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

Abstract

Alfalfa (Medicago sativa L.) is a perennial flowering plant in the legume family that is widely cultivated as a forage crop for its high yield, forage quality and related agricultural and economic benefits. Alfalfa is a photoperiod sensitive long-day (LD) plant that can accomplish its vegetative and reproductive phases in a short period of time. However, rapid flowering can compromise forage biomass yield and quality. Here, we attempted to delay flowering in alfalfa using multiplex CRISPR/Cas9-mediated mutagenesis of FLOWERING LOCUS Ta1 (MsFTa1), a key floral integrator and activator gene. Four guide RNAs (gRNAs) were designed and clustered in a polycistronic tRNA-gRNA system and introduced into alfalfa by Agrobacterium-mediated transformation. Ninety-six putative mutant lines were identified by gene sequencing and characterized for delayed flowering time and related desirable agronomic traits. Phenotype assessment of flowering time under LD conditions identified 22 independent mutant lines with delayed flowering compared to the control. Six independent Msfta1 lines containing mutations in all four copies of MsFTa1 accumulated significantly higher forage biomass yield, with increases of up to 78% in fresh weight and 76% in dry weight compared to controls. Depending on the harvesting schemes, many of these lines also had reduced lignin, acid detergent fibre (ADF) and neutral detergent fibre (NDF) content and significantly higher crude protein (CP) and mineral contents compared to control plants, especially in the stems. These CRISPR/Cas9-edited Msfta1 mutants could be introduced in alfalfa breeding programmes to generate elite transgene-free alfalfa cultivars with improved forage biomass yield and quality.

Keywords: FLOWERING LOCUS T; CRISPR/Cas9; alfalfa; biomass; genome editing; quality.

Figure 1

Schematic illustration of alfalfa FLOWERING LOCUS T (MsFTa1) gene structure with designed multiplex gRNAs–CRISPR/Cas9 vector and genome editing efficiency in alfalfa. (a) MsFTa1 gene structure, target sites in the coding region. (b) Illustration of the multiplex MsFTa1–gRNA–CRISPR/Cas9 vector. (c) Mutation efficiency (%) of different gRNAs at different target sites of MsFTa1.

Figure 2

Initial analysis of flowering time in Msfta1 CRISPR/Cas9 mutant lines. (a) Graph representing flowering time (days to flower) in all putative Msfta1 mutant lines (T0) compared to control plants harbouring empty vector (EV). Highlighted in red represents the six lines selected for further analyses. (b) Phenotype of promising putative Msfta1 mutant lines exhibiting delayed flowering and enhanced vegetative growth. Red arrow indicates developed flowers in EV.

Figure 3

Molecular analysis of MsFTa1 mutation events (deletion/insertion/substation) generated by multiplex gRNA–CRISPR/Cas9 vector. Mutations are shown at the four different target sites: MsFTa1–gRNA1, MsFTa1–gRNA2, MsFTa1–gRNA3 and MsFTa1–gRNA4. Wild‐type (WT) reference sequence is shown for each target site. Deletions are indicated by red dashed lines. Insertions or substitutions are indicated by red letters. PAMs are indicated by red underlined italicized letters. The frequency of mutations in each of the four allelic copies of the MsFTa1 gene is indicated in the right column. The four allelic copies are designated as red allele‐1 (A1), allele‐2 (A2), allele‐3 (A3) and allele‐4 (A4).

Figure 4

Analysis of agronomic and forage quality traits in selected homozygous Msfta1 mutant lines at the whole plant level. (a) Flowering time analysis of Msfta1 lines versus control plants harbouring empty vector (EV). (b) Analysis of plant height (cm) in Msfta1 lines versus EV plants. (c) Fresh and (d) dry weight analyses of Msfta1 lines versus EV plants. (e) Analysis of crude protein (%) and lignin content (%) in Msfta1 lines versus EV plants. (f) Analysis of forage digestibility parameters: ADF (acid detergent fibre), NDF (neutral detergent fibre) and IVTDMD (in vitro true dry matter digestibility) in Msfta1 lines versus EV plants. Tissue sampling was performed for each line that was harvested when it flowered. Data represent mean values (±SD; n = 6) and were analysed statistically using Student's t test (*P < 0.05; **P < 0.01; ***P < 0.001).

Figure 5

Analysis of forage quality traits in stem tissues of Msfta1 mutant lines harvested under different schemes. Batch 1 tissues were harvested when EV plants started to flower and Batch 2 tissues were harvested when the line Msfta1‐094 (exhibiting intermediate phenotype) started to flower. (a) Analysis of lignin (%) and crude protein content (%) in stem tissues of Msfta1 lines versus EV plants in Batch 1. (b) Analysis of forage digestibility traits including ADF, NDF and IVTDMD in stem tissues of Msfta1 lines versus EV plants in Batch 1. (c) Analysis of minerals content (%) in stem tissues of Msfta1 lines versus EV plants in Batch 1. (d) Analysis of lignin (%) and crude protein content (%) in stem tissues of Msfta1 lines versus EV plants in Batch 2. (e) Analysis of forage digestibility traits including ADF, NDF and IVTDMD in stem tissues of Msfta1 lines versus EV plants in Batch 2. (f) Analysis of minerals content (%) in stem tissues of Msfta1 lines versus EV plants in Batch 2. Data represent mean values (±SD; n = 6) and were analysed statistically using Student's t test (*P < 0.05; **P < 0.01; ***P < 0.001).

Figure 6

Analysis of forage quality traits in leaf tissues of Msfta1 mutant lines harvested under different schemes: Batch 1 tissues were harvested when EV plants started to flower and Batch 2 tissues were harvested when the line Msfta1‐094 (exhibiting intermediate phenotype) started to flower. (a) Analysis of lignin (%) and crude protein content (%) in leaf tissues of Msfta1 lines versus EV plants in Batch 1. (b) Analysis of forage digestibility traits including ADF, NDF and IVTDMD in leaf tissues of Msfta1 lines versus EV plants in Batch 1. (c) Analysis of minerals content (%) in leaf tissues of Msfta1 lines versus EV plants in Batch 1. (d) Analysis of lignin (%) and crude protein content (%) in leaf tissues of Msfta1 lines versus EV plants in Batch 2. (e) Analysis of forage digestibility traits including ADF, NDF and IVTDMD in leaf tissues of Msfta1 lines versus EV plants in Batch 2. (f) Analysis of minerals content (%) in leaf tissues of Msfta1 lines versus EV plants in Batch 2. Data represent mean values (±SD; n = 6) and were analysed statistically using Student's t test.