Journal of Plant Biology

Identification of Submergence-responsive Genes in two IndicaRice Genotypes Carrying SUB1A-1 but Exhibiting Differential Tolerance

Huaiyang Xiong, Jing Yang, Yangsheng Li*

State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, 430072, China

*Corresponding author. Tel:+86 27 68756631; Fax: +86 27 87214095; e-mail:

Supplementary data

SupplementaryFig. 1 Alignment of full-length coding sequences of SUB1A cDNA from FR13A and Goda Heenati

Supplementary Table1Sequences of primers for qRT-PCR or RT-PCR

SupplementaryTable2Statistics for submergence-responsiveESTs identified by combining suppression subtractive hybridizationand differential screening

Supplementary Table3Submergence-responsive genes in common between FR13A and Goda Heenati genotypes following 3-d submergence

Supplementary Table4Quantitative real-time PCR for validation of microarray data

Supplementary Table5Genes showing a significant genotype-treatment interaction effect after 3-d submergence

Supplementary Table6Genes showing differential regulation pattern between FR13A and Goda Heenati after 3-d submergence

Supplementary Table7 Metabolic pathways enriched in submergence-responsive genes in common between FR13A and Goda Heenati

SupplementaryFig. 1

Supplementary Fig. 1 Alignment of full-length coding sequences of SUB1A cDNA from FR13A and Goda Heenati

Supplementary Table1Sequences of primers for qRT-PCR or RT-PCR

Gene / GenBank accession / Former primer (5' - 3') / Reverse primer (5' - 3') / Annealing
temperature (℃) / Reference
Actin / AK058421 / ACAGGTATTGTGTTGGACTC / GCTTAGCATTCTTGGGTCC / 60 / Xu et al. 2006
G3PDH / AK062122 / GGTATGGCTTTCCGTGTTC / ACCCTGGAAGTCTGTGGAAAC / 59
SUB1A-1 / DQ011598 / CTTCTTGCTCAACGACAACG / AGGCTCCAGATGTCCATGTC / 55 / Singh et al. 2010
Os01g0369700 / AK101436 / ACCTCAGCCACTTCTCCAACATG / GGACAACAGCAAGCACACTATGG / 60
Os01g0692000 / AK064650 / AGAAGTTGTTTGGTACTGTGTCATGTG / CCTCACTATTTTGCTCTCCTTTTATTC / 58
Os03g0163300 / AK103418 / GGACTAGAGGAGGTTGGGGTG / AGAAAACGGCAGATGGTACAGC / 57
Os07g0694700 / AB053297 / GACCAACTTCCCATCCTCTCC / CCTTGTCACTCAAACCCATCTG / 57
Os10g0530500 / AF402800 / CTCCCCGCCGACCCCTAC / TTCCCGCCCGCGCACT / 62
Os11g0642800 / AB185079 / GAAGACTGCAATAGAAAAACCTGAC / CAAGTGCCTCTCCCTGCTCC / 56
Os12g0468100 / CI105466 / ACCACTGGTCCAAGCTATGTTCC / ATGGTGTCGATTTCAATTCTCGG / 60
Os04g0290000 / CI277792 / CGATCCATCGAATTCTTTTTGATAC / TCTGATTTCATCTCCATCCCCAC / 59
Os06g0185400 / AK107760 / TGGGAGGAAGCACGAGCAAG / CACACAGAGCCACAGAAAAGACATC / 60
Os03g0801000 / AK111357 / ACGGCGTGGTGGTTCATCG / GGTTTGCCTCCAGGTTACGG / 59
Os08g0374400 / CI278351 / TAGGCTGTAGCTAGGAGCAGGC / TCATGTTTTATCGCAAGGTTGTAC / 58
Os03g0334500 / AK066465 / ATATCTGTTCTTGGCACACTTGGC / CACTTCTTCAGCATCCTCTCGTTC / 59
Os06g0286500 / AK121397 / GGAGAGCATCCGCCATCTAGCA / GTGTTCGCCCCACAGGTCTTG / 60
Os12g0630200 / CI025931 / ACGAAATACTCCAAGTTCTTCAAG / ACACAAGATCACTACCCCTTATTATC / 55
Os12g0202700 / Os12g0202700 / GCTCCCTTCACTGTGTTCGTT / CAGGTCATAGTTGATGCCCCT / 56
Os09g0269900 / AK064489 / CGGAGAAAGAATTAAGGGAGCG / TTGGACGAGTGGTGTAGCGAC / 58

