Mutation Induction in Sucrose Synthase 1 to Study Cold Acclimation in Winter Wheat

Cold is one of the main abiotic stresses causing winterkill in winter wheat. Freezing tolerance is associated with the occurrence of a cold-hardening which is triggered by induction of cold responsive genes after exposure of plants to low non-freezing temperature for certain periods of time. Fourteen candidate genes with known homologies, were identified as being differentially expressed (presence/absence) between cold acclimated and non-acclimated crown and leaf tissues of two winter wheat lines (‘5899-16’ and ‘5450-1’) using cDNA-AFLP procedure. TILLING population of the same two winter wheat lines was further developed in order to create mutant forms of the candidate genes to verify their role in freezing-tolerance formation. Firstly, we optimized the dose of a mutagen EMS to achieve a substantial mutation rate while avoiding serious defects in germination and plant development. The most appropriate concentrations of EMS solution for two winter wheat genotypes were determined and a total of 2147 M2 lines were produced. Exon 8 of the identified differentially expressed Sucrose synthase 1 (Ss1) gene was chosen for mutation detection by High Resolution Melting (HRM) analysis in wheat TILLING M2 population. A total of 75.7 kb of DNA was screened resulting in an overall mutation density of one mutation per 37.8 Kb in the population. Two novel alleles of Ss1 gene were identified, of which 1 was silent and 1 nonsense (premature stop codon) mutation. qPCR analyses were performed to estimate how these mutations affect the expression level of Ss1 gene in crown and leaf tissue during cold acclimation. Putative knock-out mutant M631 had significantly lower relative expression of Ss1 gene in non-acclimated leaves as well as in crowns and leaves collected at 2, 4 and 6 weeks of cold acclimation compared with the wild type winter wheat line‘5899-16’. Further work will estimate the effect of the mutation on the freezing tolerance in winter wheat.