Anthocyanin hyperaccumulation is an important agricultural trait, associated with resistance to abiotic stress, pests, phytopathogenic fungi and bacterial diseases. B. napus with increased anthocyanin pigmentation can be generated by genome editing. Many transcription factors of the MYB family are involved in stress response and anthocyanin biosynthesis. Genes AtMYB60, AtCPC and AtMYBL2 are negative regulators of anthocyanin biosynthesis in Arabidopsis, therefore the knockout of these genes can result in increased anthocyanin pigmentation. gRNA spacers were synthesized to target the orthologs of these genes, identified in Brassica napus. Resulting genetic constructs were introduced to the plant tissues by agroinfiltration. Transient expression of gRNAs targeting DNA-binding domains of MYB transcription factors along with Cas9 nuclease successfully promoted anthocyanin hyperaccumulation. These genetic constructs can be used for genome editing and production of new colored and stress tolerant varieties of oilseed rape.

The larches of Kuzhanovo (Larix sibirica Ledeb.) are protected trees with a round crown growing in the Southern Urals. In 2020 vandals sawed the sapwood of these trees, which exposed the problem of insufficient conservation measures. Their origin and genetic characteristics have been of particular interest to breeders and scientists. The larches of Kuzhanovo were screened for polymorphisms using SSR and ISSR analyses and the sequencing of genetic markers and genes GIGANTEA and mTERF, associated with wider crown shape. A unique mutation was discovered in the atpF–atpH intergenic spacer of all protected trees, but it was absent in some of their descendants and larches with similar crown shape. Mutations were discovered in the rpoC1 and mTERF genes of all samples. Flow cytometry did not reveal any changes in genome size. Our results suggest that the unique phenotype arose from point mutations in L. sibirica, but they are yet to be found in the nuclear genome. The concurrent mutations in the rpoC1 and mTERF genes may indicate that the round crown shape originates from the Southern Urals. The atpF–atpH and rpoC1 genetic markers are not common in studies of Larix sp., but their wider use could help to establish the origin of these endangered plants. The discovery of the unique atpF–atpH mutation also allows for stronger conservation and crime detection efforts.

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Anthocyanins are well-known antioxidants that are beneficial for plants and consumers. Dihydroflavonol-4-reductase (DFR) is a key gene of anthocyanin biosynthesis, controlled by multiple transcription factors. Its expression can be enhanced by mutations in the negative regulator of anthocyanin biosynthesis myeloblastosis family transcription factor-like 2 (MYBL2). The expression profiles of the DFR gene were examined in 43 purple and green varieties of Brassica oleracea L., Brassica napus L., Brassica juncea L., and Brassica rapa L. MYBL2 gene expression was significantly reduced in purple varieties of B. oleracea, and green varieties of B. juncea. The MYBL2 gene sequences were screened for mutations that can affect pigmentation. Expression of the DFR gene was cultivar-specific, but in general it correlated with anthocyanin content and was higher in purple plants. Two single nucleotide polymorphysms (SNPs) were found at the beginning of the DNA-binding domain of MYBL2 gene in all purple varieties of B. oleracea. This mutation, leading to an amino acid substitution and the formation of a mononucleotide repeat (A)8, significantly affects RNA structure. No other noteworthy mutations were found in the MYBL2 gene in green varieties of B. oleracea and other studied species. These results bring new insights into the regulation of anthocyanin biosynthesis in genus Brassica and provide opportunities for generation of new purple varieties with precise mutations introduced via genetic engineering and CRISPR/Cas.