Mutation Induction in Sucrose Synthase 1 to Study Cold Acclimation in Winter Wheat
Poster Jul 21, 2014
Rita Armoniene, Gintaras Brazauskas
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.
Despite the developments in conventional PCR, the complexity of multiplex Real Time PCR is still limited due to the lack of sufficient detection channels. To achieve high-end multiplexing capacity on standard Real Time PCR machines, Anapa Biotech has developed the MeltPlex® technology (see box on right).READ MORE
Genome-wide association studies (GWAS) have identified more than 100 genetic loci associated with type 2 diabetes. The majority of these are located in the intergenic or intragenic regions suggesting that the implicated variants may alter chromatin conformation. This, in turn, is likely to influence the expression of nearby or more remotely located genes to alter beta cell function. At present, however, detailed molecular and functional analyses are still lacking for most of these variants. We recently analysed one of these loci and mapped five causal variants in an islet-specific enhancer cluster within the STARD10 gene locus. Here, we aimed to understand how these causal variants influence b-cell function by alteration of the chromatin structure of enhancer clusterREAD MORE
Early life stress (ELS) is highly associated with development of psychopathology
and mood disorders in adulthood. Genetic studies have identified variation in the gene calcium voltage-gated channel subunit alpha1C (CACNA1C) to increase risk for several psychiatric disorders. This poster assessed the expression of Cacna1c following prepubertal stress.
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