Gene-edited triple mutant Medicago plants do not flower.

Optimized flowering time is an important trait that ensures successful plant adaptation and crop productivity. genes encode MADS transcription factors, which are known to play important roles in flowering control in many plants. This includes the best-characterized eudicot model (Arabidopsis), where promotes flowering and functions as a floral integrator gene integrating signals from different flowering-time regulatory pathways. (Medicago) is a temperate reference legume with strong genomic and genetic resources used to study flowering pathways in legumes. Interestingly, despite responding to similar floral-inductive cues of extended cold (vernalization) followed by warm long days (VLD), such as in winter annual Arabidopsis, Medicago lacks FLC and CO which are key regulators of flowering in Arabidopsis. Unlike Arabidopsis with one gene, multiple gene duplication events have given rise to three paralogs within the genus in legumes: one Fabaceae group A gene, , and two tandemly repeated Fabaceae group B genes, and . Previously, we showed that has unique functions in floral promotion in Medicago. The retroelement insertion single mutant showed moderately delayed flowering in long- and short-day photoperiods, with and without prior vernalization, compared to the wild-type. In contrast, single mutants did not have altered flowering time or flower development, indicating that it was redundant in an otherwise wild-type background. Here, we describe the generation of triple mutant lines using CRISPR-Cas9 gene editing. We studied two independent triple mutant lines that segregated plants that did not flower and were bushy under floral inductive VLD. Genotyping indicated that these non-flowering plants were homozygous for the predicted strong mutant alleles of the three genes. Gene expression analyses using RNA-seq and RT-qPCR indicated that these plants remained vegetative. Overall, the non-flowering triple mutants were dramatically different from the single mutant and the Arabidopsis mutant; implicating multiple genes in critical overlapping roles in the transition to flowering in Medicago.