Reference Gene Selection for qPCR Analysis in Tomato-Bipartite Begomovirus Interaction and Validation in Additional Tomato-Virus Pathosystems

<div><p>Quantitative Polymerase Chain Reaction (qPCR) is currently the most sensitive technique used for absolute and relative quantification of a target gene transcript, requiring the use of appropriated reference genes for data normalization. To accurately estimate the relative expression of target tomato (<i>Solanum lycopersicum</i> L.) genes responsive to several virus species in reverse transcription qPCR analysis, the identification of reliable reference genes is mandatory. In the present study, ten reference genes were analyzed across a set of eight samples: two tomato contrasting genotypes (‘Santa Clara’, susceptible, and its near-isogenic line ‘LAM 157’, resistant); subjected to two treatments (inoculation with Tomato chlorotic mottle virus (ToCMoV) and its mock-inoculated control) and in two distinct times after inoculation (early and late). Reference genes stability was estimated by three statistical programs (geNorm, NormFinder and BestKeeper). To validate the results over broader experimental conditions, a set of ten samples, corresponding to additional three tomato-virus pathosystems that included tospovirus, crinivirus and tymovirus + tobamovirus, was analyzed together with the tomato-ToCMoV pathosystem dataset, using the same algorithms. Taking into account the combined analyses of the ranking order outputs from the three algorithms, <i>TIP41</i> and <i>EF1</i> were identified as the most stable genes for tomato-ToCMoV pathosystem, and <i>TIP41</i> and <i>EXP</i> for the four pathosystems together, and selected to be used as reference in the forthcoming expression qPCR analysis of target genes in experimental conditions involving the aforementioned tomato-virus pathosystems.</p></div

Gene stability ranking order by geNorm, NormFinder and BestKeeper; and their respective arithmetic mean ranking values, considering the entire dataset of ‘Santa Clara’-ToCMoV and ‘LAM 157’-ToCMoV samples.

<p>Gene stability ranking order by geNorm, NormFinder and BestKeeper; and their respective arithmetic mean ranking values, considering the entire dataset of ‘Santa Clara’-ToCMoV and ‘LAM 157’-ToCMoV samples.</p

Pairwise variation (V) of candidate reference genes as predicted by geNorm.

<p>Pairwise variation (Vn/Vn+1) for determination of the optimal number of reference genes predicted by geNorm and calculated between the normalization factors NFn and NFn+1, with a recommended cutoff threshold of 0.150. The pairwise variation was analyzed considering either all samples together (entire dataset) or in six separate subsets: ‘Santa Clara’ and ‘LAM 157’ (genotype subsets); control and inoculated (treatment subsets) and early and late (time after inoculation subsets).</p

Cycle of quantification (Cq) values distribution of candidate reference genes.

<p>Box-whisker plot showing Cq values distribution of each reference gene considering all samples together (entire dataset). The quartiles with medians are represented by boxes green (upper) and red (lower). Whiskers represent the maximum and minimum values.</p

Evaluation of expression stability of reference genes by geNorm, NormFinder, and BestKeeper, considering the entire dataset of ‘Santa Clara’- and ‘LAM 157’-ToCMoV samples.

<p>Evaluation of expression stability of reference genes by geNorm, NormFinder, and BestKeeper, considering the entire dataset of ‘Santa Clara’- and ‘LAM 157’-ToCMoV samples.</p

Expression quantification of target genes in four tomato-virus-pathosystems using different combinations of normalizers.

<p>The normalization of relative expression data was made with two combinations of reference genes: the two most stable genes (<i>TIP41</i> and <i>EXP</i>) and the two least stable genes (<i>GAPDH</i> and <i>EF1</i>) genes. The relative expression of two target genes [<i>Necrotic spotted lesions 1</i> (<i>NECRO</i>) and <i>HR-like lesion inducer</i> (<i>HR-Li</i>)] was evaluated in five pathosystems after virus inoculation relative to their respective mock-inoculated controls. (A) ‘Santa Clara’ 100 DAI with GRSV; (B) ‘Santa Clara’ 100 DAI with ToCV; (C) ‘LAM 144S’ 25 DAI with ToCMoV; (D)‘Moneymaker’ 18 DAI with ToBMV and (E) ‘LAM 144R’ 25 DAI with ToCMoV. Error bars represent the standard deviation of three technical replicates.</p

Gene stability ranking order by geNorm, NormFinder and BestKeeper, considering 18 samples of ‘Santa Clara’; ‘LAM 157’; ‘Moneymaker’ and ‘LAM 144’ inoculated with ToCV; GRSV; ToBMV+ TMV or ToCMoV.

