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

Stacy D Singer; Kimberley Burton Hughes; Udaya Subedi; Gaganpreet Kaur Dhariwal; Kazi Kader; Surya Acharya; Guanqun Chen; Abdelali Hannoufa

doi:10.3389/fpls.2021.774146

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

Front Plant Sci. 2022 Jan 5:12:774146. doi: 10.3389/fpls.2021.774146. eCollection 2021.

Authors

Stacy D Singer¹, Kimberley Burton Hughes¹, Udaya Subedi^{1

2}, Gaganpreet Kaur Dhariwal¹, Kazi Kader¹, Surya Acharya¹, Guanqun Chen², Abdelali Hannoufa³

Affiliations

¹ Lethbridge Research and Development Center, Agriculture and Agri-Food Canada, Lethbridge, AB, Canada.
² Department of Agricultural, Food and Nutritional Sciences, University of Alberta, Edmonton, AB, Canada.
³ London Research and Development Center, Agriculture and Agri-Food Canada, London, ON, Canada.

Abstract

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.

Keywords: Medicago sativa; crop improvement; drought tolerance; forage; genome editing; precision breeding.

Copyright © 2022 Guanqun Chen and Her Majesty the Queen in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada for the contribution of Stacy D. Singer, Kimberely Burton Hughes, Udaya Subedi, Gaganpreet Kaur Dhariwal, Kazi Kader, Surya Acharya and Abdelali Hannoufa.