g03910 2.32 OsFBX354 - F-box domain containing protein, expressed Os10g17940 0.31 OsFBX382 - F-box domain containing protein, expressed Os10g30280 3.08 OsFBX386 - F-box domain containing protein, expressed Os11g05660 0.12 OsFBX396 - F-box domain containing protein, expressed Os01g17390 0.40 OsFBX5 - F-box domain containing protein, expressed Os02g56750 0.39 OsFBX65 - F-box domain containing protein, expressed Os02g56810 0.51 OsFBX68 - F-box domain containing protein Os03g53150 2.70 OsIAA13 - Auxin-responsive Aux/IAA gene family member, expressed Os01g09450 3.03 OsIAA2 - Auxin-responsive Aux/IAA gene family member, expressed Os01g53880 0.21 OsIAA6 - Auxin-responsive Aux/IAA gene family member, expressed Os02g13520 2.69 OsIAA7 - Auxin-responsive Aux/IAA gene family member, expressed Os04g56060 3.10 protein kinase domain containing protein, expressed

29

Os11g02830 2.00 protein kinase domain containing protein, expressed Os12g40419 0.44 protein kinase domain containing protein, expressed Os03g28300 0.43 protein kinase domain containing protein, expressed Os02g43290 0.32 protein kinase domain containing protein, expressed Os04g49460 0.32 protein kinase domain containing protein, expressed Os01g10430 2.14 protein kinase family protein, putative, expressed Os11g08950 2.05 protein kinase family protein, putative, expressed Os03g03890 0.48 protein kinase family protein, putative, expressed Os05g41950 2.87 protein kinase, putative, expressed Os04g43020 2.70 protein kinase, putative, expressed Os02g02040 2.33 protein kinase, putative, expressed Os12g05394 2.23 protein kinase, putative, expressed Os03g18430 2.22 protein kinase, putative, expressed Os05g30820 2.15 protein kinase, putative, expressed Os04g43020 2.04 protein kinase, putative, expressed Os05g44030 0.54 protein kinase, putative, expressed Os01g49920 0.25 protein kinase, putative, expressed Os04g56110 0.18 protein kinase, putative, expressed Os03g42130 2.07 gibberellin 20 oxidase 2, putative, expressed Os08g44590 0.34 gibberellin 20 oxidase 2, putative, expressed Os01g55240 0.13 gibberellin 2-beta-dioxygenase, putative, expressed Os07g06880 2.40 gibberellin receptor GID1L2, putative, expressed Os05g33730 0.47 gibberellin receptor GID1L2, putative, expressed Os07g44860 0.38 gibberellin receptor GID1L2, putative, expressed Os09g28740 0.37 gibberellin receptor GID1L2, putative, expressed Os06g20200 0.34 gibberellin receptor GID1L2, putative, expressed Os07g06830 0.29 gibberellin receptor GID1L2, putative, expressed Os03g57640 0.15 gibberellin receptor GID1L2, putative, expressed

30

Table S3. Source and S5 genotypes of 83 accessions belonging to three Oryza species plus one accession of O. glumaepatula.

Species Germplasm accession no.

Locality

Subspecies* S5 genotype Haplotype ORF3 ORF4 ORF5

O. sativa (n=35)

IRGC 1012 China indica + - + H28 IRGC 17376 Indonesia indica + + + H32 IRGC 26977 Indonesia indica - + - H16 IRGC 27489 Indonesia javanica - + - H16 IRGC 30346 China indica + - + H28 IRGC 53453 China indica - + - H16 IRGC 53454 China indica - + - H18 IRGC 66515 Sri lanka indica + + n H38 IRGC 66517 Sri lanka indica + - + H28 IRGC 66524 Sri lanka indica + + + H36 IRGC 66528 Sri lanka indica + - + H28 IRGC 66560 Indonesia indica + + + H9 IRGC 66627 Indonesia indica + - + H28 IRGC 66809 Bangladesh indica - - n H14 IRGC 66813 Bangladesh indica - - n H14 IRGC 66816 Bangladesh indica - - n H14 IRGC 66831 Bangladesh indica - - n H40 IRGC 67468 Indonesia / + - + H28 IRGC 71644 Malaysia indica + - + H41 IRGC 76290 India indica + + + H24 IRGC 76404 Pakistan indica - + - H16 IRGC 77143 China indica + - + H28 IRGC 77194 Bangladesh indica + + + H1 IRGC 77196 Bangladesh indica + + + H35 IRGC 77529 India indica + - + H28 IRGC 77636 Indonesia indica + - n H21 IRGC 77645 Korea japonica - + - H16 IRGC 77665 Korea japonica - + - H16 IRGC 78244 Thailand indica + + + H35 IRGC 78269 Thailand indica + + + H32 IRGC 78595 Malaysia indica + + n H38 IRGC 80180 Laos indica + - n H21 IRGC 82097 Myanmar / + - + H28 IRGC 84154 Myanmar indica + - + H28 IRGC 113509 Myanmar / + - - H27

O. nivara (n=23)

IRGC 80622 India + - n H25 IRGC 80723 Myanmar + + n H29 IRGC 81831 India + + + H24 IRGC 81845 India + + + H24

