Fig. 4

Genetic linkage analysis of Cry1Ac resistance in the polygenic knockout strains of P. xylostella. a The crossing strategy for analysis of the linkage between polygenic knockout and Cry1Ac resistance. b To examine the genetic linkage between multiple resistance alleles and Cry1Ac resistance, 10 backcross families from the single-pair cross between C2-3KO (rr) and their F1 progeny (rs, C2-3KO×DBM1Ac-S) (top pictures), N1-3aKO (rr), and their F1 progeny (rs, N1-3aKO×DBM1Ac-S) (middle pictures), as well as C-NKO (rr) and their F1 progeny (rs, C-NKO×DBM1Ac-S) (bottom pictures) were used. Each picture contains 10 backcross families from bottom to top, the first lines 1-5 represent backcross a, and the following lines 6-10 showed backcross b. Bioassays were performed on 30 larvae from each backcross group, and a total of 300 larvae were treated with the diagnostic doses of Cry1Ac protoxin, another 300 untreated larvae were also collected as controls. The genotypes of individuals that were or were not treated with Cry1Ac were detected by the band size of the PCR products with the four primer pairs in PxABCC2 and PxABCC3 or PxAPN1 and PxAPN3a locus (shown in Additional file 1: Table S1). The number of individuals with the genotype of rr is listed on the left side of these figures, and the significant difference of genotype ratio in Cry1Ac treated or not backcross groups were calculated by the Fisher’s exact test, and the p values are shown on the right of figures, *p < 0.05, **p < 0.01, ***p < 0.001. c–e Genotype detection of F2 backcross families from crossing the polygenic knockout strains (C2-3KO, N1-3aKO, and C-NKO, respectively) and their corresponding F1 progeny. The gDNA samples of all the individuals from F2 backcross families, that were or were not exposed to Cry1Ac toxin, were extracted for PCR amplification using the four primer pairs in PxABCC2/PxABCC3 or PxAPN1/PxAPN3a loci (shown in Figs. 1c and 2b). PCR products were purified and resolved by 1.5% agarose gel electrophoresis