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.

中文翻译：

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使用 CRISPR/Cas9 基因组编辑在大麦中创建了新的大麦醇溶蛋白等位基因

在大麦中，大麦醇溶蛋白是谷物中的一种储存蛋白，被认为对麦芽质量有负面影响。本研究采用CRISPR/Cas9基因组编辑技术对春大麦品种“黄金承诺”中的大麦醇溶蛋白基因进行编辑。获得了三个转基因品系，但只有两个品系在大麦醇溶蛋白的编码区进行了修饰。通过对 T1 代进行测序分析，发现了四个新的大麦醇溶蛋白等位基因，其中有五个缺失。所有等位基因均携带Del1和Del2缺失，而Mu2、Mu3和Mu4等位基因也分别携带Del3、Del4和Del5缺失。由于Mu1的频率远远超过其他等位基因的频率，因此推断Mu2、Mu3和Mu4等位基因来源于Mu1。Mu1 等位基因是通过基因组编辑创建的，而其他等位基因被认为是体细胞突变。转录组分析表明，突变株系中大麦醇溶蛋白的转录水平低于野生型金色承诺。蛋白质 SDS-PAGE 证实了这一结果。在编辑的品系的谷物中无法检测到野生型 D 大麦醇溶蛋白。新的大麦醇溶蛋白等位基因的创建将为研究大麦醇溶蛋白的功能提供新的种质资源，并有可能培育出具有更好麦芽品质的新品种。