The wheat omega-gliadin genes: structure and EST analysis

Abstract

A survey and analysis is made of all available omega-gliadin DNA sequences including omega-gliadin genes within a large genomic clone, previously reported gene sequences, and ESTs identified from the large wheat EST collection. A contiguous portion of the Gli-B3 locus is shown to contain two apparently active omega-gliadin genes, two pseudogenes, and four fragments of the 3' portion of omega-gliadin sequences. Comparison of omega-gliadin sequences allows a phylogenetic picture of their relationships and genomes of origin. Results show three groupings of omega-gliadin active gene sequences assigned to each of the three hexaploid wheat genomes, and a fourth group thus far consisting of pseudogenes assigned to the A-genome. Analysis of omega-gliadin ESTs allows reconstruction of two full-length model sequences encoding the AREL- and ARQL-type proteins from the Gli-A3 and Gli-D3 loci, respectively. There is no DNA evidence of multiple active genes from these two loci. In contrast, ESTs allow identification of at least three to four distinct active genes at the Gli-B3 locus of some cultivars. Additional results include more information on the position of cysteines in some omega-gliadin genes and discussion of problems in studying the omega-gliadin gene family.

Fig. 1

Organization of part of a Gli-B1 locus. A section of a Gli-B1 locus containing eight ω-gliadin genes and gene fragments was cloned in BAC 419P13 (EF426565). The relative positions of the ω-gliadin sequences are shown and includes two complete ORFs (1, 2), two pseudogenes (Ψ1, Ψ2) and four 3′ ω-gliadin fragments (F1–F4). The DNA is shown in reverse orientation from the Genbank entry, and the ω-gliadin positions in EF426565 at stop codons for full-length genes and approximate positions for fragments are as follows: F1, 106,600; Ψ1, 76,749; Ψ2, 66,558; F2, 47,500; 1, 41,831; F3, 21,900; 2, 16,212; F4, 7,800. Arrows under ω-gliadin sequences indicates potential directions of transcription. The arrowhead at the left end of the DNA fragment indicates extensive DNA sequence is known and does not include more ω-gliadin sequences. The multiple arrowheads on the right indicates the direction of non-sequenced DNA that may contain more ω-gliadin sequences

Fig. 2

Comparing ω-gliadin sequences. Gene 1 from Fig. 1 is compared by dot plots to other ω-gliadin sequences to include coding and near flanking regions. a 1B ω-gliadin (AB181300) of Matsuo et al. (2005). b pseudogene Ψ2 of Fig. 1. c ω-Gliadin fragment F1 of Fig. 1. d ω-Gliadin F20b (AF280606) of Hsia and Anderson (2001). Matching criteria were 80% over a 20-base window. Down arrows indicate start codons and upward arrows indicate stop codons

Fig. 3

Full-length 1B ω-gliadin amino acid sequences. All known full-length 1B ω-gliadin DNA coding sequences are converted to amino acid sequences and aligned. Amino acid differences are shaded. Sequences are defined to be full length if they have an ORF and similar non-coding flanking DNAs, and include the two active (1, 2) and two pseudogenes (Ψ1, Ψ2) of Fig. 1 (EF426564), and includes one active (AB181300) and one pseudogene (AB181301) of Matsuo et al. (2005). The vertical bar indicates the junction of the signal peptide and the mature ω-gliadin sequences. Arrowheads below the Ψ1 sequence indicate frameshifts in the original DNA sequence. Sequences are named by Genbank accession and numbering from Fig. 1

Fig. 4

Phylogenetic tree of ω-gliadin DNA sequences. Reported ω-gliadin DNA sequences are aligned separately for the 1B and 1A/D sequences by Clustal V and the resulting two branches are shown connected by theoretical relationships to a common ancestral ω-gliadin sequence (left-most node). Names are by Genbank accession, gene name (if named), first author of report (if published) and indication of being a pseudogene (Ψ). The four B-genome gene sequences and two consensus A/D-genome gene sequences from the current report are all shown in bold. Genomes or origin are indicated by A, B, D, or A/D (1A and/or 1D). AJ937839 is indicated as a pseudogene by sequence, but may include sequence artifacts of cloning

Fig. 5

Genome assignments of ω-gliadin sequences to A, B, and D genomes. Clusters of ω-gliadin sequences as indicated in Fig. 4 were analyzed for unique DNA priming sites. Specific PCR primers were designed and used to amplify DNA from genomic DNA of wheat cv Chinese Spring nulli-tetrasomic genetic stocks. Primers are shown in Supplementary Fig. 3Sa. a Primers for branch A. b Primers for branch D′. c Primers for branch A′. Primer names are below each frame. Predicted size of amplified fragments from DNAs within each branch were used to design primers are shown below each frame. The DNA ladder was the 100-bp DNA Ladder from Invitrogen (Cat. No. 15628-019)

Fig. 6

AREL and ARQL consensus amino acid sequences. Derived amino acid sequences from the ESTs listed in Supplementary Table 1S were assembled into full-length ω-gliadin protein sequences. a The underlined sequence in the AREL consensus marks the sequence not present in Cheyenne gene F20b and the boxed region matches Cheyenne EST BE422668. b The two boxed regions of the ARQL consensus match the peptide sequences of Masci et al. (1999). Unique cysteine and methionine residues are circled

Fig. 7

Multiple 1B ω-gliadins in cultivars Chinese Spring and Recital. Derived amino acid sequences from ESTs of cultivars Chinese Spring and Recital were assembled in three and four distinct 1B ω-gliadin sequences, respectively, and compared to the derived amino acid sequences of the only three known intact 1B ω-gliadin sequences. Amino acid residue differences are shaded. Horizontal lines separate gene from EST assembly sequences. ESTs are listed in Supplementary Table 1S. Genes 1 and 2 are as in Fig. 1. The peptide sequence below the C-terminus of Recital 4 is the sequence if the frameshift were corrected