Cabbage (Brassica oleracea var. capitata) is a biennial plant with strong self-incompatibility and an obligate requirement for prolonged vernalization by exposure to low temperatures to induce flowering. These characteristics significantly increase the difficulty of exploiting novel germplasm induced by physical or chemical mutagens. In this study, we report a CRISPR/Cas9 gene-editing system based on endogenous tRNA processing to induce high efficiency and inheritable mutagenesis in cabbage. Using the phytoene desaturase gene BoPDS, the S-receptor kinase gene BoSRK, and the male-sterility-associated gene BoMS1 as the target genes, multisite and multiple gene mutations were achieved using a construct with tandemly arrayed tRNA-sgRNA architecture to express multiple sgRNAs. The BoSRK3 gene mutation suppressed self-incompatibility completely, converting the self-incompatible line into a self-compatible line. In addition, the BoMS1 gene mutation produced a completely male-sterile mutant, which was highly cross compatible with its nonmutant isoline at the flowering stage as a result of a simultaneous BoSRK3 gene mutation, enabling the economic propagation of the male-sterile line through bee-mediated cross-pollination. Interestingly, higher site mutation efficiency was detected when a guide sequence was inserted into a location in the tandemly arrayed tRNA-sgRNA architecture that was distal from the upstream Pol III promoter. In addition, mutation sites were also detected in the paralogous genes of the BoPDS and BoSRK genes that had fully consistent sequences or base mismatches but beyond the “seed” region in the spacer sequence compared with the target sgRNAs. Collectively, our results demonstrate that the CRISPR/Cas9 system, coupled with an endogenous tRNA-processing system, is an efficient tool to improve cabbage traits.
Increasing clubroot resistance (CR) of Brassica oleracea by ascertaining the molecular mechanisms has been the key focus in modern B. oleracea breeding. In order to identify the quantitative trait loci (QTLs) associated with CR in B. oleracea, 94 F2 vegetative lines which were developed by tissue culture of selfed seeds from the F1 generation between a clubroot-resistant B. oleracea inbred line and a susceptible line, were identified for disease incidence and six CR-associated traits under a lab inoculation by Plasmodiophora brassicae and were genotyped with the 60K Brassica SNP array. Significant correlations were detected for numbers of fibrous roots and P. brassicae content in roots with disease incidence. Nine linkage groups were constructed from 565 bins which covered around 3,000 SNPs, spanning 1,028 cM of the B. oleracea genome with an average distance of 1.82 cM between adjacent bins. A total of 23 QTLs were identified for disease incidence and the other two correlated traits, individually explaining 6.1–17.8% of the phenotypic variation. Several overlaps were detected among traits, including one three-traits-overlapped locus on linkage group C08 and two important overlapped regions between the two CR-associated traits on C06. The QTLs were compared with known CR loci/genes and the novelty of our QTLs was discussed.
Clubroot caused by Plasmodiophora brassicae is a devastating disease of cabbage (Brassica oleracea). To identify quantitative trait loci (QTLs) for clubroot resistance (CR) in B. oleracea, genomic resequencing was carried out in two sets of extreme pools, group I and group II, which were constructed separately from 110 and 74 F2 cloned lines derived from the cross between clubroot-resistant (R) cabbage “GZ87” (against race 4) and susceptible (S) cabbage “263.” Based on the QTL-sequencing (QTL-Seq) analysis of group I and group II, three QTLs (i.e., qCRc7-2, qCRc7-3, and qCRc7-4) were determined on the C07 chromosome. RNA-Seq and qRT-PCR were conducted in the extreme pools of group II before and after inoculation, and two potential candidate genes (i.e., Bol037115 and Bol042270), which exhibiting upregulation after inoculation in the R pool but downregulation in the S pool, were identified from the three QTLs on C07. A functional marker “SWU-OA” was developed from qCRc7-4 on C07, exhibiting ∼95% accuracy in identifying CR in 56 F2 lines. Our study will provide valuable information on resistance genes against P. brassicae and may accelerate the breeding process of B. oleracea with CR.
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