Expansion of the gamma-gliadin gene family in Aegilops and Triticum

Abstract

Background: The gamma-gliadins are considered to be the oldest of the gliadin family of storage proteins in Aegilops/Triticum. However, the expansion of this multigene family has not been studied in an evolutionary perspective.

Results: We have cloned 59 gamma-gliadin genes from Aegilops and Triticum species (Aegilops caudata L., Aegilops comosa Sm. in Sibth. & Sm., Aegilops mutica Boiss., Aegilops speltoides Tausch, Aegilops tauschii Coss., Aegilops umbellulata Zhuk., Aegilops uniaristata Vis., and Triticum monococcum L.) representing eight different genomes: Am, B/S, C, D, M, N, T and U. Overall, 15% of the sequences contained internal stop codons resulting in pseudogenes, but this percentage was variable among genomes, up to over 50% in Ae. umbellulata. The most common length of the deduced protein, including the signal peptide, was 302 amino acids, but the length varied from 215 to 362 amino acids, both obtained from Ae. speltoides. Most genes encoded proteins with eight cysteines. However, all Aegilops species had genes that encoded a gamma-gliadin protein of 302 amino acids with an additional cysteine. These conserved nine-cysteine gamma-gliadins may perform a specific function, possibly as chain terminators in gluten network formation in protein bodies during endosperm development. A phylogenetic analysis of gamma-gliadins derived from Aegilops and Triticum species and the related genera Lophopyrum, Crithopsis, and Dasypyrum showed six groups of genes. Most Aegilops species contained gamma-gliadin genes from several of these groups, which also included sequences from the genera Lophopyrum, Crithopsis, and Dasypyrum. Hordein and secalin sequences formed separate groups.

Conclusions: We present a model for the evolution of the gamma-gliadins from which we deduce that the most recent common ancestor (MRCA) of Aegilops/Triticum-Dasypyrum-Lophopyrum-Crithopsis already had four groups of gamma-gliadin sequences, presumably the result of two rounds of duplication of the locus.

Figure 1

Schematic overview of the structure of gamma-gliadins. The proteins consist of a short N-terminal signal peptide (S) followed by a unique N-terminal domain (I) and a repetitive domain (II). Domain III contains most (often 6) of the cysteines. IV is rich in glutamine. Two conserved cysteines are in V. Eight cysteine residues (indicated with vertical lines) can form four interchain disulfide bonds (indicated as connections between lines). Figure after [9].

Figure 2

Maximum-Likelihood (ML) tree of the gamma-gliadins (based on amino acid sequences). A maximum-likelihood (ML) analysis was performed with PhyML 3.0. SH-like approximate likelihood-ratio test was used for estimation of branch support. Proteins with a length in the alignment less than 200 amino acids were excluded from the analysis. The gamma-gliadins fall into six groups (1–6 on the right) in two branches (1–2 and 3-4-5-6). Key for the sequence codes in Additional file 1.

Figure 3

Occurrence and absence of genes from different ancestral groups across the Aegilops/Triticum genomes. Overview of the occurrence of genes from the six groups recognised from the maximum likelihood trees (Figure 2, Additional file 4), represented here as Gr1-Gr6 to the left, in all taxa for which we have gamma-gliadin sequences, sorted by genome (at the top). Color in a cell means present, empty means absent. Text in cells indicates additional features: ‘pseudo’ means that all sequences represent pseudogenes (i.e., with stopcodons); ‘9 cys’ indicates that all genes contain exactly 9 cysteines (all other gamma-gliadins generally contain 8 cysteines).

Figure 4

Model for the evolution of groups of gamma-gliadins inAegilops/Triticum. The six groups proposed are based on the ML tree (Figure 2, Additional file 4) and occur in genomes as summarised in Figure 3. Note that in this model the order of the groups on the chromosome is arbitrary, and duplications of genes within each group are ignored. The occurrence of pseudogenes is only indicated when it affected complete groups, but some pseudogenes may occur in all groups. Note that each genome has either group 6 gliadins or group 5 gliadins with nine cysteines and constant length.