Supplementary Table1Sequences of primers for qRT-PCR or RT-PCR(continued)

Gene / GeneBank
accession / Former primer (5' - 3') / Reverse primer (5' - 3') / Annealing
temperature (℃) / Reference
Os12g0247700 / AK066682 / ATTTGGACGACAGGAGGGAAC / ATCACAACAGGAATGATAGGGAGG / 57
Os02g0593600 / AK069976 / GCTTCGCCTCGCTTCCAAC / CAGCCTCGCCATCTCCTGC / 60
Os08g0470700 / CI456745 / GGGAGTGATCTGGACGGTTTTC / TCATGTCTGGCGATGTGTTGG / 60

Supplementary Table 2 Statistics for submergence-responsiveESTs identified by combining suppression subtractive hybridizationand differential screening

Library Name / Forward Goda Heenati / Reverse Goda Heenati / Forward FR13A / Reverse FR13A
Dot blot / 1,680 / 2,016 / 1,920 / 2,688
Sequenced / 286 / 326 / 178 / 336
Post-Filteringa / 241 / 291 / 161 / 296
Contigsb / 16 / 13 / 6 / 20
Singletonsb / 203 / 264 / 150 / 251
Nonredundant ESTsc / 219 / 277 / 156 / 271
Represented genesd / 214 / 261 / 156 / 266

aEST redundancy caused by multiple clones with the same insert was first removed by manual sequence alignment before submission to NCBIdatabase. bThe filtered sequences were then were assembled into singletons and contigs using EGassembler software ( number of nonredundant ESTs is the sum of the number of contigs plus the number of singletons.dEST identities were determined by a homology search using BLASTN against the nonredundant GenBank database

