ORIGINAL PAPER Mingjie Chen Æ Mark Wilkinson Æ Paola Tosi Guangyuan He Æ Peter Shewry Novel puroindoline and grain softness protein alleles in Aegilops species with the C, D, S, M and U genomes Received: 28 April 2005 / Accepted: 12 July 2005 / Published online: 17 August 2005 Ó Springer-Verlag 2005 Abstract 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 se- quences 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. Introduction Grain texture, or hardness, is one of the main charac- teristics that determine the processing properties of wheat, affecting both the milling and bread-making properties (Pomeranz and Williams 1990). Thus, hard wheats require more energy to mill, yielding larger par- ticles which have a higher degree of starch damage and better properties for bread making. It has been known for some time that hardness is mainly determined by a single locus, called Ha, located on the short arm of chromosome 5D (Law et al. 1978), although more recent work indicates the existence of further minor loci (Turner et al. 2004). The major structural difference between grains of hard and soft-textured wheats is the degree of adhesion between the surface of the starch granules and the ma- trix (i.e. gluten) proteins in the mature cells of the star- chy endosperm. Hence early attempts to explain the biochemical basis for hardness focussed on identifying differences between the surfaces of starch granules in hard and soft cultivars. This resulted in the identification of an M r 15,000 protein present on the surface of starch granules prepared from soft but not hard-textured types of wheat and the hypothesis that this acted as an ‘‘non- stick’’ protein which prevented adhesion between the starch granule and the gluten matrix (Greenwell and Schofield 1986). It has since been shown that this pro- tein, called ‘‘friabilin’’, actually comprises a mixture of components (Oda and Schofield 1997) including two major tryptophan-rich proteins called puroindolines (Pins) a and b (Blochet et al. 1993; Gautier et al. 1994). Furthermore, these two proteins are encoded by genes located at the Ha locus (Chantret et al. 2004), together with a gene encoding a third protein called ‘‘grain soft- ness protein’’ (GSP) (Jolly et al. 1993, 1996). The role of pins in determining grain texture is now well established, with grain hardness being determined by mutations which affect either the expression of the Pin a or Pin b genes (i.e. null alleles) or the amino acid sequence of the Pin b protein (Giroux and Morris 1997, 1998; Lillemo and Morris 2000; Turnbull et al. 2000; Morris 2002). Furthermore, direct evidence for their role has been provided by functional complementation, by expressing the Pin a and Pin b genes in transgenic rice (Krishnamurthy and Giroux 2001) and the Pin b gene in hard-textured bread wheat (Beecher et al. 2002). In both cases, the grain from the transgenic lines was softer than those from the controls. Communicated by P. Langridge M. Chen Æ G. He China-UK HUST-RRes Crop Engineering and Genomics Joint Laboratory, College of Life Science and Technology, Huazhong University of Science and Technology, 430074 Luoyu Road 1037, Wuhan, Hubei, People’s Republic of China M. Wilkinson Æ P. Tosi Æ P. Shewry (&) Æ M. Chen Rothamsted Research, Harpenden, Hertfordshire, AL5 2JQ UK E-mail: peter.shewry@bbsrc.ac.uk Theor Appl Genet (2005) 111: 1159–1166 DOI 10.1007/s00122-005-0047-7