POJ 7(6):527-539(2014) ISSN:1836-3644 Supplementary data Comparison of transcriptomes of chilling- and drought-tolerant and intolerant Nicotiana tabacum varieties and identification of genes associated with stress tolerance Dahai Hao, Wenguang Ma, Yelong Sheng, Jianbo Zhang, Yunfeng Jin, Huiqin Yang, Zhongguang Li, Shasha Wang, Ming Gong* Supplementary Figures A B Supplementary Figure 1 The length distribution of unigenes (A) and non-redundant unigenes (B) of Nicotiana. tabacum cv. Yunyan203 and MSK326. Supplementary Figure 2. Unigenes of chilling and drought stressed N. tobacco associated with glutathione metabolic pathway for revealed unigenes by KEGG (from: http://www.genome.jp/kegg/kegg1.html). Supplementary Figure 3. Gene ontology (GO) enrichment analysis. The digital gene expressions (DGEs) of MSK326 (stress tolerance) and Yunyan203 (stress intolerance) cultured under control conditions. In this plot, the p values of all GO terms were obtained using Pearson chi-square test; the gene numbers between these two DGE libraries and were all significant. Supplementary Figure 4. Gene ontology (GO) enrichment analysis of common down-regulated differentially expressed genes (DEGs) under chilling stress. Comparison of GO enrichment between the chilling stress-tolerant MSK326 and chilling stress-intolerant Yunyan203. The p values of all GO terms were obtained using Pearson chi-square test; the gene numbers of the two digital gene expression (DGE) libraries were all significant. Supplementary Figure 5. Gene ontology (GO) enrichment analysis of common differentially expressed genes (DEGs) of drought-stressed samples. Comparison of GO enrichment analysis of drought-tolerant MSK326 and drought-intolerant Yunyan203. The p values of all GO terms were obtained using Pearson chi-square test; the gene numbers of the two digital gene expression (DGE) libraries were all significant. Supplementary Figure 6. Differences between common differentially expressed genes (DEGs) of chilling and drought stress samples. The p values of all gene ontology (GO) terms were obtained using Pearson chi-square test; the gene numbers of the two digital gene expression (DGE) libraries and were all significant. 16000 160 MSK326-chilling-CYP Msk326-drought-CYP Multiple expressed genes in control Multiple expressed genes in control 18000 14000 12000 10000 8000 6000 4000 2000 140 Yunyan203-chilling-CYP Yunyan203-drought-CYP 120 100 80 60 40 20 0 0 12 h 24 h 12 h 48 h Time vs MSK326-chilling-MMT Time vs MSK326-drought-MMT 48 h 30 Multiple expressed genes in control Multiple expressed genes in control 3500 24 h Time Time 3000 2500 2000 1500 1000 500 25 Yunyan203-chilling-MMT Yunyan203-drought-MMT 20 15 10 5 0 0 12 h 24 h 48 h Time 12 h 24 h 48 h Time Supplementary Figure 7. Comparison of cytochrome P450 (CYP) and myricetin O-methyltransferase 2 (MMT) expression between control and stress-treated N. tabacum samples. 80 MSK326-chilling Yunyan203-chilling MSK326-drought Yunyan203-drought 40 FPKM of ¦Ø-3 fatty acid desaturase FPKM of ¦Ø-6 fatty acid desaturase 50 30 20 10 0 0h 12h 24h MSK326-chilling Yunyan203-chilling MSK326-drought Yunyan203-drought 70 60 50 40 30 20 48h 0h 12h Time 24h 48h Time Supplementary Figure 8. Expression pattern of fatty acid desaturase (FAD)6 and FAD3 of in chilling- and drought-treated N. tabacum samples. 