Distribution of puroindoline alleles in bread wheat cultivars of the Yellow and Huai valley of China and discovery of a novel puroindoline a allele without PINA protein

Kernel hardness is one of the most important factors determining the milling and processing quality of bread wheat (Triticum aestivum L.). In the present study, 267 wheat cultivars and advanced lines from the Yellow and Huai Valley of China, CIMMYT, Russia and Ukraine were used for identification of SKCS (Single Kernel Characterization System) hardness and puroindoline alleles. Results indicated that Pinb-D1b is the most popular genotype in wheat cultivars from the Yellow and Huai Valley, Russia and Ukraine, whereas PINA null is a predominant genotype in wheat cultivars and advanced lines from CIMMYT. Molecular characterization of PINA-null alleles indicated that one Chinese landrace Chiyacao had the allele Pina-D1l with a single nucleotide C deletion at position 265 in Pina coding region based on sequencing results, and 35 of 39 PINA-null alleles belonged to Pina-D1b according to PCR amplification with the sequence-tagged site (STS) marker Pina-N developed previously. The remaining three cultivars (Jiangdongmen, Heshangtou and Hongquanmang from China) with PINA-null alleles were characterized at the DNA level by a primer walking strategy, and the results showed that all three cultivars with PINA-null alleles possessed a uniform 10,415-bp deletion from -5,117 bp to +5,298 bp (ATG codon references zero), designated as Pina-D1r. Correspondingly, an STS marker Pina-N2 with an expected fragment size of 436-bp spanning the 10,415-bp deletion was developed for detection of the Pina-D1r allele. This study provided a useful molecular marker for straightforward detection of one of the PINA-null alleles and would also be helpful to further understand the molecular and genetic basis of kernel hardness in bread wheat.