IRGC 81928 India + + + H24

31

IRGC 86474 India + + + H24 IRGC 93186 Nepal + + + H24 IRGC 101450 China + - n H25 IRGC 103821 China + + - H20 IRGC 104684 India + + + H24 IRGC 104686 India + + + H24 IRGC 104695 India + + + H11 IRGC 104703 India + + + H13 IRGC 104706 India + + - H12 IRGC 104969 China + - + H26 IRGC 105317 India + - n H25 IRGC 105343 India + + + H22 IRGC 105360 Thailand + + + H42 IRGC 105371 Thailand + - + H34 IRGC 105409 Sri lanka + + - H23 IRGC 105459 Sri lanka + + + H10 IRGC 106355 Myanmar + - n H39 IRGC 106394 Myanmar + - + H8

O. rufipogon (n=16)

IRGC 80643 India + - n H2 IRGC 81882 India + - + H3 IRGC 83795 India + + + H32 IRGC 104624 China + + n H7 IRGC 105308 India + + + H19 IRGC 105390 Thailand - + n H17 IRGC 105884 Bangladesh + + - H30 IRGC 105887 Bangladesh + + + H35 IRGC 105902 Bangladesh + + + H6 IRGC 105948 Thailand + + + H33 IRGC 106036 Malaysia + + + H31 IRGC 106078 India + + + H5 IRGC 106452 Indonesia + - + H26 IRGC 106508 Myanmar + + + H37 IRGC 106523 Papua new guinea + + + H4 W13 China - + - H47

O. glumaepatula IRGC 105661 Brazil + + + H45 O. sativa (n=8) (representative of indica, japonica, and WCVs, or used in genetic analysis of this study)

Nanjing 11 China indica + - + H28 9311 China indica + + + H15 Minghui 63 China indica + - + H28 IR64 Philippines indica + - + H46 Balilla Italy japonica - + - H16 Nipponbare Japan japonica - + - H16 02428 China WCV + + n H43 Dular India WCV - - n H44

*Subspecies information is obtained from http://www.irgcis.irri.org:81/grc/SearchData.htm

32

Table S4. Occurrence of combinations of ORFs3-5 in 82 accessions with representatives from three rice species. Combination (ORF3, 4, 5) O. sativa O. nivara O. rufipogon Total

+, +, + 9 12 9 30 +, +, - 0 3 1 4 +, +, n 3 1 1 5 +, -, + 14 3 2 19 +, -, - 1 0 0 1 +, -, n 2 4 1 7 -, +, + 0 0 0 0 -, +, - 9 0 1 10 -, +, n 1 0 1 2 -, -, + 0 0 0 0 -, -, - 0 0 0 0 -, -, n 4 0 0 4 Total 43 23 16 82

33

Table S5. PCR-based markers used for genotyping of the S5 locus.

Marker name Corresponding gene Primer sequences (5’-3’)

PCR product size (bp) for different varieties

02428 9311 Balilla/ Nipponbare Dular Nanjing 11

S513 ORF3 Forward: ACCCACCTTGGTTCATCGTC Reverse: TTCCCGTCCTCTACCTGTTG

133 133 120 120 133

S5P50 ORF4 Forward: GAACACCTCGAATAAGCT Reverse: CTGCTGCCTCTGTGTCTA

128 128 128 117 117

S5W ORF5 Forward: GCCATTCCTTCCGATAT Reverse: GTGCTTTGCTACTTACCG

333 469 469 333 469

S5-dCAPS (Hind III)

ORF5 Forward: GCATGGATGTCAAGTACAGCG Reverse: CGTCAGTGGGCAAGCAGAAG

130 130 110 130 130

34

Table S6. Primers used for sequencing and construct preparation. Primer Purpose Primer sequence (5’-3’) 3S1F ORF3 sequencing TCTCCACATGCCCTTCACGCCT 3S1R ORF3 sequencing TCACGGCGAGGATCGTGGACC 3S2F ORF3 sequencing ACCAGGGTCTCTTGTTGCTCGCT 3S2R ORF3 sequencing ATGCGCCTCCTACACAGAAGGCA 3S3F ORF3 sequencing TTCACCACATCGTCCTCCACCCTG 3S3R ORF3 sequencing ATGGCTAAATCCCGGGCGTGACT 3S4F ORF3 sequencing ACGCTGCGACCATCGGACATAG 3S4R ORF3 sequencing ATCGAGCAAGCAAATAAAGAAAGAG SC1F ORF3 sequencing GACATTGAGCAGGCGGTATGCGCT SC1R ORF3 sequencing CGCGAGGATTCCCAAGGTTCGTCA 3CDSFa ORF3 CDS ATGGGGCGTCGTCGTCTTC 3CDSR1a ORF3 CDS TCAGATGCCTCCGACGGAAC 3CDSR2a ORF3 CDS CTACACGAGAGAGATCAGATG 4CDSFa ORF4 CDS ATGAGGGCGATGGCG 4CDSR1a ORF4 CDS TCAGAGTAGCGCTGACGTG 4CDSR2a ORF4 CDS TCATAACAGTCGTGGGGAG UPM Long ORF4 RACE CTAATACGACTCACTATAGGGCAAGCAGTG

