PCR screening and sequence analysis. Analysis of Pin sequences from Elymus and Agropyron species

The puroindoline genes located at the Ha locus on chromosome 5D of bread wheat are now considered to be the most important determinant of grain hardness. The recent identification of Pinb-2 genes on group 7 chromosomes has stressed the importance of further study of the Pin gene family and their crucial role in determining grain quality. Two diploid (Agropyron species), four tetraploid (Elymus species), five hexaploid (Elymus and Agropyron species) and their progenitor species were studied at the sequence level to identify novel variation in Pin genes and proteins which could be exploited in the improvement of cultivated wheat. This study focussed mainly on screening for the three Pin (Pina, Pinb and Pinb-2) genes. Interestingly, in all the species screened there were some differences in the frequency of distribution of all three genes. A novel sequence for the Pina gene was detected in Elymus dahuricus subsp. excelsus and Elymus nutans. New Pin b sequences was detected in Elymus burchan-buddae, Elymus dahuricus subsp. excelsus, and Elymus nutans. Novel Pinb-2v variants were also detected for Agropyron repens and Elymus repens. The putative proteins detected all showed important changes at the tryptophan-rich domain as well as changes and/or deletions of basic and hydrophobic residues for all species screened. Further Gsp-1 gene screens were carried out, where two new AGP sequences were identified in Elymus nutans and Elymus wawawaiensis. The data presented highlights the sequence diversity of this important gene family and its putative influence in determining grain texture and end use products.