Identification of allelic variations of puroindoline genes controlling grain hardness in wheat using a modified denaturing PAGE

Puroindoline genes (Pina and Pinb) form the molecular basis of wheat grain hardness or texture. Variations in either gene are reported to be associated with grain hardness in wheat. Here, a modified denaturing PAGE was used to detect Pina and Pinb allelic variations in 102 common wheat cultivars and other species related to wheat. Two variations of Pina (Pina-D1b and Pina-D1p) and five variations of Pinb (null, Pinb-D1b, Pinb-D1u, Pinb-D1v and Pinb-D1w) were identified in common (T. aestivum) and spelt wheats (T. aestivum ssp. spelta). No allelic variation was found in Tibet semi-wild wheat (T. aestivum ssp. tibetanum), club wheat (T. compactum), or Aegilops tauschii. Puroindoline genes were absent in wild␣emmer (T. turgidum var. dicoccoides). The sequencing results of the PCR fragments from Pina and Pinb (except for null type) indicated allelic variants carrying single base mutation, such as Pina-D1p, Pinb-D1u, Pinb-D1v and Pinb-D1w were novel types. Our results showed that the modified PAGE used in this study provided a satisfactory resolving power for identifying single nucleotide mutations; therefore, it is a practical and simple tool to study allelic variation of Pina and Pinb in wheat and related species.