<p>Gene stability ranking order by geNorm, NormFinder and BestKeeper, considering 18 samples of ‘Santa Clara’; ‘LAM 157’; ‘Moneymaker’ and ‘LAM 144’ inoculated with ToCV; GRSV; ToBMV+ TMV or ToCMoV.</p

Expression quantification of target genes in ‘LAM 157’-ToCMoV interaction using different combinations of normalizers.

<p>The normalization of relative expression data was made with two combinations of reference genes: the two most stable genes (<i>TIP41</i> and <i>EF1</i>) and the two least stable genes (<i>ACT</i> and <i>TUB</i>) genes. The relative expression of two target genes [<i>Necrotic spotted lesions 1</i> (<i>NECRO</i>) and <i>HR-like lesion inducer</i> (<i>HR-Li</i>)] was evaluated in ToCMoV inoculated plants of ‘LAM 157’ (resistant genotype) at early (3 and 6 DAI) and late (9, 12 and 15 DAI) times after virus inoculation relative to their respective mock-inoculated controls. Error bars represent the standard deviation of three technical replicates.</p

Early responses to dehydration in contrasting wild <i>Arachis</i> species

<div><p>Wild peanut relatives (<i>Arachis</i> spp.) are genetically diverse and were selected throughout evolution to a range of environments constituting, therefore, an important source of allelic diversity for abiotic stress tolerance. In particular, <i>A</i>. <i>duranensis</i> and <i>A</i>. <i>stenosperma</i>, the parents of the reference <i>Arachis</i> A-genome genetic map, show contrasting transpiration behavior under limited water conditions. This study aimed to build a comprehensive gene expression profile of these two wild species under dehydration stress caused by the withdrawal of hydroponic nutrient solution. For this purpose, roots of both genotypes were collected at seven time-points during the early stages of dehydration and used to construct cDNA paired-end libraries. Physiological analyses indicated initial differences in gas exchange parameters between the drought-tolerant genotype of <i>A</i>. <i>duranensis</i> and the drought-sensitive genotype of <i>A</i>. <i>stenosperma</i>. High-quality Illumina reads were mapped against the <i>A</i>. <i>duranensis</i> reference genome and resulted in the identification of 1,235 and 799 Differentially Expressed Genes (DEGs) that responded to the stress treatment in roots of <i>A</i>. <i>duranensis</i> and <i>A</i>. <i>stenosperma</i>, respectively. Further analysis, including functional annotation and identification of biological pathways represented by these DEGs confirmed the distinct gene expression behavior of the two contrasting <i>Arachis</i> species genotypes under dehydration stress. Some species-exclusive and common DEGs were then selected for qRT-PCR analysis, which corroborated the <i>in silico</i> expression profiling. These included genes coding for regulators and effectors involved in drought tolerance responses, such as activation of osmosensing molecular cascades, control of hormone and osmolyte content, and protection of macromolecules. This dataset of transcripts induced during the dehydration process in two wild <i>Arachis</i> genotypes constitute new tools for the understanding of the distinct gene regulation processes in these closely related species but with contrasting drought responsiveness. In addition, our findings provide insights into the nature of drought tolerance in wild germoplasm, which might be explored as novel sources of diversity and useful wild alleles to develop climate-resilient crop varieties.</p></div

Circos plot detailing chromosome distribution of DEGs.

<p>Distribution of the 1,602 DEGs in the ten chromosomes of <i>A</i>. <i>duranensis</i> (A01 to A10). The outer lines represent exclusive <i>A</i>. <i>duranensis</i> (red) and <i>A</i>. <i>stenosperma</i> (blue) and common (green) DEGs. The inner dots represent the distribution of Log2FC values for each up- and downregulated DEG in <i>A</i>. <i>duranensis</i> (red) and <i>A</i>. <i>stenosperma</i> (blue), with the line indicating Log2FC = 0.</p