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. 2018 Sep 10;8:13531. doi: 10.1038/s41598-018-31840-5

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Linkage analysis. Biphasic genetic linkage analysis was initiated with single-pair reciprocal crosses between F₀ adults, with either a resistant Bt4-R2 female and a susceptible APHIS-S male (shown here) or a susceptible APHIS-S female and a resistant Bt4-R2 male. The resulting F₁ progeny were backcrossed in single pairs with the resistant Bt4-R2 strain. Backcross (BC) progeny were tested either on diet containing 1 microgram Cry2Ab per mL diet or control diet. Survivors from 10 reciprocal backcross families (five F₁ ♀ X Bt4-R2 ♂ and five Bt4-R2 ♀ X F₁ ♂) were weighed and genotyped with allele-specific PCR.