The optimal number of reference genes required for effective normalization across different tissues of <i>D. latiflorus</i> Munro.

<p>(A) Different tissues, (B) Different samples from seedlings and anther-regenerated plants of different ploidy, (C) All samples. The pairwise variation (Vn/Vn+1) was analyzed between normalization factors NFn and NFn+1 by geNorm program to determine the optimal number of reference genes.</p

Identification of gene specificity and amplicon size.

<p>(A) Agarose gel (2%) electrophoresis showing amplification of a specific PCR product of the expected size for each gene. (B) Melting curves of twelve candidate reference genes and two target genes showing single peaks.</p

Expression stability values of the candidate reference genes calculated using BestKeeper.

*<p>Coeff. of corr (r) is the shortage of Coefficient of correlation.</p

Ranking of the candidate reference genes according to their stability value using NormFinder.

<p>Ranking of the candidate reference genes according to their stability value using NormFinder.</p

Gene expression stability of the twelve candidate genes of <i>D. latiflorus</i> Munro as predicted by geNorm.

<p>(A) Different tissues, (B) Different samples from seedlings and anther-regenerated plants of different ploidy, (C) All samples. Lower average expression stability (M value) indicates more stable expression. The least stable genes are on the left, and the most stable on the right.</p

Description of twelve candidate reference genes for RT-qPCR in <i>D. latiflorus</i> Munro.

<p>Description of twelve candidate reference genes for RT-qPCR in <i>D. latiflorus</i> Munro.</p

RT-qPCR Ct values for reference genes.

<p>Ct values for each reference gene in all Hyperaccumulating ecotype of <i>S. alfredii</i> samples. A line across the box depicts the median. The box indicates the 25% and 75% percentiles, whiskers caps represent the maximum and minimum values, dots represent outliers.</p

Pairwise variation (V) calculated by geNorm to determine the optimal number of reference genes.

<p>The average pairwise variations (Vn/Vn+1) were analyzed to measure the effect of adding additional reference gene on the RT-qPCR normalization for all samples.</p

Confirmation of primer specificity and amplicon size.

<p>(a) Melting curves of ten candidate reference genes and one target gene showing single peaks. (b) Agarose gel (2%) showing specific RT-qPCR products of the expected size for each genes. M1 and M2 represent 2000bp and 500bp DNA marker respectively.</p

Expression stability (<i>M</i>) of ten reference genes across all 31 samples calculated by geNorm.

<p>(a) all 31 samples, (b) different tissue/organs, (c) Cd²⁺ treatment, (d) Pb²⁺ treatment, (e) Zn²⁺ treatment, (f) Cu²⁺ treatment.</p