Identification of a chemically induced point mutation mediating herbicide tolerance in annual medics (Medicago spp.)

Sulfonylurea (SU) herbicides are used extensively in cereal-livestock farming zones as effective and cheap herbicides with useful levels of residual activity. These residues can persist beyond the cropping year, severely affecting legumes in general, and annual medics in particular, resulting in reduced dry matter production, lower seed yields and decreased nitrogen fixation. A strand medic cultivar, Medicago littoralis 'Angel', has been developed via chemical mutagenesis with tolerance to SU soil residues. Identifying the molecular basis of the observed tolerance was the aim of this study.

Two F(2) populations were generated from crosses between 'Angel' and varieties of intolerant M. truncatula, the male-sterile mutant tap and the cultivar 'Caliph'. Genetic mapping with SSR (single sequence repeat) and gene-based markers allowed identification of the trait-defining gene. Quantitative gene expression studies showed the activity of the respective alleles.

Segregation ratios indicated the control of SU-herbicide tolerance by a single dominant gene. SU herbicides inhibit the biosynthesis of the branched-chain amino acids by targeting the acetolactate synthase enzyme, allowing the choice of a mapping approach using acetolactate synthase (ALS) gene homologues as candidates. SSR-marker analysis suggested the ALS-gene homologue on chromosome 3 in M. truncatula. The ALS-gene sequences from 'Angel' and intolerant genotypes were sequenced. In 'Angel', a single point mutation from C to T translating into an amino acid change from proline to leucine was identified. The polymorphism was used to develop a diagnostic marker for the tolerance trait. Expression of the mutant ALS allele was confirmed by quantitative RT-PCR and showed no differences at various seedling stages and treatments to the corresponding wild-type allele.

The identification of the trait-defining gene and the development of a diagnostic marker enable efficient introgression of this economically important trait in annual medic improvement programs.