Molecular characterization of allelic variations at Pina and Pinb loci in Shandong wheat landraces, historical and current cultivars

Grain hardness is very important in determining the milling and end-use quality of bread wheat. The objectives of this study were to develop gene specific primers for the allele Pina-D1b, evaluate the method for the identification of Pinb-D1p with restriction enzyme PflMI and characterize allelic variations at Pina and Pinb loci in Shandong wheat germplasm. Based on the nucleotide sequences of Ha locus reported previously (CR626934 and CR626926), 121 primer sets were developed to test the Pina-D1b allele. Sequence alignment showed that the promoter region of Pina-D1b allele was highly conserved in the region from −1134 to −23 bp relative to the transcription start codon ATG. Pinb-D1p was first reported in Chinese wheat landraces, with a base A deletion at position 213 in the coding region. Restriction analysis of Pinb-D1p indicated that the base A deletion resulted in the missing of the cleavage site with enzyme PflMI and the digestion with PflMI was validated as a reliable tool for the identification of Pinb-D1p allele. A total of 523 wheat accessions from Shandong province including 431 landraces, 63 historical and 29 current cultivars were chosen for the test of SKCS hardness and identification of puroindoline alleles using DNA markers developed in this study and those reported previously. Frequencies of soft, mixed and hard genotypes were 3.9%, 20.4% and 75.6% in Shandong landraces; 68.3%, 19.0% and 12.7% in historical cultivars; and 27.6%, 58.6% and 13.8% in current cultivars, respectively. Frequencies of Pina-D1b, Pinb-D1b and Pinb-D1p were 38.0%, 0.9% and 59.6% in hard landraces; and 37.5%, 37.5% and 25% in hard historical cultivars, respectively, whereas the Pinb-D1b was the only genotype in hard current cultivars. A novel Pinb allele with double mutations at the positions 96 (C to A) and 213 (deletion of A) was found in three landraces and designated as Pinb-D1aa.