Supplementary Table4Quantitative real-time PCR forvalidation of microarray dataa

Gene / Real-time PCR / Microarray / Real-time PCR / Microarray
FR13A_Ctrl / FR13A_Sub / FC(FR13A_Sub/
FR13A_Ctrl) / FC(FR13A_Sub/
FR13A_Ctrl) / Goda Heenati_Ctrl / Goda Heenati_Sub / FC(Goda Heenati_Sub/
Goda Heenati_Ctrl) / FC (Goda Heenati_Sub/
Goda Heenati_Ctrl)
Mean Cp / STD Cp / Mean Cp / STD Cp / Mean Cp / STD Cp / Mean Cp / STD Cp
G3PDH / 21.17 / 0.038 / 19.71 / 0.15 / 1.00 / 23.52 / 0.042 / 19.91 / 0.18 / 1.00
Os01g0353400 / 27.76 / 0.048 / 29.07 / 0.47 / 0.15 / 0.19 / 32.45 / 0.046 / 28.62 / 0.48 / 1.16 / 2.55
Os01g0369700 / 27.25 / 0.33 / 28.94 / 0.26 / 0.11 / 0.24 / 36.73 / 0.65 / 33.14 / 0.84 / 0.99 / 1.25
Os01g0692000 / 28.97 / 0.45 / 31.08 / 0.34 / 0.084 / 0.16 / 30.96 / 0.21 / 33.15 / 0.16 / 0.018 / 0.06
Os03g0163300 / 27.82 / 0.14 / 28.53 / 0.48 / 0.22 / 0.57 / 29.03 / 0.13 / 28.14 / 0.077 / 0.15 / 0.34
Os05g0116100 / 22.65 / 0.063 / 23.62 / 0.33 / 0.19 / 0.43 / 24.05 / 0.12 / 24.44 / 0.057 / 0.062 / 0.15
Os07g0694700 / 21.42 / 0.057 / 22.36 / 0.025 / 0.19 / 0.26 / 23.47 / 0.085 / 22.41 / 0.049 / 0.17 / 0.21
Os10g0530500 / 24.64 / 0.16 / 29.05 / 0.34 / 0.017 / 0.026 / 26.76 / 0.023 / 30.68 / 0.54 / 0.0054 / 0.0092
Os11g0642800 / 27.41 / 0.49 / 25.72 / 0.21 / 1.17 / 0.89 / 38.57 / 0.72 / 34.62 / 0.11 / 1.27 / 2.07
Os12g0468100 / 26.16 / 0.25 / 29.56 / 0.51 / 0.034 / 0.303 / 32.28 / 0.32 / 29.24 / 0.28 / 0.67 / 1.23
Os04g0290000 / 25.05 / 0.12 / 24.51 / 0.072 / 0.53 / 1.02 / 29.89 / 0.086 / 31.42 / 0.58 / 0.028 / 0.15
Os06g0185400 / 33.95 / 0.46 / 33.32 / 0.62 / 0.56 / 0.99 / 29.89 / 0.17 / 24.58 / 0.11 / 3.26 / 4.63
Os03g0801000 / 14.36 / 0.041 / 13.14 / 0.14 / 0.85 / 0.96 / 16.7 / 0.18 / 12.11 / 0.061 / 1.97 / 2.45
G3PDH / 13.56 / 0.32 / 12.60 / 0.56 / 1.00 / 16.55 / 0.24 / 12.65 / 0.19 / 1.00
Os08g0374400 / 35.6 / 0.16 / 35.36 / 0.61 / 0.61 / 0.98 / 36.13 / 1.13 / 31.04 / 1.61 / 2.28 / 4.92
Os03g0334500 / 38.6 / 1.25 / 38.44 / 0.95 / 0.57 / 1.23 / 35.23 / 0.82 / 37.81 / 0.61 / 0.011 / 0.71
Os06g0286500 / 29.69 / 0.069 / 29.09 / 0.23 / 0.78 / 1.16 / 35.78 / 1.41 / 35.6 / 0.69 / 0.076 / 0.12

aResults from real-time qPCR of 15 target genes. The G3PDH gene was used as an internal control. The crossing points (Cp) were determined for the target genes and the internal control based on‘Fit Point Method’ in the LightCycler software 3.3 (Roche Diagnostics). Fold changes (FC) in expression were computed as described by Livak & Schmittgen (2001)

Supplementary Table 7Metabolic pathways enriched in submergence-responsive genes in common betweenFR13A and Goda Heenati

Metabolic pathwaya / P-valueb / Enzymec / Gene IDd
Pathways involving up-regulated genes
Valine, leucine and isoleucinebiosynthesis / 0.023 / Pyruvate dehydrogenase E1alpha subunit / Os04g0119400
Branched-chain-amino-acid aminotransferase / Os04g0559400
Carbohydrate transporter/ sugar porter / Os10g0360100
Leucyl-tRNA synthetase / Os05g0241100
Stilbene, coumarine and lignin biosynthesis / 0.051 / Peroxidase 1 precursor / Os01g0326100
Peroxidase 2 precursor / Os04g0688300
Peroxidase 52 precursor / Os06g0546500
Peroxidase 16 precursor / Os06g0695200
Peroxidase 1 precursor / Os07g0104500
Peroxidase 54 precursor / Os10g0109300
Peroxidase N precursor / Os10g0109600
Caffeoyl-CoA O-methyltransferase 2 / Os08g0498600
Trans-cinnamate 4-monooxygenase / Os02g0467600
Cyanogenic beta-glucosidase precursor / Os01g0813800
Periplasmic beta-glucosidase precursor / Os02g0131400
Non-cyanogenic beta-glucosidase / Os04g0474800
Metabolismofxenobiotics by cytochrome P450 / 0.067 / Cytochrome P450 family protein / Os07g0644600
Cytochrome P450 86A1 / Os10g0524700
Chloroplastic quinone-oxidoreductase / Os04g0358000
Alcohol dehydrogenase 1 / Os11g0210300
Alcohol dehydrogenase 2 / Os11g0210500
Carbon fixation / 0.17 / Pyruvate, phosphate dikinase / Os03g0432100
Phosphoglycerate kinase / Os06g0668200
Aspartate aminotransferase / Os01g0760600
Aspartate aminotransferase / Os02g0236000
Starch and sucrose metabolism / 0.18 / Pectinesterase / Os01g0788400
Glucan endo-1,3-beta-glucosidase GV / Os01g0946500
Glucan endo-1,3-beta-glucosidase GV / Os01g0946700
Beta-1,3-glucanase-like protein / Os07g0539900
Glucan 1,3-beta-glucosidase precursor, / Os10g0370500
Cyanogenic beta-glucosidase precursor / Os01g0813800
Periplasmic beta-glucosidase precursor / Os02g0131400
Non-cyanogenic beta-glucosidase / Os04g0474800
CSLC9, cellulose synthase-like family C / Os03g0770800
Glycogen synthase / Os01g0720600
1,4-alpha-glucan branching enzyme IIB / Os02g0528200

