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Supplemental Information

Supplementary table S1 Stability evaluation of 13 candidate reference genes by four statistical algorithms in Saccharum spp

SV, stability value

DOI: 10.7287/peerj.preprints.1487v1/supp-1

Supplementary table S2 The general information of ScMTP, ScMT2-1-3, ScMPP and ScHMA1 for quantitative real time PCR

DOI: 10.7287/peerj.preprints.1487v1/supp-2

Figure 1 The optimal combination of reference genes for gene expression normalization under heavy metal stresses in sugarcane

The pairwise variation (Vn/Vn+1) was analyzed between normalization factors NFn and NFn+1 by geNorm program to determine the optimal combination of reference genes for accurate normalization in samples from different sugarcane cultivar samples.

DOI: 10.7287/peerj.preprints.1487v1/supp-3

Figure 2 Expression analysis of ScMTP, ScMT2-1-3, ScMPP and ScHMA1 genes based on selected reference gene / genes under cadmium stress

ScMTP (Sugarcane metal tolerance protein gene, GenBank Accession No. KP864146), ScMT2-1-3 (Sugarcane metallothionein, GenBank Accession No. JQ627644), ScMPP (sugarcane metallophosphoesterase, GenBank Accession No. CA267392.1) and ScHMA1 (sugarcane heavy metal transporting ATPase 1, GenBank Accession No. CA156665.1) were heavy metal stress response genes in sugarcane. In this study, the normalization of ScMTP (A), ScMT2-1-3 (B), ScMPP (C) and ScHMA1 (D) employed a single reference gene, UBQ, APRT, GAPDH or 25S rRNA, or the reference gene set, CAC + CUL as reference control under cadmium chloride (CdCl2) treatment. Using 2-ΔΔCt to normalize the ScMTP, ScMT2-1-3, ScMPP and ScHMA1, the control sample were converted into 1. Significant different expression of ScMTP, ScMT2-1-3, ScMPP and ScHMA1 were marked with p value (p < 0.01 level (highly significant) and p < 0.05 level (significant)) on the line respectively when comparing the normalization by UBQ, APRT, GAPDH and 25S rRNA with the normalization by CAC + CUL.

DOI: 10.7287/peerj.preprints.1487v1/supp-4

Figure 3 Expression analysis of ScMTP, ScMT2-1-3, ScMPP and ScHMA1 genes based on selected reference gene / genes under copper stress

ScMTP (Sugarcane metal tolerance protein gene, GenBank Accession No. KP864146), ScMT2-1-3 (Sugarcane metallothionein, GenBank Accession No. JQ627644), ScMPP (sugarcane metallophosphoesterase, GenBank Accession No. CA267392.1) and ScHMA1 (sugarcane heavy metal transporting ATPase 1, GenBank Accession No. CA156665.1) were heavy metal stress response genes in sugarcane. In this study, the normalization of ScMTP (A), ScMT2-1-3 (B), ScMPP (C) and ScHMA1 (D) employed a single reference gene, UBQ, APRT, GAPDH or 25S rRNA, or the reference gene set, CAC + CUL as reference control under copper chloride (CuCl2) treatment. Using 2-ΔΔCt to normalize the ScMTP, ScMT2-1-3, ScMPPand ScHMA1, the control sample were converted into 1. Significant different expression of ScMTP, ScMT2-1-3, ScMPP and ScHMA1 were marked with p value (p < 0.01 level (2-tailed) and the 0.05 level (1-tailed) on the line respectively when comparing the normalization by UBQ, APRT, GAPDH and 25S rRNA wtih the normalization by CAC + CUL.

DOI: 10.7287/peerj.preprints.1487v1/supp-5

Additional Information

Competing Interests

The authors have declared that no competing financial interests exist.

Author Contributions

Hui Ling performed the experiments, analyzed the data, wrote the paper, prepared figures and/or tables.

Ning Huang performed the experiments, analyzed the data, prepared figures and/or tables.

Liping Xu conceived and designed the experiments, reviewed drafts of the paper.

Long Huang performed the experiments.

Qibin Wu contributed reagents/materials/analysis tools.

Jinlong Guo conceived and designed the experiments.

Yachun Su contributed reagents/materials/analysis tools.

Youxiong Que conceived and designed the experiments, reviewed drafts of the paper.

Data Deposition

The following information was supplied regarding data availability:

The research in this article did not generate any raw data.The research in this article did not generate any raw data.

Funding

This work was funded by the earmarked fund for the Modern Agriculture Technology of China (CARS-20). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


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