Citrus reference genes ranked according to their expression stability as determined by geNorm.

<p>*UNK: Unknown protein.</p><p>M stability values calculated by geNorm considering all tissues and experimental conditions (total) and each subset (tissue or organ; fungal or oomycete stress; bacterial stress and viral stress). M values are ranked from the most stable pair of genes to the least stable gene.</p

Expression profile of the target genes SvAP3/PI and SvPEPC.

<p>The plot corresponding to the expression profile of the MADS-box gene SvAP3/PI (magenta bars) and the enzyme SvPEPC (green bars) responsible for the initial carbon fixation on C4 photosynthesis is shown in (A). The developmental dataset was normalized using the Si034613 and Si035045 genes. It can be observed in (B) that the expression profile of SvPEPC on the leaf gradient was normalized using the gene pair that included Si021373 and Si034613. This result is compared with the transcriptome data (light blue line) for the same gene (personal communication with Todd Mockler). The small plots correspond to the same described target genes normalized with the poorly ranked reference genes: Si018607 and Si025395 for the developmental set and Si000245 and Si018607 for leaf gradient. The reference samples are indicated with an asterix.</p

Citrus candidate reference gene description and comparison with Arabidopsis orthologs.

<p>Eleven of the fifteen candidate citrus reference genes were selected according to their similarity to reference genes identified in <i>Arabidopsis</i>. Citrus sequences were retrieved from the citrus database (CitEST). Sequences used to design primer pairs for <i>ADP-ribosylation factor</i> (<i>ADP</i>), <i>cathepsin</i> (<i>CtP</i>) and <i>cyclophilin</i> (<i>CYP</i>) were retrieved from HarvEST Citrus according to Carvalho et al. (2010).</p>a<p><i>Arabidopsis</i> Gene Initiative (AGI) locus identifier number.</p>b<p>Unigene identifier according to CitEST database. Abbreviations: CS- <i>Citrus sinensis</i>; CR- <i>C. reticulata</i>; PT- <i>Poncirus trifoliata</i>.</p

Transcript level of the <i>WRKY70</i> transcription factor gene in citrus under infection with <i>C.</i> Liberibacter asiaticus.

<p>Error bars show mean standard error calculated from two biological replicates. Normalization factors were calculated as the geometric mean of the expression levels of the three most stable reference genes (<i>DIM1</i>, <i>GAPC2</i> and <i>PTB1</i>) and the two most unstable (<i>CYP</i> and <i>TUB</i>). A control uninoculated sample was used as calibrator.</p

Average expression stability values (M) calculated by geNorm.

<p>M values of the remaining candidate citrus reference genes during stepwise exclusion of the least stable citrus reference gene in the different subsets. The ranking of the reference genes is in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031263#pone-0031263-t002" target="_blank">Table 2</a>. A lower M value indicates more stable expression. (a) Total citrus samples examined, (b) Different citrus tissues or organs, (c) Fungal or oomycete stress, (d) Bacterial stress and (e) Viral stress.</p

Pairwise variation (V) to determine the optimal number of reference genes for each subset.

<p>(a) Total citrus samples examined, (b) Different citrus tissues or organs, (c) Fungal or oomycete stress, (d) Bacterial stress and (e) Viral stress. The ranking of the reference genes is in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031263#pone-0031263-t002" target="_blank">Table 2</a>.</p

Summary of biotic stress assays used to select candidate citrus genes for normalization in RT-qPCR.

<p>ai⁽¹⁾ = after inoculation.</p><p>CVC⁽²⁾ = Citrus variegated chlorosis.</p

Expression levels of candidate reference genes in different experimental sets.

<p>Box plot graphs of Cq values for each reference gene tested in all citrus samples and subsets. Cq values are inversely proportional to the amount of template and are shown as the first and third quartile. Vertical lines indicate range of values, and median values are indicated by the black lines. Circles indicate outliers. (a) Total citrus samples examined, (b) Different citrus tissues or organs, (c) Fungal or oomycete stress, (d) Bacterial stress and (e) Viral stress.</p

RNA integrity profile.

<p>The assessment of the best methodology for RNA extraction for each representative tissue: seedling (A); shoot (B); root (C), inflorescence (D) and axis (E). Electropherograms were obtained using an Agilent 2100 Bioanalyzer. RNA quality is expressed as the RNA integrity number (RIN).</p

Candidate genes ranked according to their expression stability as determined by NormFinder.

<p>*UNK: Unknown protein.</p><p>Stability values are listed from the most stable to the least stable gene.</p