Polyphenol oxidase (PPO) in grain is regarded as a major factor resulting in time-dependent darkening of wheat end products, particularly for Asian noodles and steamed bread. Breeding wheat cultivars with low PPO activity using efficient and reliable markers is one of the best ways to reduce the undesirable darkening. In the present study, we developed a gene-specific marker (PPO05) for low PPO activity from the sequence AY515506. This marker detected double PCR fragments (\750 and [750 bp) in the cultivars with low PPO activity and a single PCR fragment (\750 bp) in the cultivars with high PPO activity. Screening of this marker on 235 Chinese wheat micro-core collections showed that the double fragments were present in 113 genotypes and the single fragments in the remaining 122 genotypes. Statistic analysis revealed that the cultivars with the double fragments had significantly lower mean PPO activity than those with single fragments. Through sequence analysis and blast search in NCBI, we found that the cultivars with the double fragments contained the PPO-2Ab allele, while the cultivars with the single fragments contained the PPO-2Aa allele. The PPO-2Ab and PPO-2Da alleles were associated with the low grain PPO activity and the PPO-2Aa and PPO-2Db alleles associated with the high PPO activity. The genotypes carrying both PPO2Ab and PPO-2Da showed the lowest PPO activity, while the genotypes carrying both PPO-2Aa and PPO-2Db showed the highest PPO activity. Comparison of PPO05 and STS01 with the STS markers PPO18 and PPO29 showed that the larger and small fragments of PPO05 were equivalent to the 876-and 685-bp fragments of PPO18, respectively, and that STS01 was the complementary marker of PPO29. Thus, the STS markers PPO05 and STS01 along with PPO18 and PPO29 are the efficient and reliable markers for the evaluation of PPO activity and can be used in wheat breeding programs to improve the quality of noodles and other end products.

The grain hardness index (HI) is one of the important reference bases for wheat quality and commodity properties; therefore, it is essential and useful to identify loci associated with the HI in wheat breeding. The grain hardness index of the natural population including 150 common wheat genotypes was measured in this study. The phenotypic data diversity of HI based on four environments and the best linear unbiased prediction (BLUP) was analyzed. The results showed that the grain HI of the natural population ranged from 15.00 to 83.00, the variation range was from 5.10% to 24.44%, and the correlation coefficient was 0.872–0.980. BLUP value was used to grade and assign the grain HI to hard wheat, mixed wheat, and soft wheat, and the assigned phenotypes were used for genome-wide association analysis. Two types of grain hardness index phenotypic values were used for genome-wide association analysis (GWAS) using a 55K SNP array. A total of five significant association loci (p < 0.001) were excavated, among which four loci could be detected in three or more environments. They were distributed on chromosomes 1A and 7D, and the phenotypic contribution rate was 7.52% to 10.66%. A total of 48 sites related to grain hardness were detected by the assignment method, among which five were stable genetic sites, distributed on chromosomes 1A(2), 3B(1), 4B(1), and 7D(1), with phenotypic contribution rates ranging from 7.63% to 11.12%. Of the five loci detected by the assignment method, two stable loci were co-located in the phenotypic mapping results of the hardness index. One of the loci was consistent with previous reports and located on chromosome 1A, and one locus was unreported on chromosome 7D. Therefore, it may be a feasible attempt to use the assignment method to conduct genome-wide association analysis of the grain hardness index. In this study, a total of five genetic loci for grain hardness stability were excavated, and two of the loci were located in the two phenotypic values, two of which were not reported.