Table_4_In-Depth Characterization of greenflesh Tomato Mutants Obtained by CRISPR/Cas9 Editing: A Case Study With Implications for Breeding and Regulation.DOCX

Gene editing has already proved itself as an invaluable tool for the generation of mutants for crop breeding, yet its ultimate impact on agriculture will depend on how crops generated by gene editing technologies are regulated, and on our ability to characterize the impact of mutations on plant phenotype. A starting operational strategy for evaluating gene editing-based approaches to plant breeding might consist of assessing the effect of the induced mutations in a crop- and locus-specific manner: this involves the analysis of editing efficiency in different cultivars of a crop, the assessment of potential off-target mutations, and a phenotypic evaluation of edited lines carrying different mutated alleles. Here, we targeted the GREENFLESH (GF) locus in two tomato cultivars (‘MoneyMaker’ and ‘San Marzano’) and evaluated the efficiency, specificity and mutation patterns associated with CRISPR/Cas9 activity for this gene. The GF locus encodes a Mg-dechelatase responsible for initiating chlorophyll degradation; in gf mutants, ripe fruits accumulate both carotenoids and chlorophylls. Phenotypic evaluations were conducted on two transgene-free T2 ‘MoneyMaker’ gf lines with different mutant alleles (a small insertion of 1 nucleotide and a larger deletion of 123 bp). Both lines, in addition to reduced chlorophyll degradation, showed a notable increase in carotenoid and tocopherol levels during fruit ripening. Infection of gf leaves and fruits with Botrytis cinerea resulted in a significant reduction of infected area and pathogen proliferation compared to the wild type (WT). Our data indicates that the CRISPR/Cas9-mediated mutation of the GF locus in tomato is efficient, specific and reproducible and that the resulting phenotype is robust and consistent with previously characterized greenflesh mutants obtained with different breeding techniques, while also shedding light on novel traits such as vitamin E overaccumulation and pathogen resistance. This makes GF an appealing target for breeding tomato cultivars with improved features for cultivation, as well as consumer appreciation and health.

基因编辑已被证实为作物育种突变体创制的极为宝贵的工具，然而其对农业的最终影响，将取决于基因编辑技术培育作物的监管方式，以及我们能否解析突变对植物表型的影响。评估基于基因编辑的作物育种策略的初始操作方案，可采用作物及基因座特异性方式评估诱导突变的效应：具体包括分析作物不同栽培品种的编辑效率、评估潜在脱靶突变，以及对携带不同突变等位基因的编辑株系进行表型鉴定。本研究以两个番茄栽培品种（‘MoneyMaker’与‘San Marzano’）的GREENFLESH（GF）基因座为靶向目标，评估了该基因的CRISPR/Cas9编辑活性相关的编辑效率、特异性及突变模式。GF基因座编码负责启动叶绿素降解的镁脱螯合酶；在gf突变体中，成熟果实可同时积累类胡萝卜素与叶绿素。本研究对两个携带不同突变等位基因的无外源基因T2代‘MoneyMaker’ gf株系进行了表型鉴定：其中一个株系为单核苷酸小片段插入，另一个为123 bp的大片段缺失。相较于野生型，这两个株系不仅延缓了叶绿素降解，还在果实成熟过程中显著提升了类胡萝卜素与生育酚的含量。相较于野生型（WT），gf株系的叶片与果实经灰葡萄孢（Botrytis cinerea）侵染后，病斑面积与病原菌增殖量均显著降低。本研究数据表明，利用CRISPR/Cas9介导的番茄GF基因座突变具有高效、特异且可重复的特点，所获得的表型稳定可靠，与此前通过其他育种技术创制的greenflesh突变体表型一致；同时还揭示了维生素E过量积累、抗病性增强等全新性状。这表明GF基因座可作为优良番茄育种的理想靶向目标，助力培育出兼具更优栽培性状、更佳消费品质与健康价值的番茄栽培品种。