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

Abstract

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.

Gene editing in Brassica napus for basic research and trait development

“…As was the case for BnaTT8, homozygous BnaTT2 double mutants were yellow-seeded due to significantly reduced flavonoid content, especially epicatechin and isorhamnetin, increased oil content and improved fatty acid composition with more polyunsaturated fatty acids (Xie et al 2020). Yellow seeds with homozygous BnaTT2 double LOF mutants also had reduced lignin and synaptic acid (Xie et al 2020).…”

“…In B. rapa and B. juncea, natural LOF mutants in these genes lead to a yellow seeded trait (Li et al 2012a(Li et al , 2012bPadmaja et al 2014). Xie et al (2020) used hypocotyl transformation of cv. J9712 with Agrobacterium tumefaciens, using CRISPR/Cas9 with gRNAs designed to target LOF alleles in the R2 and R3 domain of the Myb123 transcription factors BnaA08.TT2 (BnaA08g29930D) and BnaC08.TT2 (BnaC08g07960D) towards achieving a yellow-seeded canola.…”

“…J9712 with Agrobacterium tumefaciens, using CRISPR/Cas9 with gRNAs designed to target LOF alleles in the R2 and R3 domain of the Myb123 transcription factors BnaA08.TT2 (BnaA08g29930D) and BnaC08.TT2 (BnaC08g07960D) towards achieving a yellow-seeded canola. Following selection, 81 transformants had PCR products from the target sites sequenced identifying a total of 4 plants with targeted mutant alleles, three alleles for each of BnaA08.TT2 and BnaC08.TT2 (Xie et al 2020). In the T 0 generation, 2 plants had a mutant allele in either BnaA08.TT2 or BnaC08.TT2 and the other two with biallelic mutations in either BnaA08.TT2 or BnaC08.TT2 and hemizygous mutations in the other allele; no homozygous mutant lines were obtained (Xie et al 2020).…”

“…Following selection, 81 transformants had PCR products from the target sites sequenced identifying a total of 4 plants with targeted mutant alleles, three alleles for each of BnaA08.TT2 and BnaC08.TT2 (Xie et al 2020). In the T 0 generation, 2 plants had a mutant allele in either BnaA08.TT2 or BnaC08.TT2 and the other two with biallelic mutations in either BnaA08.TT2 or BnaC08.TT2 and hemizygous mutations in the other allele; no homozygous mutant lines were obtained (Xie et al 2020). As was the case for BnaTT8, homozygous BnaTT2 double mutants were yellow-seeded due to significantly reduced flavonoid content, especially epicatechin and isorhamnetin, increased oil content and improved fatty acid composition with more polyunsaturated fatty acids (Xie et al 2020).…”

“…In the T 0 generation, 2 plants had a mutant allele in either BnaA08.TT2 or BnaC08.TT2 and the other two with biallelic mutations in either BnaA08.TT2 or BnaC08.TT2 and hemizygous mutations in the other allele; no homozygous mutant lines were obtained (Xie et al 2020). As was the case for BnaTT8, homozygous BnaTT2 double mutants were yellow-seeded due to significantly reduced flavonoid content, especially epicatechin and isorhamnetin, increased oil content and improved fatty acid composition with more polyunsaturated fatty acids (Xie et al 2020). Yellow seeds with homozygous BnaTT2 double LOF mutants also had reduced lignin and synaptic acid (Xie et al 2020).…”