Introduction
Environmental stresses, such as salinity, are the cause of great losses in crop yields every year all over the globe (Boyer, Reference Boyer1982). Therefore, salt-responsive or salt-tolerance mechanisms have been intensively studied from biological and genetic perspectives. Signal transduction pathways regulate reactive oxygen species, damage repair and ion homeostasis, while maintaining a low Na+/K+ ratio is important for plant survival under salt stress (Kader et al., Reference Kader, Seidel, Golldack and Lindberg2006). Molecules that have a function in the adaptation to environmental stresses can be divided into two groups: the effector molecules and the regulatory molecules (Hasegawa and Bressan, Reference Hasegawa and Bressan2000). In particular, WRKY factors are one of the largest families of transcriptional regulators in plants and form integral parts of signalling webs that modulate many plant processes, including the responses to abiotic stresses, such as drought, cold and salt stresses. The WRKY family is among the ten largest families of transcription factors in higher plants and is found throughout the green lineage (Rushton et al., Reference Rushton, Torres, Parniske, Wernert, Hahlbrock and Somssich1996). Recent studies have confirmed that WRKY proteins often act as repressors as well as activators and that the members of the family play a role in both repression and depression of important plant processes (Ishiguro and Nakamura, Reference Ishiguro and Nakamura1994). Furthermore, it may be that a single WRKY transcription factor is involved in regulating several seemingly disparate processes. The defining feature of WRKY transcription factors is their DNA-binding domain, called WRKY after the most unvaried WRKY amino-acid sequence at the N terminus (Rushton et al., Reference Rushton, Torres, Parniske, Wernert, Hahlbrock and Somssich1996). The WRKY domain consists of a four-stranded β-sheet, with the zinc coordinating Cys/His residues forming a zinc-binding pocket, enabling access to the major DNA groove and contacting with the DNA. High-resolution melting (HRM) analysis has been developed to detect single-nucleotide polymorphisms (SNPs) in small PCR amplicons because it is an easy and low-cost method. Up to now, it has been principally used for scanning mutations of large multiple exon genes to identify disease-related mutations in humans (Kennerson et al., Reference Kennerson, Warburton, Nelis, Brewer, Polly, De Jonghe, Timmerman and Nicholson2007). In the area of plant biology, the use of this technique is still limited and has been applied exclusively for constructing linkage map (Chagné et al., Reference Chagné, Carlisle, Blond, Volz, Whitworth, Oraguzie, Crowhurst, Allan, Espley, Hellens and Gardiner2007; Croxford et al., Reference Croxford, Rogers, Caligari and Whilkinson2008). This technique measures temperature-induced strand separation, and is therefore able to detect variations as small as one base difference between samples. The evolution of the HRM technique from traditional melting curve analysis has been implemented through the invention of new-generation intercalating dyes which can saturate-bind to the double-stranded DNA without PCR inhibition. In this study, we have searched for the presence of SNPs in durum wheat varieties with different degrees of tolerance to salt stress. Conserved portions of WRKY-1 gene were selected through a multi-alignment of homologue sequences of the gene in wild relatives of the species; specific primers have been designed in order to obtain amplicons not longer than 100 bp analysed by HMR technique.
