Abstract:

【Objective】The goal of this work is to establish an optimized EcoTILLING protocol for identification of natural variations within target genes and specified regions of genomes in barley. 【Method】The universal adapter M13 was labeled with fluorescent dyes, the nested PCR with specific primer combination and M13 adapter was performed for the targeted region. The EcoTILLING protocol for barley was optimized, including the template amount and annealing temperature in PCR amplification, CELⅠamount and reaction time in cleavage of mispaired heteroduplex DNA. 【Result】The results showed that the optimal amount of genomic DNA in a 10 μL of reaction system was 20 ng as template for the first round of PCR, and the first PCR product was diluted 3-fold and used as a template for the second round. The proper annealing temperature for both first- and second-round PCRs was 58℃. Optimum amount of CELⅠ enzyme was 0.4 μL (3 U•μL-1) in a 20 μL of digestion reaction system, and 17.5 min at 45℃ was the optimal time for the cleavage of heteroduplex DNAs. 【Conclusion】The experiment indicates that the optimized EcoTILLING protocol can effectively detect DNA polymorphisms for natural population of barley with lower cost, and it could provide a foundation for larger scale efforts in reverse genetics and characterization of natural nucleotide variation in barley.

Key words: barley, EcoTILLING, optimization, point mutation