*4.2. Real-Time Quantitative PCR*

RNA was extracted from 50 mg of the plant tissue using Lira reagent (Biolabmix, Novosibirsk, Russia). cDNA was synthesized with OT-M-MuLV-RH kit (Biolabmix, Novosibirsk, Russia) using oligo(dT) primer.

Real-time quantitative PCR (RT-qPCR) was performed on Quant studio 5 Real-Time PCR System (Thermo Fisher Scientific, USA) using BioMaster HS-qPCR Lo-ROX SYBR mix (Biolabmix, Novosibirsk, Russia) with the primers 50 -CGGATCTGCAGGTTTAACTG-30 and 5 0 -GCCGACATGTGACTATCGG-30 for the *DFR* gene and two primer pairs 5 0 -AGAGGGAGAAAGTACGCAAAG-30 and 50 -CGGTTCGTCCAGGCAATCTT-30 ; 50 -TTC ATGCACTTCTTGGCAAT-30 and 50 -CGGTTCGTCCAGGCAATCTT-30 for the *MYBL2* gene. Primers were designed using NCBI Primer BLAST tool [46]. Actin7 (primers 5 0 -AGGAATCGCTGACCGTATGAG-30 and 50 -GCTGAGGGATGCAAGGATGGA-30 ) was used as a reference gene. This gene is stably expressed in normal conditions and under biotic and abiotic stress [47].

#### *4.3. DNA Sequencing*

DNA was extracted from plant tissue using CTAB method [48].

*MYBL2-1* gene was amplified using primers 50 -AGAGGGAGAAAGTACGCAAAG-30 and 50 -AGAAGTGTTTCTTGACTCGTTGA-30 . PCR products were purified with ExoSAP-IT™ PCR Product Cleanup Reagent (Thermo Fisher Scientific, Waltham, MA, USA), prepared using BigDye™ Terminator v3.1 Cycle Sequencing Kit and subjected to Sanger sequencing via Applied Biosystems 3500 genetic analyzer (Thermo Fisher Scientific, Waltham, MA, USA).

#### *4.4. Total Anthocyanin Content*

Anthocyanin pigment concentration was measured by the spectrophotometric pH differential method using a Perkin Elmer LS 55 Luminescence Spectrometer (Perkin Elmer, Waltham, MA, USA) and expressed as cyanidin-3-glucoside equivalents. Anthocyanins in *Brassicaceae* are known to be cyanidin derivatives. Extracts from 15 mg of dried leaf tissue in pH 1.0 and pH 4.5 buffers were prepared and analyzed according to Lee et al. [49]. Measurements were performed in three technical and three biological replicates in 96-well plates.

#### *4.5. Statistical Analysis*

Three samples of each variety were used for DNA and RNA extraction. Primers were designed using NCBI Primer BLAST tool [46]. RT-qPCR was carried out for three replicates of each sample. Results of RT PCR were assessed by 2−∆∆CT method. Sequencing was performed using forward and reverse primers for each sample. Sequences were aligned to the reference sequence of the *MYBL2-1* gene of *B. rapa* (JN379102) and analyzed using SnapGene software. Functional domains were analyzed via InterPro [50]. Search for gRNAs was performed in CRISPOR web tool [51]. RNA structure was predicted by RNAfold web tool [52]. For all experiments, means and standard deviation (*p* < 0.05) were compared by analysis of variance (ANOVA), and Pearson correlation coefficient was calculated using LibreOffice v.

**Author Contributions:** DNA and RNA extraction and PCR, E.K.; sequencing, Y.S. and A.A.; methodology, analysis, and supervision, E.V.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Russian Science Foundation, grant number 20-74-10053.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The sequence of the mutant genotype was submitted to the NCBI database (ON464161).

**Conflicts of Interest:** The authors declare no conflict of interest.
