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

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.

Multiplex CRISPR/Cas9‐mediated mutagenesis of alfalfa FLOWERING LOCUS Ta1 (MsFTa1) leads to delayed flowering time with improved forage biomass yield and quality

“…In general, small deletions (1–11 bp) were predominant mutations (83% frequency) (Figure 3 , Figure S1 ). CRISPR/Cas9 system has been shown to induce similar mutation events (indels) with large truncations for other genes in alfalfa, soybean and rice (Cai et al ., 2018 ; Chen et al ., 2020 ; Singer et al ., 2022 ; Stritzler et al ., 2022 ; Wolabu et al ., 2020a ; Wu et al ., 2020 ).…”

“…The development of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR‐associated protein 9) system for genome editing has paved the way for precise engineering of genomes for basic and applied research, including the trait‐driven improvement of important crop species (Cai et al ., 2019 ; Chen et al ., 2020 ; Kumar et al ., 2022 ; Singer et al ., 2022 ; Wolabu et al ., 2020a , b ). Recently, this system has been used to generate delayed flowering time mutants in different species.…”

Deciphering the role of SPL12 and AGL6 from a genetic module that functions in nodulation and root regeneration in Medicago sativa

“…It was also reported that miR156 targets at least eleven SPL genes, including SPL12 , for silencing by transcript cleavage (Gao et al 2016 ; Feyissa et al 2021 ). Whereas the role of some of the targeted SPL transcription factors, such as SPL13, SPL9, SPL8 and SPL20 have been characterized in alfalfa (Arshad et al 2017 ; Gao et al 2018a ; Feyissa et al 2019 , 2021 ; Matthews et al 2019 ; Hanly et al 2020 ; Ma et al 2021 ; Singer et al 2021 ), the specific functions of SPL12 remain elusive, as no studies have been conducted on the possible role of miR156/SPL12 module in the development of underground tissues. In the present study, we analyzed transgenic plants with altered transcript levels of SPL12 and AGL6 , including SPL12 -RNAi, 35S::SPL12 , GFP-tagged SPL12 and AGL6 -RNAi to investigate the role of SPL12 in root architecture.…”

Application of CRISPR/Cas9 technology in forages

“…CRISPR technology has been used for trait modification in alfalfa. Singer et al (2022) used the binary pKSE401 vector to validate the function of MsSPL8 (Gou et al, 2018) in alfalfa. In the vector, the expression of sgRNAs was driven by the Arabidopsis U6 polymerase Ⅲ promoter and the expression of a Zea mays codon‐optimized Cas9 was driven by the CaMV35S promoter.…”

“…In the vector, the expression of sgRNAs was driven by the Arabidopsis U6 polymerase Ⅲ promoter and the expression of a Zea mays codon‐optimized Cas9 was driven by the CaMV35S promoter. The resulting genotypes displayed morphological alterations, even with the presence of up to two wild‐type MsSPL8 alleles, including early flowering, reduced leaf size, reduced plant height, and decreased biomass (Singer et al, 2022). By CRISPR editing of the MsGA3ox1 gene from alfalfa, Zheng et al (2022) obtained three lines that carried mutations in all four alleles in the T 0 generation.…”