Introduction
TdDRF1 (T. durum dehydration responsive factor 1), a DREB2-related gene that was isolated and characterized in durum wheat (Latini et al., Reference Latini, Rasi, Sperandei, Cantale, Iannetta, Dettori, Ammar and Galeffi2007), belongs to the AP2 gene family and is highly homologous to the barley HvDRF1 gene (Xue and Loveridge, Reference Xue and Loveridge2004) and to the bread wheat wdreb2 gene (Egawa et al., Reference Egawa, Kobayashi, Ishibashi, Nakamura, Nakamura and Takumi2006). These genes share a complex gene structure, consisting of four exons and three introns, and produce three transcript variants by means of alternative splicing. In the present investigation, the different parts of the gene have been systematically analysed in several accessions of durum wheat, triticale, wheat genome donors (A. speltoides, A. tauschii and Triticum urartu) as well as in other related plants.
Material and methods
Eight T. durum genotypes (total 67 accessions), a few accessions of T. urartu, A. tauschii and triticale, as well as 69 accessions of A. speltoides v speltoides, A. speltoides v ligustica and other Aegilops were grown in the greenhouse and used for DNA extraction.
The amplified fragments (PCR thermal cycle reaction: initial denaturation 94°C for 5 min, 94°C for 1 min, 55°C for 1 min, 72°C for 1 min and 30 s and final extension at 72°C for 7 min, then at 4°C for storage) were gel-purified, cloned (pCR®II-TOPO® vector by Invitrogen, USA) and sequenced (ABI 3730 DNA analyzer; Applied Biosystems, USA) following standard procedures.
An exon 1 region of 150 bp at the beginning of protein CoDing Sequence (CDS), was sequenced in 100 genotypes (E1 For: 5′-AAGTCGACGCGGCGAA 3′; SSR1 Rev: 5′-CCGGGATCTCGAAGGGTG 3′).
An exon 4 part, from 400 bp to 1 kb long, including the whole AP2 domain coding region, was sequenced in 168 samples of Aegilops (E4 FOR: 5′-ATGATCCACAGGGTGCAA 3′; E4 Rev: 5′-GGTCCACCATTTGATCTTCATT 3′).
Six full-length DRF1 gene sequences, namely FJ858188, FJ858187, FJ843102, EU089819, EU197052 and GU017675, were submitted to Genbank and used for transposon identification and analysis.
Several computational tools were used for phylogenetic and molecular evolutionary analyses (Rozas et al., Reference Rozas, Sanchez-Delbarrio, Messeguer and Rozas2003; Excoffier et al., Reference Excoffier, Laval and Schneider2005; Huson and Bryant, Reference Huson and Bryant2006; Tamura et al., Reference Tamura, Dudley, Nei and Kumar2007), sequence analyses as well as identification of repeats and palindromes (Kurz et al., Reference Kurtz, Choudhuri, Ohlebusch, Schleiermacher, Stoye and Giegerich2001; Kohany et al., Reference Kohany, Gentles, Hankus and Jurka2006), secondary structure prediction and modelling (for details see Supplementary Table S1, available online only at http://journals.cambridge.org).
Results and discussion
The various analyses provided interesting insights into the DRF1 gene which are outlined below, separately, for each exon.
Exon 1
Simple sequence repeat (SSR) genetic polymorphism occurs due to variations in the number of repeated units, probably due, in turn, to slippage during DNA replication (Levinson and Gutman, Reference Levinson and Gutman1987; Taylor et al., Reference Taylor, Durkin and Breden1999). An SSR was identified in the DRF1 gene (Fig. 1). This SSR is located at the N terminus of exon 1, position 10, and codes for a stretch of alanine residues variable in length (from 3 to 7). Another study on SSR loci in expressed sequences suggested the potential correlation between mutational events within SSR repeats and the evolutionary relationships across taxa (Rossetto et al., Reference Rossetto, McNally and Henry2002). It is worthwhile pointing out that the shortest SSRs belong mainly to the ancestor of Triticum and to Brachypodium distachyon.
Fig. 1 Sequences and frequencies of the ALA-stretch in T. durum, A. speltoides, T. urartu, A. tauschii and in two related plants. (Brachypodium d.: Bd2:29505646.29515646, http://www.brachybase.org/blast/; Leymus c.: GenBank accession EU999998.1).
Variability was also observed, resulting from a single nucleotide mutation (ALA to SER or THR), exclusively at the beginning or at the end of the stretch. The ALA stretch is present also in Leymus chinensis and B. distachyon, suggesting that this SSR represents a shared feature of the Pooideae subfamily (Poaceae family), and is not present in other subfamilies such as Ehrhartoideae (Oryza sativa) and Panicoideae (Zea mays). A double mutation changing ALA to LEU was observed in L. chinensis, while a single change, GLU to VAL, was found in B. distachyon.
Further investigations are needed to better understand the possible linkage of this SSR and its variability, both at genomic and transcriptomic level, in relationship with maps and phenotypic traits.
