Expression profile of two storage-protein gene families in hexaploid wheat revealed by large-scale analysis of expressed sequence tags

Abstract

To discern expression patterns of individual storage-protein genes in hexaploid wheat (Triticum aestivum cv Chinese Spring), we analyzed comprehensive expressed sequence tags (ESTs) of common wheat using a bioinformatics technique. The gene families for alpha/beta-gliadins and low molecular-weight glutenin subunit were selected from the EST database. The alignment of these genes enabled us to trace the single nucleotide polymorphism sites among both genes. The combinations of single nucleotide polymorphisms allowed us to assign haplotypes into their homoeologous chromosomes by allele-specific PCR. Phylogenetic analysis of these genes showed that both storage-protein gene families rapidly diverged after differentiation of the three genomes (A, B, and D). Expression patterns of these genes were estimated based on the frequencies of ESTs. The storage-protein genes were expressed only during seed development stages. The alpha/beta-gliadin genes exhibited two distinct expression patterns during the course of seed maturation: early expression and late expression. Although the early expression genes among the alpha/beta-gliadin and low molecular-weight glutenin subunit genes showed similar expression patterns, and both genes from the D genome were preferentially expressed rather than those from the A or B genome, substantial expression of two early expression genes from the A genome was observed. The phylogenetic relationships of the genes and their expression patterns were not correlated. These lines of evidence suggest that expression of the two storage-protein genes is independently regulated, and that the alpha/beta-gliadin genes possess novel regulation systems in addition to the prolamin box.

Figure 1.

Phylogenetic tree of α/β-gliadin genes. Accession numbers indicate the genes registered in the DDBJ (Table I). Numbers in open boxes indicate genes previously assigned to the chromosome. Contig numbers in colored boxes indicate genes identified by EST analysis in this study. Boxes in red, blue, and green indicate genes from 6AS, 6BS, and 6DS chromosomes, respectively. Yellow circle indicates cluster groups, namely, A, B, C, D, and E. Specific primers are indicated by gli-AS_1 to 10 (Table II) for the genes in the gray bar. Assigned chromosome locations are given in parentheses. Accession numbers marked with asterisks indicate genes obtained from genomic DNA, and ψ indicates genes determined to be pseudogenes. γ-Gliadin (M11077) is used as the out-of-group gene.

Figure 2.

Example of haplotype-specific PCR using aneuploid lines of CS wheat. A, B, and C indicate haplotype-specific PCR using primer sets gli-AS_1, gli-AS_2, and gli-AS_3, respectively (Table II). 1, N6AT6B; 2, N6BT6A; 3, N6DT6B; 4, DT6AL; 5, DT6BL; 6, DT6DL; 7, CS; and M, pGEM DNA markers (Promega).

Figure 3.

Phylogenetic tree of LMW-GS genes. Accession numbers indicate genes registered in the DDBJ (Table I). Numbers in open boxes indicate genes previously assigned to chromosomes. Contig numbers in colored boxes indicate genes identified by EST analysis in this study. Boxes in red, blue, and green represent genes from 1AS, 1BS, and 1DS chromosomes, respectively. Yellow circles indicate cluster group, namely A, B, C, and D. Specific primers are indicated by Glu-AS_1 to 4 (Table II) for the genes in the gray bar. Assigned chromosome locations are given in parentheses. Accession numbers marked with asterisks indicate genes obtained from genomic DNA. α/β-Gliadins (U51307) is used as the out-of-group gene.

Figure 4.

Hierarchical clustering of gene expression patterns at 12 stages of wheat life cycle. A, Thirty-six α/β-gliadin genes. B, Fifteen LMW-GS genes. C, Reference genes (glyceraldehyde-3-P dehydrogenase and cyclophilin A) as constantly expressed throughout the life cycle. Contig names colored in red, blue, and green indicate the assigned to A, B, and D genomes, respectively. Letters on the right of the contig names indicate clusters grouped by evolutional relationship in Figures 1 (α/β-gliadins) and 3 (LMW-GS).

Figure 5.

Gene expression patterns of multigenes encoding two storage proteins during seed development. A, Total number of ESTs for α/β-gliadins and LMW-GS expressed during the course of seed maturation. B, Number of ESTs corresponding to each locus for α/β-gliadin and LMW-GS genes. C, Number of ESTs corresponding to each multigene for α/β-gliadin and LMW-GS gene loci from three genomes. Number of ESTs presented in the figure was normalized to equal the original size of cDNA libraries.