Molecular characterization of waxy mutations in wheat

Abstract

To date, few mutations in wheat have been characterized at the molecular level. In this study, the mutations in the three waxy alleles in waxy wheat (Wx-A1b,Wx-B1b and Wx-D1b) were characterized, and waxy gene expression was compared in several wheat lines, including hexaploid and tetraploid waxy lines of wheat. Southern analysis showed that the Wx-B1b allele had sustained a deletion which included the entire coding region of the Wx-B1 gene. DNA homologous to waxy gene sequences was still present in the Wx-A1b and Wx-D1b alleles of the hexaploid waxy mutant. Transcripts of waxy alleles were also detected in both hexaploid and tetraploid mutants at 10 days post-anthesis, but the transcript level was dramatically reduced compared to that found in non-waxy lines. Isolation of cDNAs showed that transcripts were produced by both the Wx-A1b and Wx-D1b alleles. A 23-bp deletion sustained by the Wx-A1b allele at an exon-intron junction results in the use of a cryptic splice site during mRNA processing. The deduced translation product encoded by the Wx-A1b cDNA lacks 39 amino acids, including the putative ADP-glucose binding site and a portion of the transit peptide. The C-terminal region of the deduced protein encoded by the Wx-D1b cDNA lacks the last 30 amino acids. Comparison of the genomic sequences of the null and wild-type Wx-D1 alleles indicated that 588 bp were deleted in the Wx-D1b mutation, and that the last 261 bp of the Wx-D1b cDNA originated from the normally non-transcribed 3′ flanking region. Like several deletion mutations characterized in other plant species, both Wx-A1b and Wx-D1b alleles contain small DNA insertions, or filler DNA, between the deletion end-points.