Abstract
BackgroundDue to cowpea ability to fix nitrogen in poor soils and relative tolerance to drought and salt stresses, efforts have been directed to identifying genes and pathways that confer stress tolerance in this species. Real-time quantitative PCR (qPCR) has been widely used as the most reliable method to measure gene expression, due to its high accuracy and specificity. In the present study, nine candidate reference genes were rigorously tested for their application in normalization of qPCR data onto roots of four distinct cowpea accessions under two abiotic stresses: root dehydration and salt (NaCl, 100 mM). In addition, the regulation of four target transcripts, under the same referred conditions was also scrutinized.ResultsgeNorm, NormFinder, BestKeeper, and ΔCt method results indicated a set of three statistically validated RGs for each stress condition: (I) root dehydration (actin, ubiquitin-conjugating enzyme E2 variant 1D, and a Phaseolus vulgaris unknown gene—UNK), and (II) salt (ubiquitin-conjugating enzyme E2 variant 1D, F-box protein, and UNK). The expression profile of the target transcripts suggests that flavonoids are important players in the cowpea response to the abiotic stresses analyzed, since chalcone isomerase and chalcone synthase were up-regulated in the tolerant and sensitive accessions. A lipid transfer protein also participates in the cowpea tolerance mechanisms to root dehydration and salt stress. The referred transcript was up-regulated in the two tolerant accessions and presented no differential expression in the sensitive counterparts. Chitinase B, in turn, generally related to plant defense, was an important target transcript under salt stress, being up-regulated at the tolerant, and down-regulated in the sensitive accession.ConclusionsReference genes suitable for qPCR analyses in cowpea under root dehydration and salt stress were identified. This action will lead to a more accurate and reliable analysis of gene expression on this species. Additionally, the results obtained in this study may guide future research on gene expression in cowpea under other abiotic stress types that impose osmotic imbalance. The target genes analyzed, in turn, deserve functional evaluation due to their transcriptional regulation under stresses and biotechnological potential.
Highlights
Due to cowpea ability to fix nitrogen in poor soils and relative tolerance to drought and salt stresses, efforts have been directed to identifying genes and pathways that confer stress tolerance in this species
The initial screening of nine candidate reference genes (RGs) and four target transcripts (Table 1) by quantitative PCR (qPCR) showed that all evaluated primer pairs were functional in all cowpea samples, amplifying a single band as indicated by the presence of a single peak in melting curves (Additional file 5: S2 Appendix)
Considering, preliminarily, a stringent Cqs standard deviation (SD) < 1 associated to Coefficient of variance (CV), all potential RGs were constitutively expressed in the treatments evaluated, with the exception of EF1-α (11.36 ± 1.90), β-TUB (6.70 ± 1.33), and VuUBQ10 (6.64 ± 1.04), concerning samples under salt stress (Table 2)
Summary
Due to cowpea ability to fix nitrogen in poor soils and relative tolerance to drought and salt stresses, efforts have been directed to identifying genes and pathways that confer stress tolerance in this species. Using different methodological approaches (i.e. ESTs, HT-SuperSAGE and RNA-Seq) this effort resulted in the generation of millions of transcripts obtained from cowpea plants under different abiotic [root dehydration and salt (NaCl, 100 mM)] and biotic (infection by Cowpea Aphid-borne Mosaic Virus—CABMV and Cowpea Severe Mosaic Virus—CPSMV) stresses [4]. These data provide a good start for identifying putative genes and gene families associated with resistance/tolerance to such challenging conditions [4]
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