Semi-dwarfism and lodging tolerance in tef (Eragrostis tef) is linked to a mutation in the α-Tubulin 1 gene

Abstract

Genetic improvement of native crops is a new and promising strategy to combat hunger in the developing world. Tef is the major staple food crop for approximately 50 million people in Ethiopia. As an indigenous cereal, it is well adapted to diverse climatic and soil conditions; however, its productivity is extremely low mainly due to susceptibility to lodging. Tef has a tall and weak stem, liable to lodge (or fall over), which is aggravated by wind, rain, or application of nitrogen fertilizer. To circumvent this problem, the first semi-dwarf lodging-tolerant tef line, called kegne, was developed from an ethyl methanesulphonate (EMS)-mutagenized population. The response of kegne to microtubule-depolymerizing and -stabilizing drugs, as well as subsequent gene sequencing and segregation analysis, suggests that a defect in the α-Tubulin gene is functionally and genetically tightly linked to the kegne phenotype. In diploid species such as rice, homozygous mutations in α-Tubulin genes result in extreme dwarfism and weak stems. In the allotetraploid tef, only one homeologue is mutated, and the presence of the second intact α-Tubulin gene copy confers the agriculturally beneficial semi-dwarf and lodging-tolerant phenotype. Introgression of kegne into locally adapted and popular tef cultivars in Ethiopia will increase the lodging tolerance in the tef germplasm and, as a result, will improve the productivity of this valuable crop.

Keywords: Ergarostis tef; kegne; lodging; microtubules; semi-dwarf; tef; α-Tubulin..

Fig. 1.

Kegne plants twist in the right-hand direction, and are semi-dwarf and lodging tolerant. Nine-day-old shoots of (A) WT and (B) kegne, and 3-day-old coleoptiles of (C) WT and (D) kegne in a growth room. (E) The angle of tilting was significantly higher for kegne compared with the WT (P=1.83 E-76 after ANOVA, n=96, error bars indicate 1 SD). (F) The height of both genotypes from germination to harvest maturity; error bars indicate 1 SD (n=10). Two-month-old (G) WT and (H) kegne plants in the greenhouse. Scale bars in A and B=1cm, in C and D=500 μM, in G and H=10cm.

Fig. 2.

Plants grown in the field in Ethiopia. (A) Plant height and (B) lodging index of the WT compared with the kegne mutant at maturity (n=30, height P=1.70E-33, lodging P=2.43E-54 after ANOVA, error bars indicate 1 SD).

Fig. 3.

The response of kegne to propyzamide and oryzalin. (A) The twisting phenotype of kegne was induced in 3-day-old WT plants treated with 2 μM propyzamide. Scale bar=1cm. Effect of different concentrations of oryzalin on the height of (B) WT and (C) kegne plants grown for 10 d in the light. Values with the same letters are not significantly different at the P<0.05 level after Tukey test (n=15). Error bars indicate 1 SD. The height of seedlings transferred to the medium without oryzalin and grown for an additional 6 d to recover from (D) 0.05 μM (n=15) and (E) 0.1 μM oryzalin (n=15).

Fig. 4.

The orientation of cortical microtubules in coleoptiles of the WT and kegne. Distributions of microtubule orientations (in degrees) in epidermal cells (n=56 cells for the WT, and n=59 cells for kegne) of 5-day-old dark-grown seedlings.

Fig. 5.

Overview of the structure of the EtTUA1 gene and the position of the point mutation in a single genomic copy of the kegne mutant. Black boxes indicate the four exons while the lines show the three introns. The total size of the CDS of EtTUA1 is 1353bp or 451 amino acids. The single mutation discovered in kegne was at 1596bp on the genomic sequence or 198 amino acids on the CDS. The C to T change in kegne introduced a unique site for the ClaI restriction enzyme which can be used as a CAPS marker and leads to an amino acid substitution from a threonine (T) in the WT to an isoleucine (I) in kegne.

Fig. 6.

Plant height and angle of twisting in progeny of kegne and the Quncho cross. (A) Plant height (n=121) and (B) angle of twisting (total n=114) of F₂ plants. (C) The height of three classes of F₂ plants grouped based on the ClaI marker after 10 d of oryzalin treatment followed by 6 d of recovery (n=93). (D) The height of F₃ plants derived from a known F₂ heterozygous line after receiving the same treatment. ***P<0.001; *P<0.05; n.s., not significant after ANOVA. Values with the same letters are not significantly different at P<0.05 after Tukey test. Error bars indicate 1 SD.