Genetic Screening to Identify Candidate Resistance Alleles to Cry1F Corn in Fall Armyworm Using Targeted Sequencing

SIMPLE SUMMARY: Monitoring of resistance alleles is critical to the sustainability of transgenic crops producing insecticidal Cry proteins. Highly sensitive and cost-effective DNA-based methods are needed to improve current bioassay-based resistance screening. Our goal was to evaluate the use of targeted sequencing in detecting known and novel candidate resistance alleles to Cry proteins. As a model, we used field-collected fall armyworm (Spodoptera frugiperda) from Puerto Rico, the first location reporting continued practical field-evolved S. frugiperda resistance to corn producing the Cry1F insecticidal protein, and sequenced the SfABCC2 gene previously identified as critical to Cry1F toxicity. Targeted sequencing of SfABCC2 detected a previously reported Cry1F resistance allele and mutations originally identified in populations from Brazil. Importantly, targeted sequencing also identified nonsynonymous and frameshift mutations as novel candidate resistance alleles. These results advocate for the use of targeted sequencing in screening for resistance alleles to Cry proteins and support potential gene flow, including resistance alleles, between S. frugiperda from Brazil and the Caribbean. ABSTRACT: Evolution of practical resistance is the main threat to the sustainability of transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt crops). Monitoring of resistance to Cry and Vip3A proteins produced by Bt crops is critical to mitigate the development of resistance. Currently, Cry/Vip3A resistance allele monitoring is based on bioassays with larvae from inbreeding field-collected moths. As an alternative, DNA-based monitoring tools should increase sensitivity and reduce overall costs compared to bioassay-based screening methods. Here, we evaluated targeted sequencing as a method allowing detection of known and novel candidate resistance alleles to Cry proteins. As a model, we sequenced a Cry1F receptor gene (SfABCC2) in fall armyworm (Spodoptera frugiperda) moths from Puerto Rico, a location reporting continued practical field resistance to Cry1F-producing corn. Targeted sequencing detected a previously reported Cry1F resistance allele (SfABCC2mut), in addition to a resistance allele originally described in S. frugiperda populations from Brazil. Moreover, targeted sequencing detected mutations in SfABCC2 as novel candidate resistance alleles. These results support further development of targeted sequencing for monitoring resistance to Bt crops and provide unexpected evidence for common resistance alleles in S. frugiperda from Brazil and Puerto Rico.