Characterizing HMW-GS alleles of decaploid Agropyron elongatum in relation to evolution and wheat breeding

Abstract

Bread wheat quality is mainly correlated with high molecular weight glutenin subunits (HMW-GS) of endosperm. The number of HMW-GS alleles with good processing quality is limited in bread wheat cultivars, while there are plenty of HMW-GS alleles in wheat-related grasses to exploit. We report here on the cloning and characterization of HMW-GS alleles from the decaploid Agropyron elongatum. Eleven novel HMW-GS alleles were cloned from the grass. Of them, five are x-type and six y-type glutenin subunit genes. Three alleles Aex4, Aey7, and Aey9 showed high similarity with another three alleles from the diploid Lophopyrum elongatum, which provided direct evidence for the Ee genome origination of A. elongatum. It was noted that C-terminal regions of three alleles of the y-type genes Aey8, Aey9, and Aey10 showed more similarity with x-type genes than with other y-type genes. This demonstrates that there is a kind of intermediate state that appeared in the divergence between x- and y-type genes in the HMW-GS evolution. One x-type subunit, Aex4, with an additional cysteine residue, was speculated to be correlated with the good processing quality of wheat introgression lines. Aey4 was deduced to be a chimeric gene from the recombination between another two genes. How the HMW-GS genes of A. elongatum may contribute to the improvement of wheat processing quality are discussed.

Fig. 1

PCR amplification of HMW-GS coding sequences from genomic DNA of A. elongatum. M lambda DNA digested by EcoR I + Hind III, Ag amplicon of A. elongatum

Fig. 2

Expression of the modified ORFs of five alleles Aex1, Aex4, Aey2, Aey7, and Aey9 in E. coli and SDS-PAGE analysis of expressed products. The modified ORFs were prepared by removing the signal peptide sequence from each of the sequences by mutagenesis. Protein extracts were prepared by dissolving cells directly in SDS-PAGE sample buffer. The glutenin proteins synthesized in E. coli directed by Aex1, Aex4, and Aey7 under IPTG induction showed identical electrophoretic mobility to those from seeds of A. elongatum (shown by arrows). No proteins from seeds of A. elongatum displayed similar mobility with those directed by Aey2 and Aey9 in bacteria (shown by arrows). CK proteins extracted from bacteria harboring pET–Aex1 without IPTG induction for control, Ag proteins extracted from seeds of A. elongatum

Fig. 3

Comparison of primary structure of five x-type subunits from A. elongatum with that of three representative x-type subunits from common wheat. The N- and C-terminal regions were boxed. The tailed arrows indicated the cysteine residues and the additional cysteine residues of 1Dx5, Aex2, and Aex4 were underlined. The glutamine (Q) residues conserved in N-terminal domain of x-type subunits but absent from most y-type subunits were shown by non-tailed arrows. The in-frame stop codon was represented by asterisk. The Genbank accession numbers of these sequences were displayed in Table 1

Fig. 4

Comparison of primary structure of all the ten y-type subunits from A. elongatum and that of three representative y-type subunits from common wheat. The N- and C-terminal regions were boxed. The tailed arrows indicated the cysteine residues and additional cysteine residues of Aey1, Aey3, and Aey10 were underlined. The sequences of Aey1, Aey4, and Aey5 were rectified to diminish the influence of frame shift. The revised amino acid residues were substituted by underlined X. The in-frame stop codon was represented by asterisk. The extra glutamine (Q) residues in N-terminal domain of Aey4, Aey8, Aey9, and Aey10 were shown by non-tailed arrows. The Genbank accession numbers of these sequences were displayed in Table 1

Fig. 5

Phylogenetic analysis of HMW-GS from A. elongatum and some other wheat-related grass. a Neighbor-Joining tree of full length sequences of Glu-1 genes from A. elongatum and some other wheat-related grass. b Neighbor-Joining tree of N-terminal regions of HMW-GS from A. elongatum. c Neighbor-Joining tree of C-terminal regions of HMW-GS from A. elongatum. This work was done under the help of MEGA program (Version 3.1)