Precise editing of CLAVATA genes in Brassica napus L. regulates multilocular silique development

Multilocular silique (多室角果) is a desirable agricultural trait with great potential for the development of high-yield varieties of Brassica (芸苔属). To date (到目前为止), no spontaneous (天然的) or induced multilocular mutants have been reported in Brassica napus (欧洲油菜), which likely reflects its allotetraploid (异源四倍体) nature and the extremely low probability of the simultaneous random mutagenesis of multiple gene copies with functional redundancy. Here, we present evidence for the efficient knockout of rapeseed (油菜籽) homologues of CLAVATA3 (CLV3) for a secreted peptide (分泌肽) and its related receptors CLV1 and CLV2 in the CLV signalling pathway using the CRISPR/Cas9 system and achieved stable transmission of the mutations across three generations. Each BnCLV gene has two copies located in two subgenomes. The multilocular phenotype can be recovered only in knockout mutations of both copies of each BnCLV gene, illustrating that the simultaneous (同时的) alteration of multiple gene copies by CRISPR/Cas9 mutagenesis has great potential in generating agronomically important mutations in rapeseed. The mutagenesis efficiency varied widely from 0% to 48.65% in T0 with different single-guide RNAs (sgRNAs), indicating that the appropriate selection of the sgRNA is important for effectively generating indels in rapeseed. The double mutation of BnCLV3 produced more leaves and multilocular siliques with a significantly higher number of seeds per silique and a higher seed weight than the wild-type and single mutant plants, potentially contributing to increased seed production. We also assessed the efficiency of the horizontal transfer (水平转移) of Cas9/gRNA cassettes by pollination. Our findings reveal the potential for plant breeding strategies to improve yield traits in currently cultivated rapeseed varieties.

多室角果是一个非常理想的农艺性状，对于开发高产的芸苔属物种有潜在的帮助。到目前为止，还没有天然的或者人工诱导的多室角果欧洲油菜突变体，这可能是由于欧洲油菜本身是异源四倍体的特性所导致的，进而同时对存在功能冗余的多个基因拷贝同时进行诱变变得希望渺茫。本文报道了利用CRISPR/Cas9系统有效敲除油菜CLV3的同源基因，以及其在CLV信号通路中相关的受体CLV1和CLV2基因，并获得了连续三代的突变稳定遗传株系。每个BnCLV基因都有两个拷贝，位于不同的两个亚基因组上。多室角果的表型只有通过同时敲掉BnCLV基因的两个拷贝，进一步表明了CRISPR/Cas9诱变系统可以通过同时突变多个基因拷贝进而获得重要的农艺性状突变体油菜籽。利用不同的sgRNAs获得的T0代植株突变的效率从0％到48.65%不等，表明对于sgRNA的合适的选择是油菜高效突变的重要前提。BnCLV3基因的双突变会产生更多的叶子以及多室的角果，并且每个角果产生的种子数量也显著的增加，并且种子重量也要较野生型和单突变体植株要重，而这有潜力用于提升种子的产量。作者同时还通过授粉作用评估Cas9/gRNA表达盒水平转移的效率。本文的研究为现有油菜栽培种产量性状改良的植物育种策略提供参考。

通讯：范楚川 (http://cpst.hzau.edu.cn/info/1015/1659.htm)

个人简介：2006年，华中农业大学，博士，方向：生化与分子生物学专业；2008年3月-9月，美国Donald Danforth Plant Science Center，访问学者。

doi: 10.1111/pbi.12872