Table_4_Functional Analysis of the teosinte branched 1 Gene in the Tetraploid Switchgrass (Panicum virgatum L.) by CRISPR/Cas9-Directed Mutagenesis.xlsx

Tillering is an important biomass yield component trait in switchgrass (Panicum virgatum L.). Teosinte branched 1 (tb1)/Branched 1 (BRC1) gene is a known regulator for tillering/branching in several plant species; however, its role on tillering in switchgrass remains unknown. Here, we report physiological and molecular characterization of mutants created by CRISPR/Cas9. We successfully obtained nonchimeric Pvtb1a and Pvtb1b mutants from chimeric T0 mutants using nodal culture. The biallelic Pvtb1a-Pvtb1b mutant plants produced significantly more tillers and higher fresh weight biomass than the wild-type plants. The increased tiller number in the mutant plants resulted primarily from hastened outgrowth of lower axillary buds. Increased tillers were also observed in transgene-free BC1 monoallelic mutants for either Pvtb1a-Pvtb1b or Pvtb1b gene alone, suggesting Pvtb1 genes act in a dosage-dependent manner. Transcriptome analysis showed 831 genes were differentially expressed in the Pvtb1a-Pvtb1b double knockdown mutant. Gene Ontology analysis revealed downregulation of Pvtb1 genes affected multiple biological processes, including transcription, flower development, cell differentiation, and stress/defense responses in edited plants. This study demonstrates that Pvtb1 genes play a pivotal role in tiller production as a negative regulator in switchgrass and provides opportunities for further research aiming to elucidate the molecular pathway regulating tillering in switchgrass.

分蘖是柳枝稷（Panicum virgatum L.）重要的生物量产量构成性状。玉米分枝1基因（Teosinte branched 1，tb1）/分枝1基因（Branched 1，BRC1）是多个植物物种分蘖/分枝的已知调控因子，但其在柳枝稷分蘖中的功能尚未明确。本研究报道了经CRISPR/Cas9技术创制的突变体的生理与分子特征。我们通过节点培养从嵌合型T0代突变体中成功获得了非嵌合的Pvtb1a与Pvtb1b突变体。双等位Pvtb1a-Pvtb1b突变体植株的分蘖数显著高于野生型植株，鲜重生物量亦显著提升。该突变体植株分蘖数的增加主要源于下部腋芽的提早萌发生长。在仅针对Pvtb1a-Pvtb1b或单独Pvtb1b基因的无转基因BC1代单等位基因突变体中，同样观察到分蘖数增加的表型，提示Pvtb1基因以剂量依赖的方式发挥调控作用。转录组分析显示，Pvtb1a-Pvtb1b双敲低突变体中共存在831个差异表达基因。基因本体论（Gene Ontology，GO）分析表明，Pvtb1基因的下调会影响编辑植株的多个生物学过程，包括转录、花发育、细胞分化以及胁迫与防御响应。本研究证实，Pvtb1基因作为负调控因子在柳枝稷分蘖产生过程中发挥关键作用，同时为进一步解析柳枝稷分蘖调控的分子通路提供了研究契机。