Comparison of the expression stability of 13 reference genes in <i>Saccharum officinarum</i> as calculated by geNorm, Normfinder and deltaCt.

<p>*β-actin,</p><p>**β-tubulin.</p

The optimal number of reference genes required for effective normalization in each of four experimental sets in sugarcane.

<p>The pairwise variation (V_n/V_n+1) was analyzed between normalization factors NF_n and NF_n+1 by geNorm program to determined the optimal number of reference genes for accurate normalization in samples from different sugarcane cultivar samples (1^st set), sugarcane hormone-treated (2^nd set), abiotic-treated (3^rd set) and treatments (hormone-& abiotic-treated, 4^th). <i>ACT</i> stand for “β-actin” and <i>TUB</i> stand for “β-tubulin”.</p

Gene expression stability of 13 candidate genes in sugarcane as predicted by geNorm.

<p>Average expression stability (M) following stepwise exclusion of the least stable gene across all the samples within an experimental set. The least stable gene is on the left, and the most stable on the right. The name <i>eIF-4a</i> in the figure stands for <i>eIF-4α. ACT</i> stands for “β-actin” and <i>TUB</i> stands for “β-tubulin”.</p

Thirteen reference genes surveyed in this work with their amplification and expression characteristics in <i>Saccharum officinarum.</i>

<p>Mean Ct values and RNA concentration were used for calculating slopes and correlation coefficients (R²). According to the formula [E = (10^(−1/slope)−1)×100%, qPCR efficiencies (E) were calculated based on the standard curves. Mean Ct value (mean), Standard deviation (SD) and Covariance (CV) were calculated by Microsoft Excel 2003 and the Ct values from all of the samples were used. The sequence numbers were obtained from <a href="http://www.ncbi.nlm.nih.gov/nucest/?term=sugarcane" target="_blank">www.ncbi.nlm.nih.gov/nucest/?term=sugarcane</a>.</p

Correlation coefficients based on the visualizing reference genes ranked by geNorm, Normfinder and deltaCt.

<p>The p-value indicated by asterisks (***<0.0001; **<0.05).</p

Expression stability of 13 reference genes in <i>Saccharum officinarum</i> as calculated by Normfinder.

<p>*β-actin,</p><p>**β-tubulin.</p

Expression levels and variation of 13 reference genes across four experimental sets in <i>Saccharum officinarum.</i>

<p>Mean Ct value (Mean), Standard deviation (SD) and Covariance (CV) were calculated by Microsoft Excel 2003. 1^st set: leaf, leaf sheath, stem epidermis,stem pith and bud from “ROC”20, “ROC”22, FN40, liucheng03-182 and YC05-179; 2^nd set: ABA, MeJA and SA, treated-samples from “ROC”20, FN40, Liucheng03-182 and YC05-179; 3^rd set: H₂O₂, NaCl, PEG, CuCl₂ and CdCl₂ treated-samples from “ROC”20, FN40, liucheng03-182 and YC05-179; 4^th set: 2ⁿset+3^rd set.</p

Additional file 2 of Systematic identification of miRNA-regulatory networks unveils their potential roles in sugarcane response to Sorghum mosaic virus infection

Additional file 2: Table S1. The statistic result of sRNA-seq data in 12 samples. Table S2. The statistic result of annotation and classification of the small RNAs (%). Table S3. The differentially expressed miRNAs in sugarcane varieties ROC22 and FN39 infected by SrMV. Table S4. List of 653 annotated predicted target genes corresponding to 132 identified miRNAs. Table S5. List of predicted target genes corresponding to the differentially expressed miRNAs. Table S6. The quantitative primers ofthe 12 selected miRNAs. Table S7. The quantitative primers of the 15 predicted target genes. Table S8. Primers used for cloning the candidate miRNAs and expression analysis of the Nicotiana benthamiana immune-related marker genes

Additional file 1 of Systematic identification of miRNA-regulatory networks unveils their potential roles in sugarcane response to Sorghum mosaic virus infection

Additional file 1: Figure S1. The targeting sites of the selected 12 miRNAs and their 15 target genes predicted by TargetFinder v1.6 software. When the score of miRNA that matches the mRNA is less than or equal to 3, the transcript sequence is considered as the miRNA target gene. Range represents the interval of mRNA used for alignment. Black lines indicate matched RNA base pairs, and two dots show a GU mismatch whereas none dot represents other types of mismatch. The gene ID follows the name of miRNAs and their target genes. Figure S2. The original image of PCR products of the three candidate sugarcane pre-miRNAs detected by electrophoresis. M, DNA marker 15,000 + 2,000 bp; 1, pre-nov_3741; 2, pre-nov_22650; and 3, pre_nov_40875

Phylogenetic trees based on catalase amino acid sequences, showing the phylogenetic relationships between ScCAT1 (KF664183) and the catalases from other plant species.

<p>Neighbor-joining method was used.</p