Materials and methods
Four durum wheat varieties, Cham I (moderately salt tolerant), Jennah Khetifa (salt tolerant), Belikh 2 (moderately salt tolerant) and Trinakria (salt susceptible) were used for SNPs discovery and validation in the WRKY-1 gene. Plants were grown with a nutritive solution for 7 d, after which NaCl was added at different concentrations: 0 M (as a negative control), 0.75 and 1.5 M. RNA were then extracted from leaf and root material using TRIzol® reagent (Invitrogen) following the manufacturer's protocol. The cDNA was synthesized using oligo(dT)-primers and the SuperScript™ III reverse transcriptase (Invitrogen), according to the manufacturer's instructions; the reactions were subsequently treated with RNaseH (Invitrogen). The cDNA obtained was used in HRM amplifications. Primers employed in HRM analysis were designed utilizing the software Primer3, starting from a multi-alignment of the sequences, which characterize the conserved domain of the WRKY-1 gene close to WRKY site, in 14 wild relative species. Two primer pairs were designed to cover a conserved domain of 250 bp. PCR amplifications were performed on a Rotor-Gene 6000 realtime PCR Thermocycler (Corbett Research, Australia). For data quality control, PCR amplification was analysed through the assessment of the threshold cycle (Ct) value, endpoint fluorescence level and the amplification efficiency. The melting data were normalized by adjusting the beginning and end fluorescence signals to the same level. HRM curve analysis was performed using the HRM analysis module. Different plots of the melting data were visualized by selecting a genotype for comparison and negative first-derivative melting curves were produced from the fluorescence versus temperature plots. Representative genotypes were chosen for sequencing, using an ABI 3130xl sequencing platform. Sequences obtained were blasted with the corresponding sequences present in genes databases through BLAST2 Sequences program and multi-aligned to identify mutations.
Results and discussions
A total of 14 sequences relative to a conserved WRKY-1 domain were multi-aligned for primer design. There were 36 amplicons corresponding to the cDNA extracted from leaves and roots of the different varieties treated with the different salt concentrations. HRM analysis revealed that eight amplicons showed polymorphic melting curves when assayed against non-treated samples (negative controls), while the remaining 28 were monomorphic (Fig. 1). Nine amplicons, in two replicates, showing both mono- and polymorphic melting curves, and analysed on normalised melting curves and through different plots, were chosen, purified and sequenced. Among these, two SNPs were found in salt-tolerant lines Cham I and J. Ketifa both in leaves and roots. The first SNPs present in an A/T transversion were located close (only one amino acid distant) to the WRKY domain. The A/T transversions are commonly considered to be the SNPs variations most difficult to resolve by melting analysis. Despite this, the current study showed the amplicon with the A/T transversion was distinctly differentiated (Fig. 1). Moreover, this SNP was present in the salt-tolerant line, Cham I, even in the NaCl-untreated condition. A second SNP, consisting of a G/C transversion, was located on the other resistant line, J. Ketifa, treated with the maximum concentration (1.5 M) of NaCl. Also, this SNP was located close (only two amino acid distant, adjacent to the first SNP) to the WRKY domain. This SNP also showed a distinctive melting curve profile (Fig. 1). Both of the identified SNPs created two corresponding amino acid substitutions, Q/L for the first SNP and K/N for the second (Q = Gln = Glutamine, L = Leu = Leucine, K = Lys = Lysine, N = Asn = Asparagine), both close to the WRKY domain (Fig. 2). The presence of these two altered amino acids so close to WRKY may cause a functional modification in the biochemistry of the WRKY-1 promoter (considering that this protein site is involved in DNA binding) that could explain and contribute to the resistance of these two durum wheat lines to salt stress. The presence of the first SNP in the Cham I line, in the untreated condition, may be explained by WRKY-1 being part of one of the largest families of transcription factors. These factors are involved in regulating several seemingly disparate processes where some proteins act as repressors as well activators (Rushton et al., Reference Rushton, Somssich, Ringler and Shen2010). Furthermore, understanding the different mechanisms of WRKY transcription factors is straightforward, as reported by Ishiguro and Nakamura (Reference Ishiguro and Nakamura1994) and Rushton et al. (Reference Rushton, Somssich, Ringler and Shen2010). Nevertheless, the localization of both SNPs in both the resistant lines confirms their value for future breeding activities. In addition, this work confirms that HRM represents a feasible means for SNP detection and genotyping, being a simple, accurate, high-throughput and low-cost method.
Fig. 1 WRKY-1 melting curve profiles.
Fig. 2 Amino acid and nucleotide sequences of WRKY-1 with WRKY domain (grey box) and the two different SNPs found for Cham I (round box) and J. Ketifa (squarely box).
