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Materials and methods Genetic materials The backcross breeding method was used to create various combinations of the nic1 and nic2 alleles derived from LAFC53 [30], and the mutated myc2a allele derived from TI 313 [1], in the genetic background of elite flue-cured tobacco cultivar K326. During the backcrossing effort, plants carrying the 5 bp myc2a mutation were identified via a cleaved amplified polymorphic sequence (CAPS) marker assay