TILLING For A Spaced-induced Mutant Pool In Wheat

TILLING has been successfully applied in chemical-induced plant population and good progess on high throughput screening of mutations have been made. This study aims to explore the feasibility of applying TILLING on wheat by using the space-induced SP2 population of wheat varity Xinmai 18. The CELâ… -based TILLING facility will be established, and the new alleles of targeted genes associated with wheat quality, stress tolerance and disease resistance will be screened. The deletion mutations of the wheat Lr34 genes will be also screened based on PCR amplification. The main results were as following:1. Through prolonging the reaction time of KAc and raising the grinding frequence in genome DNA extraction, adjusting the concentration of dNTP, Mg²⁺ and primers, ultrapure water substituting for CELâ… buffer and improving air humidity to 40%, we setted up the CELâ… -based high-throughput TILLING platform.2. Five targeted genes of Pinb, DREB1 and Lr34 were screened in 2304 SP2 individual plants. Alleles were found on Pinb and DREB1 genes. The other three targeted genes were not found point mutations. The allele of Pinb(P679)was a base A deletion and the mutation frequency was 1/3073 Kb. The mutation induced the increase of seed hardness in P679. Two mutants(D49 and D157) were founded on DREB1 locus of wheat genome A and the mutation frequency was 1/1713 Kb. D49 was a base T deletion. D157 produced base T deletion and T/C missense mutations at the same time.3. Twelve samples were founded to produce deletion mutations of Lr34 targeted genes in 2304 SP2 plants. The result of deletion mutants verification showed that wild type of Xinmai 18 could amplify Lr34 targeted genes, but not for deletion mutants in the same PCR and electrophoresis conditions. The deleted fragments were about 6Kb and the mutation frequency was 0.52%.It was concluded that TILLING could be applied in space-induced wheat population. The frequency of point mutations in space-induced populations was lower than the EMS populations. But with the results of deletion mutations based on PCR detection, it showed that the frequency of deletion mutations in space-induced populations was relatively higher, implying that spaceflight environment mainly induces deletion mutations.