The cigar tobacco variety Beinhart 1000 is well known to have high level of partial resistance to black shank caused by Phytophthora nicotianae. The researchers at the North Carolina State University reported that it's quantitative trait loci (QTLs) associated with the resistance and the major/novel QTL on linkage group 15 among them is closely linked with NtCPS2 gene which encodes an enzyme for the biosynthesis of cis-abienol that is undesirable for the flavour of flue-cured and Burley tobacco. The linkage between the major QTL and NtCPS2 allele must be broken to utilise in breeding.

The objective of this research was to produce a useful resistance source by inactivating the NtCPS2 allele linked with the major QTL. For the efficient screening of mutated non-functional NtCPS2 allele, the F1 seeds from a cross of Beinhart 1000 and K326 were treated with ethyl methanesulphonate. The NtCPS2 locus of F1 plant consists of functional and non-functional NtCPS2 allele derived from Beinhart 1000 and K326 respectively.

The cis-abienol production in leaves of F1 plants was analysed by thin-layer chromatography. As the result, 27 F1 plants which did not accumulate cis-abienol were selected from approximately 4,000 F1 plants. Two independent F1 plants harbouring a mutation-introduced NtCPS2 allele were identified among them by DNA sequence analysis. Each sequence of NtCPS2 allele in the selected two plants had a nonsense mutation and a missense mutation.