Novel puroindoline and grain softness protein alleles in Aegilops species with the C, D, S, M and U genomes

DNA from six hexaploid, tetraploid and diploid species of Aegilops with the C, D, S, M and U genomes was amplified with specific PCR primers to identify sequences encoding puroindolines (Pins) a and b and grain softness protein (GSP), all of which are encoded by genes at the Ha (hardness) locus, with Ae. tauschii (DD) and bread wheat (T. aestivum) (AABBDD) cv Hiline being studied as controls. Seven new allelic forms of Pin a and Pin b were identified, including forms with mutations within or close to the tryptophan motif. In addition, five new forms of GSP were detected. In all species both genomic DNA from leaves and cDNA from developing grain were analysed. This revealed the presence of both silent genes (with premature stop codons) and multiple genes, with the latter being confirmed by Southern blot analysis. Freeze fracture analysis demonstrated that all except one accession (Ae. sharonensis) were soft textured. However, this difference cannot be accounted for by the sequences of the Pin alleles present in this line.