Data_Sheet_1_The CRISPR/Cas9-Mediated Modulation of SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 8 in Alfalfa Leads to Distinct Phenotypic Outcomes.docx

Alfalfa (Medicago sativa L.) is the most widely grown perennial leguminous forage and is an essential component of the livestock industry. Previously, the RNAi-mediated down-regulation of alfalfa SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 8 (MsSPL8) was found to lead to increased branching, regrowth and biomass, as well as enhanced drought tolerance. In this study, we aimed to further characterize the function of MsSPL8 in alfalfa using CRISPR/Cas9-induced mutations in this gene. We successfully generated alfalfa genotypes with small insertions/deletions (indels) at the target site in up to three of four MsSPL8 alleles in the first generation. The efficiency of editing appeared to be tightly linked to the particular gRNA used. The resulting genotypes displayed consistent morphological alterations, even with the presence of up to two wild-type MsSPL8 alleles, including reduced leaf size and early flowering. Other phenotypic effects appeared to be dependent upon mutational dosage, with those plants with the highest number of mutated MsSPL8 alleles also exhibiting significant decreases in internode length, plant height, shoot and root biomass, and root length. Furthermore, MsSPL8 mutants displayed improvements in their ability to withstand water-deficit compared to empty vector control genotypes. Taken together, our findings suggest that allelic mutational dosage can elicit phenotypic gradients in alfalfa, and discrepancies may exist in terms of MsSPL8 function between alfalfa genotypes, growth conditions, or specific alleles. In addition, our results provide the foundation for further research exploring drought tolerance mechanisms in a forage crop.

紫花苜蓿（Medicago sativa L.）是全球栽培面积最广的多年生豆科牧草，亦是畜牧业生产不可或缺的核心饲草资源。此前研究表明，通过RNA干扰（RNAi）介导下调紫花苜蓿SQUAMOSA启动子结合蛋白样8（MsSPL8）的表达，可提升植株分枝能力、再生特性与生物量积累，并增强其耐旱性。本研究拟借助CRISPR/Cas9技术诱导该基因发生突变，以进一步解析MsSPL8在紫花苜蓿中的生物学功能。我们成功在初代转化植株中，针对最多4个MsSPL8等位基因中的3个，在靶位点获得了携带小插入/缺失（indels）突变的紫花苜蓿基因型。基因编辑效率与所使用的向导RNA（gRNA）密切相关。所得突变体基因型均表现出稳定的形态学改变：即使携带最多2个野生型MsSPL8等位基因，仍可观察到叶面积缩小与开花提前的表型。其余表型变化则依赖于突变等位基因的剂量——携带突变MsSPL8等位基因数量最多的植株，其节间长度、株高、地上与地下生物量及根长均显著下降。此外，与空载体对照株系相比，MsSPL8突变体的耐旱能力显著提升。综上，本研究结果提示，等位基因突变剂量可诱导紫花苜蓿产生梯度表型，且不同紫花苜蓿基因型、生长条件或特定等位基因间的MsSPL8功能可能存在差异。本研究同时为解析饲草作物的耐旱机制奠定了研究基础。