ABSTRACT

Genome editing technology, which enables researchers to modify specific genomic loci, may be useful for accelerating the breeding of many fruit crops. The aim of this study was to evaluate the CRISPR/Cas9-mediated editing of the blueberry (Vaccinium spp.) genome. We first optimised the plant regeneration system to increase the genetic transformation efficiency for ‘Blue Muffin’ and ‘O’Neal’. We also tested the utility of the axillary bud transformation technique for modifying blueberry genes. We revealed that the axillary bud transformation method accelerated the blueberry transformation process and increased the transformation rate. Of the 47 transgenic lines obtained for two cultivars, six lines contained a mutated CENTRORADIALIS (CEN) region. A sequence analysis revealed 1- to 2-bp insertions/deletions in CEN alleles, with an average mutated allele ratio of 19% and 22% for gRNA1 and gRNA2, respectively. Two of four gRNAs (gRNA 3, 4) did not produce mutations, suggesting that selecting appropriate gRNA sequences is critical for genome editing. The growth phenotypes of the CEN-mutated lines imply a non-functional CEN allele in the blueberry genome may restrict vegetative growth. The results described herein confirm the utility of the CRISPR/Cas9 genome editing protocol for functionally characterising blueberry genes.

摘要

使研究人员能够修改特定基因组基因座的基因组编辑技术，对于加速许多水果作物的育种可能很有用。这项研究的目的是评估CRISPR / Cas9介导的蓝莓（Vaccinium spp。）基因组编辑。我们首先优化了植物再生系统，以提高“蓝松饼”和“奥尼尔”的遗传转化效率。我们还测试了腋芽转化技术用于修饰蓝莓基因的效用。我们发现腋芽转化法加速了蓝莓转化过程并提高了转化率。在两个品种获得的47个转基因品系中，六个品系包含一个突变的CENTRORADIALIS（CEN） 地区。序列分析显示在CEN等位基因中有1到2 bp的插入/缺失，gRNA1和gRNA2的平均突变等位基因比率分别为19％和22％。四个gRNA中的两个（gRNA 3、4）没有产生突变，这表明选择合适的gRNA序列对于基因组编辑至关重要。CEN突变株系的生长表型暗示蓝莓基因组中无功能的CEN等位基因可能会限制营养生长。本文描述的结果证实了CRISPR / Cas9基因组编辑协议可用于功能性表征蓝莓基因。