Targeted Knockout of <i>BnTT2</i> Homologues for Yellow-Seeded <i>Brassica napus</i> with Reduced Flavonoids and Improved Fatty Acid Composition

Abstract

<i>Brassica napus</i> is one of the important oil crops grown worldwide, and oil quality improvement is a major goal in rapeseed breeding. Yellow seed is an excellent trait, which has great potential in improving seed quality and economic value. In this study, we created stable yellow seed mutants using a CRISPR/Cas9 system and obtained the yellow seed phenotype only when the four alleles of two <i>BnTT2</i> homologues were knocked out, indicating that the two <i>BnTT2</i> homologues had conserved but redundant functions in regulating seed color. Histochemical staining and flavonoid metabolic analysis proved that the <i>BnTT2</i> mutation hindered the synthesis and accumulation of proanthocyanidins. Transcriptome analysis also showed that the <i>BnTT2</i> mutation inhibited the expression of genes in the phenylpropanoid and flavonoid biosynthetic pathway, which might be regulated by the complex of BnTT2, BnTT8 and BnTTG1. In addition, the homozygous mutants of <i>BnTT2</i> homologues increased oil content and improved fatty acid composition with higher linoleic acid (C18:2) and linolenic acid (C18:3), which could be used for the genetic improvement of rapeseed. Overall, this research showed that the <i>BnTT2</i> mutation can be used for yellow seed breeding and oil improvement, which is of great significance in improving the economic value of rapeseeds.