DNA based approaches to characterise the chromosomal region containing the grain hardness (Ha) locus in wheat (Triticum aestivum L.)

Abstract

Grain hardness is an important quality and end use determinant of wheat grain. The precise factor controlling grain hardness is not known but it is thought to be controlled at the starch granules-protein matrix interface in the endosperm tissue. The work described in this thesis consists of three broad approaches. The first approach involves isolation of candidate hardness regulating gene sequences from Triticum tauschii, a soft grained diploid relative of wheat. Sequences of the candidate genes and surrounding areas were obtained. A D genome specific GSP-1 sequence was identified in these studies. Sequence differences between A genome and D genome GSP-1 genes were observed. This is an important aspect of the study because grain hardness is regulated by chromosome 5D in wheat. wGSPlD-13, a sequence downstream of the GSP-1 coding region from Triticum tauschii, is not associated with A genome GSP-1 sequences. This was used successfully in mapping studies involving the hardness locus. The coding region sequences may later be used in transformation studies to clarify their involvement in the control of grain hardness. A second approach was concerned with characterisation of members of the GSP-1/ puroindoline family ( candidate hardness controlling genes) that are expressed in developing wheat endosperm. An aim of this approach was to ascertain whether new members of the family could be identified that may be responsible for grain hardness control or could assist in future high resolution mapping applied to the region surrounding the grain hardness locus. A novel clone was identified that contained an entire GSP-1 coding region and upstream sequence, this upstream sequence showed homology to an element in a high molecular weight glutenin subunit gene. Further work is required to confirm this result. If the sequence is expressed in typical wheat endosperm it may have an involvement in regulating grain hardness. The third approach made no assumption about the mode of grain hardness control and assumed only that there is a genetic difference responsible for variation in the character. RAPD analysis was performed on DNA bulks generated from material differing only in grain hardness that is controlled by chromosome SD. Variations to the RAPD protocol were assessed and polymorphic bands were cloned. An attempt was made to create STS markers from sequence information obtained from the cloned polymorphic bands. One primer pair produced a polymorphism when used on soft and hard DNA bulks, but difficulty reproducing the polymorphism was experienced with new primer aliquots. DNA sequence information and cloned sequences generated in these studies will provide a valuable contribution to clarifying an involvement of candidate genes in grain hardness control and to high resolution mapping studies that will precisely define the grain hardness locus.