CRISPR/Cas9-mediated tetra-allelic mutation of the 'Green Revolution' SEMIDWARF-1 (SD-1) gene confers lodging resistance in tef (Eragrostis tef)

Abstract

Tef is a staple food and a valuable cash crop for millions of people in Ethiopia. Lodging is a major limitation to tef production, and for decades, the development of lodging resistant varieties proved difficult with conventional breeding approaches. We used CRISPR/Cas9 to introduce knockout mutations in the tef orthologue of the rice SEMIDWARF-1 (SD-1) gene to confer semidwarfism and ultimately lodging resistance. High frequency recovery of transgenic and SD-1 edited tef lines was achieved in two tef cultivars by Agrobacterium-mediated delivery into young leaf explants of gene editing reagents along with transformation and regeneration enhancing morphogenic genes, BABY BOOM (BBM) and WUSCHEL2 (WUS2). All of the 23 lines analyzed by next-generation sequencing had at least two or more alleles of SD-1 mutated. Of these, 83% had tetra-allelic frameshift mutations in the SD-1 gene in primary tef regenerants, which were inherited in subsequent generations. Phenotypic data generated on T₁ and T₂ generations revealed that the sd-1 lines have reduced culm and internode lengths with no reduction in either panicle or peduncle lengths. These characteristics are comparable with rice sd-1 plants. Measurements of lodging, in greenhouse-grown plants, showed that sd-1 lines have significantly higher resistance to lodging at the heading stage compared with the controls. This is the first demonstration of the feasibility of high frequency genetic transformation and CRISPR/Cas9-mediated genome editing in this highly valuable but neglected crop. The findings reported here highlight the potential of genome editing for the improvement of lodging resistance and other important traits in tef.

Keywords: BABY BOOM; Eragrostis tef; SEMIDWARF-1; WUSCHEL; CRISPR/Cas9; lodging.

Figure 1

Tef and rice GA 20‐oxidases. The four tef GA 20‐oxidases (EtGA20ox1‐4) and corresponding rice orthologs (a), phylogenetic tree showing grouping of each of the tef GA 20‐oxidases with corresponding rice orthologs (b). Note, rice SD‐1 grouped with EtGA20ox2. Multiple sequence alignment of tef GA 20‐oxidases along with rice SD‐1 showing conservation of critical amino acid residues (c). Multiple sequence alignment was run using Clustal Omega (Madeira et al., 2019), and aligned sequence was edited in sequence alignment viewer Jalview (Waterhouse et al., 2009). The highly conserved HX(D/E)X_nH triad motif (marked with red star), essential for binding Fe(II) and the two residues (marked with green star) involved in 2‐oxoglutarate binding (c) common to other 2‐oxoglutarate/Fe(II)‐dependent dioxygenases (2‐ODDs) described by Farrow and Facchini (2014) are shown.

Figure 2

Tef plant transformation and recovery of transgenic events. Two‐ to three‐week‐old tef seedlings grown aseptically (a) portion of young leaves (folded young leaves or stems) used as a source of explants (b) excised and longitudinally sectioned young leaf explants ready for Agrobacterium‐mediated transformation (c), white light and UV‐light images of sectors of callus expressing green fluorescent protein (GFP) after 4–5 weeks culture on callus induction media (d, e), regenerating tef plantlets developing on regeneration media (f) and white light (g) and UV‐light (h) pictures of transgenic tef panicle showing T₁ seeds segregating for GFP expression obtained from 12‐week‐old T₀ plants grown in the growth chamber. Scale bars in a, b and c = 1 cm, in d and e = 1 mm.

Figure 3

Mutations in the tef SD‐1 gene at two target sites (gRNA1 and gRNA2). Schematic presentation of tef SD‐1 showing relative positions of gRNA1 and gRNA2 (a), on exon‐1 and exon‐2, respectively; insertion (+) deletion (−) generated at the two target sites (red font) in T₀ Magna edited plants (b) T₀ Ada edited plants (c) T₂ Ada edited plants (d). Read percent of each of the mutation calls were determined from next‐generation sequencing (NGS) data generated at each respective generation for the target site. Nucleotides in blue are the protospacer adjacent motive (PAM) site. Note chimera seen at gRNA2 site.

Figure 4

Tef plant height at vegetative growth stage in T₁ generation plants after 5 weeks of growth in the growth chamber. Performance of tetra‐allelic sd‐1 mutated lines generated from Magna (a, c) and Ada (b, d) with respective wild‐type controls. Plant height was measured from stem base at soil level to the topmost collar of the main stem. Bars show mean ± SD, n = 18–36. ***, denotes for significant differences compared with the wild‐type cultivar at P < 0.001. Scale bars in (c) and (d) = 10 cm.

Figure 5

Tef plant height and internode, culm and panicle lengths in T₂ tetra‐allelic sd‐1 lines and wild‐type Magna. Plant height at 5 weeks after planting (a), plant height (b) panicle length (c), culm length (d) and lengths of each of the internodes from internode number 2 through 7 (e) measured at harvest 13 weeks after planting from staked plants. Internode length is the length of internodes between two consecutive nodes. Culm length is the sum of the lengths of all internodes without internode #1 (peduncle) and panicle lengths. Plant height was recorded by adding the sum of all internodes including peduncle and panicle lengths. Measurements were taken on 10 plants per line, three stems per plant (pot). *, ** and ***, indicate significant differences compared with the wild‐type Magna at P < 0.05, P < 0.01, P < 0.001. ns, stands for nonsignificant differences at P < 0.05.

Figure 6

Internode diameter of sd‐1 tef lines and Magna control. Internode diameter of internode number 9 (first internode above the soil) (a) and Internode 8 (second internode above the soil) (b) of sd‐1 tef lines and the wild‐type Magna control. Data were collected using caliper for external diameter. Bars are SD, n = 5–7. **, *** and **** stand for significant differences, respectively, at P ≤ 0.01, P ≤ 0.001 and P ≤ 0.0001. The Student's t‐test was used for comparison.

Figure 7

Tef lodging analyses using PlantCV. Tef plant pictures were collected on weekly basis from the 4th to 8th weeks after planting. Lodging was estimated by analyzing collected images using PlantCV from plant height, width and computing height to width ratio. Average normalized plant height (a); width (b) and plant height to width ratio (c) of two semidwarf sd‐1 tef lines and the wild‐type Magna cultivar. Representative images of sd‐1 tef line collected at 6 (d) and 8 (f), and wild‐type Magna at 6 (e) and 8 (g) weeks after planting. Note lodging in the wild‐type control.