Strains with MC <50% were checked by PCR using the primer sets listed in Kim et al. (13) to confirm proper gene deletions. PCR was performed with two primer sets for both 5′ and 3′ deletions. The 5′ deletions were checked with cp5, a gene-specific primer outside the deletion cassette and with either CPN10 or CPN1, which are primers that bind inside the cassette (fig. S6A). Similarly, the 3′ deletion was checked with cp3, as an outside primer, and either CPC3 or CPC1, as inside primers (fig. S6B). LA Taq DNA polymerase (Takara Bio Inc.) was used for PCR following the manufacturer’s instructions. Nine strains failed to show amplification bands, confirming deletion, and were removed (fig. S6). Genes associated with the remaining 85 deletions were considered GZE.

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