GTATCAACGCAGAGT UPM Short ORF4 RACE CTAATACGACTCACTATAGGGC NUPM ORF4 RACE AAGCAGTGGTATCAACGCAGAGT G4-2F ORF4 RACE TTGCTGTCATGTCAGGAGACCA G4-2R ORF4 RACE ACAGGCCAGATGTCGAGCGAGT G4-5F ORF4 RACE GCTCCTGCACATGAGGATAGGT G4-5R ORF4 RACE GTGGCAAGATCGAAGGAAGGCT G4-10F ORF4 RACE CCTTCCTTCGATCTTGCCACTA G4-10R ORF4 RACE CTGCTTCTGCTTCTTCCTCCTC G4-6F ORF4 RT-PCR GCGGCAAGAAGAATAAGACGAG G4-6R ORF4 RT-PCR GAGCTGTTGTTTGAGCTGGGAT G4-12F ORF4 RT-PCR CATGCATTTATCTTGTGCACTG G4-13R ORF4 RT-PCR TTCCCCCTAGCTAATACTAG G4-8F ORF4 RT-PCR AGACGAGGAGGAAGAAGCAGAA F18 ORF4 sequencing TGTCAACGCCGCATGGTTCTGAGA R10 ORF4 sequencing CAGGCAGTCAAACGTAGGAAAGGA F18-01 ORF4 sequencing CGAGATAAACCTTGGCAAACGCGT R10-01 ORF4 sequencing CTTCCTTCGATCTTGCCACTACCT F18-02 ORF4 sequencing AACGATGCTCATGCATGCTGAGGT R10-02 ORF4 sequencing CCTCTGCTGCCTCTGTGTCTACGT F18-03 ORF4 sequencing GTGACGTCGAGATAAACCTTGGCA R10-03 ORF4 sequencing TTCGGTCGCACAATGGACGCAACA aPrimers used for CDS amplification. 3CDSF/3CDSR1 and 3CDSF/3CDSR2 were used for ORF3- and ORF3+, respectively; 4CDSF/4CDSR1 and 4CDSF/4CDSR2 were used for ORF4+ and ORF4-, respectively.

35

Table S7. Primers used in qPCR analysis of ER stress-responsive and PCD related genes and OsbZIP50-S detection.

Gene Loci of RGAP Forward (5' to 3') Reverse (5' to 3') OsBiP1 Os02g02410 CTGTCGTGACTGTTCCTGC GTTGGCTCATTGATGATCC OsBiP2 Os03g50250 AGTTGCAGATGGGGCGAAGGGA TGATCCGGTTGCCCTGGTCGTT OsBiP3 Os05g30480 TCAGGGACTACTTCGGCGGCAA AGGACGACCATGTCGCGGGTTT OsBiP4 Os05g35400 CAAGGAGGAGTACGAGGAGAAG CACACTTTCGATCGAATCCAAAC OsBiP5 Os08g09770 AACACGCTCGGCGGCAAGAT TCTGGTAGACCGCCGACATGA CRT1 Os07g14270 AATGGAAGGCACCGATGA GTCTCCTCTGCGAATGTCT CRT2-1 Os01g67054 CGACTCTTCCTCCGATTCAT CTTCCTGCCGTGTGTTTG CRT2-2 Os03g61670 GTTCAGCAGCAAGGACAAG GTGGCATACCCGCAGATA PDIL1-1 Os11g09280 TGTCATCGCTAAGATGGATGC AGCTCATCCTTGAGAGGCTCG OsbZIP50 Os06g41770 TGGATGGCGATGATCCCATGAGC TGCGCAGCACTGAAGCGCGTAG SAR1B-like Os06g12090 TTCTACATGCTCTCACAAG ACCACGTAAACTACTGCATC ERdj3B-like Os05g06440 GCGACCTATATGTAACTTTCGAG TACTGCTGACTGTTGTACAACCAG Ero1 Os03g52340 GAACCATTTGAATCAATCACTC GCACTTACTATTTCACGATTCAG SDF2-like Os08g17680 CATTACGGGAAATTTAGAGGTTAG GTGTACTTCCTATCATGGCTATG Fes1-like Os09g33780 GAGAGAAGTCCAAATTCTGTTCCAAG TAGTGCAGGGAATGATGAATTAACC OsBI1 Os02g03280 CTACATCAAGCACGCACTC ACCTCTTCTTCCTCTTCTTCTC OsCP1 Os04g57490 TCAAGAACCAGGGCCAGTG CCAGCTCCTGCTCCGACA OsKOD1 Os04g42889 TCAAGCCATTCATCTTCCAT ATCAGCAACCTCGTCAAG Hsr203j Os01g06220 CGGCGAGGCTGAAGGAGAT GATTCAGACCACATGAGAACACCA Profilin Os06g05880 GAGGAGATTACTGGCATCA GTCATCGGCTCATCATAGA OsbZIP50-S Os06g41770 GTGCCGCCACAGCCATGCAG GATGCTCACGAGCCAAAGCAG

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