Supplementary Table 7Metabolic pathways enriched in submergence-responsive genes in common betweenFR13A and Goda Heenati(continuded)

Metabolic pathwaya / P-valueb / Enzymec / Gene IDd
Pathways involving down-regulated genes
Flavonoid biosynthesis / 0.016 / 3-ketoacyl-CoA synthase / Os02g0731900
Isoflavone reductase-like protein / Os01g0106400
Leucoanthocyanidin reductase / Os04g0630300
Leucoanthocyanidin reductase / Os04g0630400
Cytokinin-O-glucosyltransferase 1 / Os01g0638600
Anthocyanidin 3-O-glucosyltransferase / Os01g0865400
Dihydroflavonol-4-reductase / Os01g0283700
Dihydroflavonol-4-reductase / Os08g0277200
Cytochrome P450 71A4 / Os01g0227500
Cytochrome P450 / Os02g0504000
Cytochrome P450 76C4 / Os10g0144700
Photosynthesis / 0.023 / Vacuolar ATP synthase subunit B isoform 1 / Os01g0711000
Vacuolar ATP synthase subunit F / Os02g0824700
Ferredoxin--NADP reductase / Os03g0784700
Cyanoaminoacid metabolism / 0.081 / Nitrilase / Os06g0206000
Beta-glucosidase / Os04g0513400
Beta-primeverosidase / Os06g0320200
Gamma-glutamyltranspeptidase family protein / Os04g0457500
L-asparagine amidohydrolase / Os04g0650700
Oxidative phosphorylation / 0.15 / Cytochrome c1, heme protein / Os01g0935700
Vacuolar ATP synthase subunit B isoform 1 / Os01g0711000
Vacuolar ATP synthase subunit F / Os02g0824700
Soluble inorganic pyrophosphatase / Os02g0704900

aPathway analysis was performed by theMicro Array Data Interface for Biological Annotationweb tool (Law et al. 2008).

bFisher's exact test was used to calculate a P-value determining the probability that the association between the enzymes and the pathway is explained by chance alone. A pathway with P-value0.05 is considered assignificant.

cEnzymes mapped onto the pathway and encoded by submergence-responsive genes.

dIdentifiers of genes encoding the corresponding enzymes, based on the Rice Annotation Project Database

References

Xu K, Mackill DJ (1996) A major locus for submergence tolerance mapped on rice chromosome 9. Mol Breed 2:219–224

Singh N, Dang TTM, Vergara GV, Pandey DM, Sanchez D, Neeraja CN, Septiningsih EM, Mendioro M, Tecson-Mendoza EM, Ismail AM, Mackill DJ, Heuer S(2010) Molecular marker survey and expression analyses of the rice submergence-tolerance gene SUB1A. Theor Appl Genet 121:1441–1453

Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2 (-Delta Delta C(T)) method. Methods 25:402–408