9000 MSK326-Chilling Yunyan203-Chilling MSK326-Drought Yunyan203-Drought 8000 FPKM of aquaporins 7000 6000 5000 4000 3000 2000 1000 0h 12h 24h 48h Time Supplementary Figure 9. The sum of FPKM for aquaporins in the 14 digital gene expressions (DGEs) of N. tabacum. FPKM, fragments per kilobase of exon per million fragments mapped. 1000 MSK326-chilling Yunyan203-chilling MSK326-drought Yunyan203-drought FPKM of LEA 5 800 600 400 200 0 0h 12 h 24 h 48 h Time Supplementary Figure 10. The sum of FPKM for late embryogenesis abundant (LEA) 5 proteins in 14 digital gene expressions (DGEs) of N. tabacum. FPKM, fragments per kilobase of exon per million fragments mapped. 60000 MSK326-chilling Yunyan203-chilling MSK326-drought Yunyan203-drought FPKM of dehydrin 50000 40000 30000 20000 10000 0 0h 12 h 24 h 48 h Time Supplementary Figure 11. The sum of FPKM for dehydrin (DHN) proteins in the 14 digital gene expressions (DGEs) of N. tabacum. FPKM, fragments per kilobase of exon per million fragments mapped. 1000 MSK326-chilling Yunyan203_chilling MSK326-drought Yunyan203_drought FPKM of P5CS 800 600 400 200 0 0h 12h 24h 48h Time Supplementary Figure 12. The FPKM for â–³1-pyrroline-5-carboxylate synthase (P5CS) in the 14 digital gene expressions (DGEs) of N. tabacum. FPKM, fragments per kilobase of exon per million fragments mapped. FPKM of trehalose-6-phosphate synthase 500 MSK326-chilling Yunyan203-chilling MSK326-drought Yunyan203-drought 400 300 200 100 0 0h 12 h 24 h 48 h Time Supplementary Figure 13. The FPKM for trehalose-6-phosphate synthase in the 14 digital gene expressions (DGEs) of N. tabacum. FPKM, fragments per kilobase of exon per million fragments mapped. Yunyan203-chilling-SOD Yunyan203-drought-SOD Yunyan203-chilling-APX Yunyan203-drought-APX 2200 2200 MSK326-chilling-SOD MSK326-drought-SOD MSK326-chilling-APX MSK326-drought-APX 2000 1800 2000 1800 FPKM of Unigenes FPKM of Unigenes 1600 1600 1400 1200 1000 800 1400 1200 1000 800 600 600 400 400 200 200 0h 12 h 24 h 0 48 h 0h 12 h 24 h 48 h Time Time 6000 6000 MSK326-chilling-CAT MSK326-drought-CAT Yunyan203-chilling-CAT Yunyan203-drought-CAT 5000 FPKM of Unigenes FPKM of Unigenes 5000 4000 3000 2000 4000 3000 2000 1000 1000 0 0 0h 12 h 24 h 0h 48 h 12 h 24 h 48 h Time Time Supplementary Figure 14. The FPKM for superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT) in the 14 digital gene expressions (DGEs) of N. tabacum. FPKM, fragments per kilobase of exon per million fragments mapped. 3000 MSK326-chilling Yunyan203-chilling MSK326-drought Yunyan203-drought FPKM of DREBs 2500 2000 1500 1000 500 0 0h 12 h 24 h 48 h Time Supplementary Figure 15. The FPKM for dehydration responsive element binding (DREB) proteins in the 14 digital gene expressions (DGEs) of N. tabacum. FPKM, fragments per kilobase of exon per million fragments mapped. 1800 MSK326-chilling-NAC Yunyan203-chilling-NAC MSK326-drought-NAC Yunyan203-drought-NAC 1600 FPKM of Unigenes 1400 1200 1000 800 600 400 200 0 0h 12 h 24 h 48 h Time Supplementary Figure 16. The FPKM of NAC in the 14 digital gene expressions (DGEs) of N. tabacum. FPKM, fragments per kilobase of exon per million fragments mapped. Supplementary Figure 17. Some common reaction of N. tabaccum cv. Yunyan203 (intolerant) and MSK326 (tolerant) to drought/chilling stress.
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