The CRISPR/Cas9 site-directed gene-editing system offers great advantages for identifying gene function and crop improvement. The circadian clock measures and conveys day length information to control rhythmic hypocotyl growth in photoperiodic conditions, to achieve optimal fitness, but operates through largely unknown mechanisms. Here, we generated core circadian clock evening components, Brassica rapa PSEUDO-RESPONSE REGULATOR (BrPRR) 1a, 1b, and 1ab (both 1a and 1b double knockout) mutants, using CRISPR/Cas9 genome editing in Chinese cabbage, where 9–16 genetic edited lines of each mutant were obtained. The targeted deep sequencing showed that each mutant had 2–4 different mutation types at the target sites in the BrPRR1a and BrPRR1b genes. To identify the functions of BrPRR1a and 1b genes, hypocotyl length, and mRNA and protein levels of core circadian clock morning components, BrCCA1 (CIRCADIAN CLOCK-ASSOCIATED 1) and BrLHY (LATE ELONGATED HYPOCOTYL) a and b were examined under light/dark cycles and continuous light conditions. The BrPRR1a and 1ab double mutants showed longer hypocotyls, lower core circadian clock morning component mRNA and protein levels, and a shorter circadian rhythm than wildtype (WT). On the other hand, the BrPRR1b mutant was not significantly different from WT. These results suggested that two paralogous genes may not be associated with the same regulatory function in Chinese cabbage. Taken together, our results demonstrated that CRISPR/Cas9 is an efficient tool for achieving targeted genome modifications and elucidating the biological functions of circadian clock genes in B. rapa, for both breeding and improvement.

Glucosinolates (GSLs) are secondary metabolites providing defense against pathogens and herbivores in plants, and anti-carcinogenic activity against human cancer cells. Profiles of GSLs vary greatly among members of genus Brassica. In this study, we found that a reference line of Chinese cabbage (B. rapa ssp. pekinensis), ‘Chiifu’ contains significantly lower amounts of total GSLs than the oilseed-type B. rapa (B. rapa ssp. trilocularis) line ‘LP08’. This study aimed to identify the key regulators of the high accumulation of GSLs in Brassica rapa plants using transcriptomic and linkage mapping approaches. Comparative transcriptome analysis showed that, in total, 8,276 and 9,878 genes were differentially expressed between ‘Chiifu’ and ‘LP08’ under light and dark conditions, respectively. Among 162 B. rapa GSL pathway genes, 79 were related to GSL metabolism under light conditions. We also performed QTL analysis using a single nucleotide polymorphism-based linkage map constructed using 151 F5 individuals derived from a cross between the ‘Chiifu’ and ‘LP08’ inbred lines. Two major QTL peaks were successfully identified on chromosome 3 using high-performance liquid chromatography to obtain GSL profiles from 97 F5 recombinant inbred lines. The MYB-domain transcription factor gene BrMYB28.1 (Bra012961) was found in the highest QTL peak region. The second highest peak was located near the 2-oxoacid-dependent dioxygenase gene BrGSL-OH.1 (Bra022920). This study identified major genes responsible for differing profiles of GSLs between ‘Chiifu’ and ‘LP08’. Thus, our study provides molecular insights into differences in GSL profiles between vegetative- and oilseed-type B. rapa plants.