Improving wheat grain hardness by knocking out pina and pinb genes

Common wheat (Triticum aestivum L) is one of the most important and widely cultivated staple crops in the world. With the increasing growth of the economy and continuous improvement of people's living standards, the demand for high-quality specialized wheat varieties is growing. In recent years, wheat grain quality improvement has become a main breeding goal. Grain hardness is one of the most important quality characteristics of cultivated wheat (Triticum aestivum L.), which was primarily controlled by the pina and pinb genes on the 5D chromosome. The two genes constitute the molecular genetic basis of wheat grain hardness together. Pina-D1a/pinb-D1a is the most common genotype in wild-type wheat, and the mutation in one of the two genes can increase grain hardness. There are various types of mutations in the pina and pinb genes in common wheat in China, including the deletion of the PINA protein caused by pina mutations and pinb nucleotide mutations, and the variations could change the grain hardness. At present, there are many discovered Pin alleles, except for the deletion of pina, all of which belong to single base mutations. Most wheat materials containing these mutation types have similar grain hardness, with an SKCS hardness index between 40 and 60, which is difficult to meet the requirements of high-quality bread production for grain hardness and flour water absorption. CRISPR/Cas9-mediated (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9) genome editing technology has been widely applied in wheat, which promotes the research on wheat gene function. In this study, CRISPR/Cas9 technology was utilized to knock out pina and pinb genes. Two specific sgRNAs for each gene were designed according to the sgRNA design principles. Single and double gene editing vectors were constructed and transformed into the winter wheat cultivar K35 by the Agrobacterium-mediated transformation. Through the successive screening across T0, T1, and T2 generations, pina and pinb mutants, as well as pinapinb double mutants were obtained. The homozygous mutants were further confirmed by Sanger sequencing. The obtained mutants with elevated grain hardness did not affect the plant growth and other agronomic traits. The cross-section of grains was observed using scanning electron microscopy. The results showed that the binding of starch particles became more compact. The grain hardness of the mutants was all higher than the non-transgenic plants. Transcriptome sequencing identified differentially expressed genes, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. These results suggested that the formation and variation of wheat grain hardness involved a complex interaction of multiple metabolic pathways. The creation of homozygous mutants with novel allelic variations offers precise insights into grain hardness research and provides valuable germplasm resources for breeding high-quality specialized wheat. This study is of great significance for functional investigations and wheat quality improvement.