Exons 2–3
The six available full-length gene sequences from T. durum and Aegilops were analysed in order to identify possible transposable elements. Repeats, palindromes, tandem and inverted repeats were investigated. All the identified elements, namely a terminal inverted repeat (TIR-32 bp), a target site duplication (TSD-2 bp), an internal TSD (ITSD-4 bp), direct and reverse repeats and a variable number of tandem repeats revealed the presence of a transposable element in the DRF1 gene which had never been described before.
Thus, a novel transposable element, approximately 1.5 kb in length, was identified between intron 1 and intron 3, including exon 2 and exon 3. Since no sequence for transposase was found in the transposon identified, it was classified as a non-autonomous transposon. Furthermore, the presence of nested TIR-18bp was also observed, the sequences of which are highly homologous to the longest ones (Supplementary Fig. S1, available online only at http://journals.cambridge.org).
The new transposon is now included in Repbase (see Report at http://www.girinst.org/2009/vol9/issue3/AsDRF1.html; Thiyagarajan et al., Reference Thiyagarajan, Latini, Galeffi and Porceddu2009).
All analysed genes showed the above observations, with minor mismatch/deletion, mainly located at 3′ Terminal Inverted Repeat (TIR) that appears to be the most variable part of the transposable element.
The transposon is inserted into a poor CG (about 40%) region and its core (exon 2+intron 2+exon 3 and few bps of intron 1 and intron 3 at flanking sides) appears to be very similar (85% identity, 372 bp fragment, E = 10− 69) to a sequence from B. distachyon, suggesting that the transposon might have played a vital role in moving these exons during evolution of the Pooideae subfamily. Furthermore, it is tempting to hypothesize its possible involvement in the mechanism of the alternative splicing regulation of the DRF1 gene.
Exon 4
Even if most of the variations intra- and inter- species are selectively neutral (Nei, Reference Nei1987), there is an increasing interest in detecting genes, or genomic regions, that affect the fitness of the organisms (Nielsen, Reference Nielsen2005). As the exon 4 sequence of DRF1 gene contains the region codifying the AP2 domain, responsible for DNA recognition and binding, 168 sequences (137 from A. speltoides and 31 from other Aegilops species) were analysed to evaluate variability (Supplementary Fig. S2, available online only at http://journals.cambridge.org).
Overall, 279 sites were analyzed (excluding gaps/missing data) for all 168 sequences; 67 polymorphic sites were identified (25 singleton variable sites, two variants; 35 parsimony informative sites, two variants; and seven parsimony informative sites, three variants). Out of 67 polymorphic sites, five were located in the AP2 domain and their effect was investigated in a structural model. The AP2 local geometry was not affected (data not shown).
To investigate the evolutionary relationships among the 168 sequences, a minimum spanning tree was built. Two main groups were observed, lineage I and lineage II, separated by 21 mutation steps (Fig. 2). The neighbour-joining tree, estimated using a Kimura-2 parameter model, clustered in the same way, a 70% bootstrap value (data not shown).
Fig. 2 Minimum spanning tree of 168 genotypes of Aegilops calculated by an exon 4 region of the DRF1 gene. A. speltoides ssp ligustica is shown in light grey (
), A. speltoides ssp speltoides is shown in black (●), A. speltoides is shown in black (•) and other Aegilops are shown in dark grey (
).
Lineage I is a complex network, with two main groups, ‘star-like’ pattern, including all except three A. speltoides accessions. These three genotypes, classified as A. speltoides (2065_3, 2065_4 and 2065_5), cluster in the lineage II, together with all Aegilops genotypes. Possibly, a reconsideration of the taxonomic assignment could be proposed, even if more loci have to be analysed before reaching a final conclusion.
A geographic subdivision was applied to investigate geographic frequency patterns, but the design, based on three clusters (east, centre and west) was unable to reveal substantial differences in polymorphic patterns, thus suggesting a neutral selection.
A design based on taxonomy was also tested; the variability observed in the analysed DNA region was unable to distinguish ssp speltoides from ssp ligustica, while it was able to distinguish Aegilops from A. speltoides (data not shown). Analysis of molecular variance (AMOVA) was carried out testing the genetic structure (two groups and four populations). The F-statistic was shown to be quite significant (F-statistic, i.e. specific population index (F st) P value = 0.00) and a substantial variation between A. speltoides and Aegilops species was found, accounting for 62.34% of the total variation, even if not significant, P>0.1. Due to missing data, locus-by-locus AMOVA provided a better estimate and, indeed, the data were significant (54.7%, P = 0.00).
In conclusion, the detailed analysis of the sequence of the DRF1 gene and its variability intra- and inter-species highlighted some interesting features characterizing the complex structure of this gene, clearly contributing to our overall knowledge and thus opening new perspectives regarding the evolution and regulation of this gene.
Acknowledgements
Authors thank Dr. K. Ammar (CIMMYT) for valuable suggestions during this investigation and Mrs. Marian Boreham for the revision of English language.
