New D hordein alleles were created in barley using CRISPR/Cas9 genome editing

Abstract

In barley, D hordein is one of the storage proteins in the grain, which is thought to have a negative effect on malting quality. In this study, the CRISPR/Cas9 genome editing technology was employed to edit the D hordein gene in the spring barley cultivar, ‘Golden Promise’. Three transgenic lines were obtained but only two lines were modified in the coding region of D hordein. Four new D hordein alleles, with five deletions, were discovered through sequencing analysis of the T1 generation. All alleles carried the Del1 and Del2 deletions, while the Mu2, Mu3 and Mu4 alleles also carried the Del3, Del4 and Del5 deletions, respectively. As the frequency of Mu1 far exceeded the frequency of the other alleles, it was inferred that the Mu2, Mu3 and Mu4 alleles were derived from Mu1. The Mu1 allele was created by genome editing, whilst the other alleles were considered to be somatic mutations. Transcriptome analysis showed that the transcript level of D hordein was lower in the mutated lines than in wild type Golden Promise. Protein SDS-PAGE confirmed this result. Wild type D hordein protein could not be detected in the grain of the edited lines. The creation of new D hordein alleles will provide a new germplasm resource for studying the function of D hordein and may allow the breeding of new cultivars with better malt quality.