Transcriptome Profiling of Wild Arachis from Water-Limited Environments Uncovers Drought Tolerance Candidate Genes

Abstract

Peanut (Arachis hypogaea L.) is an important legume cultivated mostly in drought-prone areas where its productivity can be limited by water scarcity. The development of more drought-tolerant varieties is, therefore, a priority for peanut breeding programs worldwide. In contrast to cultivated peanut, wild relatives have a broader genetic diversity and constitute a rich source of resistance/tolerance alleles to biotic and abiotic stresses. The present study takes advantage of this diversity to identify drought-responsive genes by analyzing the expression profile of two wild species, Arachis duranensis and Arachis magna (AA and BB genomes, respectively), in response to progressive water deficit in soil. Data analysis from leaves and roots of A. duranensis (454 sequencing) and A. magna (suppression subtractive hybridization (SSH)) stressed and control complementary DNA (cDNA) libraries revealed several differentially expressed genes in silico, and 44 of them were selected for further validation by quantitative RT-PCR (qRT-PCR). This allowed the identification of drought-responsive candidate genes, such as Expansin, Nitrilase, NAC, and bZIP transcription factors, displaying significant levels of differential expression during stress imposition in both species. This is the first report on identification of differentially expressed genes under drought stress and recovery in wild Arachis species. The generated transcriptome data, besides being a valuable resource for gene discovery, will allow the characterization of new alleles and development of molecular markers associated with drought responses in peanut. These together constitute important tools for the peanut breeding program and also contribute to a better comprehension of gene modulation in response to water deficit and rehydration.

Keywords: 454 Sequencing; Differential gene expression; Dry-down; Peanut wild relatives; SSH libraries; qRT-PCR.

Fig. 1

Normalized transpiration ratio (NTR) of A. duranensis and A. magna during dry-down and after soil re-watering. Day 0 corresponds to dry-down start. Collecting points (1 to 3) are showed in blue for A. duranensis and in red for A. magna

Fig. 2

a–f Roots of A. duranensis from two WW plants (a) and from two DS plants (b) collected at point 3 (NTR 0.28) and from two DS plants (c) collected at point 5 (72 h after soil re-watering). Each root corresponds to an independent individual. Leaves of A. magna from one WW plant (d) and from one DS plant (e) collected at point 3 (NTR 0.25) and from one DS plant (f) collected at point 4 (5 h after soil re-watering). Each aerial part corresponds to an independent individual. For both species, DS roots (b) and leaves (e) show a phenotype strongly affected by drought imposition when compared to normal WW plants (a, d), such as a dehydrated appearance, roots with evident desiccation, brownish and brittle appearance (b), and wilting leaflets with chlorotic areas (e). The phenotype of re-watered roots (c) and leaves (f) is partially recovered (WW alike) after 72 and 5 h, respectively

Fig. 3

a, b Distribution by plant species of 12,792 A. duranensis contigs (a) and 757 A. magna unigenes (b), according to BLAST hits. Similarities to plant species are determined and affected by the data available in the relevant public databases. Bar values correspond to the percentage of unigenes in relation to the total number of annotated sequences of A. duranensis 454 and A. magna SSH sequences

Fig. 4

Gene Ontology (GO) classification of the predicted A. magna unigenes according to biological process, cellular component, and molecular function using Blast2GO

Fig. 5

Expression analysis of 13 transcription factors (TFs) and 18 other candidate genes (CGs) by qRT-PCR in A. duranensis plants submitted to a gradual water deficit. Axis X represents the relative quantification (RQ) between the DS and WW gathered samples, and the error bars indicate standard deviation of three replicates. Significantly (p < 0.05) upregulated or downregulated genes are indicated by asterisks

Fig. 6

Expression analysis of 13 candidate genes by qRT-PCR in A. magna plants submitted to a gradual water deficit. Axis X represents the relative quantification (RQ) between the DS and WW gathered samples, and the error bars indicate standard deviation of three replicates. Significantly (p < 0.05) upregulated or downregulated genes are indicated by asterisks

Fig. 7

a–p Expression analysis of eight candidate genes by qRT-PCR in leaves and roots of A. duranensis and A. magna submitted to a gradual water deficit and rehydration, at different collecting points. Axis X represents the relative quantification (RQ) between the DS and WW pools and the mean value of three replicates. Significantly (p < 0.05) upregulated or downregulated genes are indicated by asterisks