Genome-wide association study of grain hardness and novel Puroindoline alleles in bread wheat

Grain hardness is a critical quality trait that determines the processing properties and commercial value of wheat, playing an essential role in both food security and the high-quality development of the wheat industry. In this study, we used an association panel of 233 wheat accessions to measure the grain hardness index (HI) across four different environments. Based on the re-sequencing data, we performed a genome-wide association study (GWAS) to identify the genetic loci associated with wheat grain hardness. We detected two environmentally stable QTLs named QHa.hzau-5D and QHa.hzau-6B.1, which explained 11.27%-18.75% and 11.44%-15.54% of the phenotypic variation, respectively. QHa.hzau-5D co-localized with the known grain hardness locus (Ha), confirming that Ha locus plays a central role in regulating grain hardness. Sequence variability analysis of Pina and Pinb genes in the tested population revealed seven alleles for each gene, forming 12 distinct haplotypes. Among them, the haplotypes Pina-D1a/Pinb-D1b (n = 99) and Pina-D1a/Pinb-D1a (n = 80) were the most prevalent. A novel PIN allele with double-deletion allelic variation was detected in the cultivar Kenda4. The novel haplotype was characterized by an approximately 27-kb fragment deletion at the Ha locus. Additionally, we successfully developed five kompetitive allele-specific PCR (KASP) markers for four PIN allelic variations, including Pina-D1n, Pinb-D1ac (two SNPs), Pinb-D1d, and Pinb-D1u. Within the QHa.hzau-6B.1 interval, two grain-hardness-associated SNPs (SNP-49222665 and SNP-49239367) were identified in genes TraesCS6B02G247300 and TraesCS6B02G247700. Based on expression profiling and gene annotation, these two genes were proposed as key candidate genes to regulate wheat grain hardness. Furthermore, SNP-49222665 and SNP-49239367 were converted into a CAPS marker and a KASP marker, respectively, for subsequent genotyping. This study provides new insights into the molecular regulatory mechanism of wheat grain hardness and offers a novel theoretical basis and practical molecular markers for wheat quality breeding.

The online version contains supplementary material available at https://doi.org/10.1007/s11032-026-01727-9.