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

Brassica
napus 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 BnTT2 homologues were knocked out, indicating that the two BnTT2 homologues had conserved but redundant functions in
regulating seed color. Histochemical staining and flavonoid metabolic
analysis proved that the BnTT2 mutation hindered
the synthesis and accumulation of proanthocyanidins. Transcriptome
analysis also showed that the BnTT2 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 BnTT2 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 BnTT2 mutation can be used
for yellow seed breeding and oil improvement, which is of great significance
in improving the economic value of rapeseeds.

油菜（Brassica napus）是全球重要的油料作物之一，油质的提升是油菜育种的重要目标。黄籽性状作为一种优异的特征，在提升种子品质与经济价值方面具有巨大潜力。在本研究中，我们利用CRISPR/Cas9系统创建了稳定的黄籽突变体，并仅在敲除两个BnTT2同源基因的四个等位基因后获得黄籽表型，这表明这两个BnTT2同源基因在调控种子颜色方面具有保守但冗余的功能。组织化学染色和类黄酮代谢分析证实，BnTT2突变阻碍了原花青素的合成与积累。转录组分析亦显示，BnTT2突变抑制了苯丙素和类黄酮生物合成途径中基因的表达，这可能与BnTT2、BnTT8和BnTTG1的复合体调控相关。此外，BnTT2同源基因的纯合突变体增加了油含量并改善了脂肪酸组成，具有更高的亚油酸（C18:2）和亚麻酸（C18:3）含量，这可用于油菜的遗传改良。总体而言，本研究表明BnTT2突变可应用于黄籽育种和油质改良，对于提升油菜的经济价值具有重要意义。