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K326, TN 90, and TN 90 SRC were each initially hybridized with an individual plant in which the
BBL-a,
BBL-b, and
BBL-c truncation mutations were initially assembled in triple homozygous conditions (
Lewis et al., 2015). Each F1 was then backcrossed to their respective recurrent parents, and selection for all three mutations was carried out using Kompetitive Allele Specific PCR (KASP) markers. The backcross breeding method was carried out to the BC7F1 generation for each of the three lines, at which time triple heterozygous mutant genotypes were self-pollinated to produce the BC7F2 generation. BC7F2 individuals were genotyped to identify all eight possible homozygous genetic combinations (
Supplementary Table 1), which were then self-pollinated to produce BC7F3 seed of the nearly isogenic lines (NILs) for each genetic background. Numerical nomenclature is hereafter used to refer to each of these genotypes, where a “0” indicates the homozygous “wild-type” condition at a particular locus, “2” designates the homozygous mutant condition at a particular locus, and “1” indicates a heterozygote (
Supplementary Table 1). For example, the designation “K326 (210)” indicates a line homozygous for the mutant genotype at the
BBL-a locus, heterozygous for the mutant genotype at the
BBL-b locus, and homozygous for the wild type allele at the
BBL-c locus.
The eight K326-derived NILs were evaluated for yield, physical cured leaf quality, and cured leaf chemistry in comparison with K326, NC95, and corresponding NC95 isolines MAFC5 (
Nic1/
Nic1 nic2/
nic2) (
Chaplin, 1986), LMAFC34 (
nic1/
nic1 Nic2/
Nic2) (
Chaplin and Burk, 1984), and LAFC53 (
nic1/
nic1 nic2/
nic2) (
Chaplin, 1975). An F1 hybrid between K326 and K326 (222), designated as K326 (111) was also included. Experiments were carried out at three North Carolina field locations (Upper Coastal Plain Research Station, Rocky Mount; Cunningham Research Station, Kinston; and the Oxford Tobacco Research Station, Oxford) during 2016 and 2017, for a total of six field environments. Experimental units at each location consisted of single 20-plant rows managed according to standard flue-cured production practices for North Carolina. Intra-row spacing was 56 cm at all three locations, while inter-row spacing was 122 cm at the Oxford and Rocky Mount locations and 112 cm at the Kinston locations.
