Gene editing of authentic Brassica rapa flavonol synthase 1 generates dihydroflavonol-accumulating Chinese cabbage

Flavonols are the major class of flavonoids of green Chinese cabbage(Brassica rapa subsp.pekinensis).The B.rapa genome harbors seven flavonol synthase genes(BrFLSs),but they have not been functionally characterized.Here,transcriptome analysis showed four BrFLSs mainly expressed in Chinese cabbage.Among them,only BrFLS1 showed major FLS activity and additional flavanone 3β-hydroxylase(F3H)activity,while BrFLS2 and BrFLS3.1 exhibited only marginal F3H activities.We generated BrFLS1-knockout(BrFLS1-KO)Chinese cabbages using CRISPR/Cas9-mediated genome editing and obtained transgene-free homozygous plants without off-target mutation in the T1 generation,which were further advanced to the T2 generation showing normal phenotype.UPLC-ESI-QTOF-MS analysis revealed that flavonol glycosides were dramatically decreased in the T2 plants,while dihydroflavonol glycosides accumulated concomitantly to levels corresponding to the reduced levels of flavonols.Quantitative PCR analysis revealed that the early steps of phenylpropanoid and flavonoid biosynthetic pathway were upregulated in the BrFLS1-KO plants.In accordance,total phenolic contents were slightly enhanced in the BrFLS1-KO plants,which suggests a negative role of flavonols in phenylpropanoid and flavonoid biosynthesis in Chinese cabbage.Phenotypic surveys revealed that the BrFLS1-KO Chinese cabbages showed normal head formation and reproductive phenotypes,but subtle morphological changes in their heads were observed.In addition,their seedlings were susceptible to osmotic stress compared to the controls,suggesting that flavonols play a positive role for osmotic stress tolerance in B.rapa seedling.In this study,we showed that CRISPR/Cas9-mediated BrFLS1-KO successfully generated a valuable breeding resource of Chinese cabbage with distinctive metabolic traits and that CRISPR/Cas9 can be efficiently applied in functional Chinese cabbage breeding.

基于辣椒基因组测序探究辣味在种群中的进化

辣椒(Capsicum annuum)是美洲最古老的驯化作物之一,在全世界被广泛种植。本研究对CM334(墨西哥地方品种)进行了全基因组测序和组装(覆盖度为186.6×),同时还对另外两个栽培种和一个野生种分别进行了重测序和从头(de novo)测序。结果表明辣椒基因组大小约为番茄的4倍,辣椒基因组上存在大量gypsy转座子和花椰菜病毒家族元件的积累。结合转录组综合分析认为,辣椒素合成酶基因表达模式的改变及新功能化是导致辣椒素生物合成的原因,研究还发现辣椒与番茄在乙烯合成及果实成熟调控方面存在明显不同的分子模式。辣椒参考基因组的发布为辣椒营养品质和药用价值的改善提供了重要平台。