Endosperm texture, i.e. the hardness or softness of the grain, is an important quality criterion in cereals because it determines many grain end-use properties. Grain softness is the dominant trait and is mainly controlled by the Ha locus on the short arm of chromosome 5D in hexaploid bread wheat. Genes for puroindoline a (Pina-D1), puroindoline b (Pinb-D1), and grain softness related protein (Gsp-D1) have been shown to be linked to the Ha locus in different mapping populations and have been associated with the expression of grain softness. The study of the linkage relationships among these genes has been limited by the low level of polymorphism in the D genome of hexaploid Triticum aestivum. In the present study, a highly polymorphic Triticum monococcum mapping population was used to analyze linkage relationships among these three genes. Gsp-Am1 and Pina-Am1 were found to be completely linked and lie 0.14 cM distal to Pinb-Am1 in the distal region of the short arm of chromosome 5Am. The tight genetic linkage among these three genes was paralleled by their physical proximity within a single 105-kb clone isolated from a T. monococcum bacterial artificial chromosome (BAC) library. A restriction map of this BAC clone showed that Pina-Am1 is located between Pinb-Am1 and Gsp-Am1. Partial sequences of the T. monococcum genes showed a high degree of similarity with their T. aestivum counterparts (> or =94%). Marker-assisted selection strategies based on the tight linkage among Ha-related genes are discussed.