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The fall armyworm, Spodoptera frugiperda (J. E. Smith), is a highly polyphagous pest native to the tropical Americas that has recently spread to become a global super-pest threatening food and fiber production. Transgenic crops producing insecticidal Cry and Vip3Aa proteins from Bacillus thuringiensis (Bt) are used for control of this pest in its native range. The evolution of practical resistance represents the greatest threat to sustainability of this technology and its potential efficacy in the S. frugiperda invasive range. Monitoring for resistance is vital to management approaches delaying S. frugiperda resistance to Bt crops. DNA-based resistance screening provides higher sensitivity and cost-effectiveness than currently used bioassay-based monitoring. So far, practical S. frugiperda resistance to Bt corn-producing Cry1F has been genetically linked to mutations in the SfABCC2 gene, providing a model to develop and test monitoring tools. In this study, we performed targeted SfABCC2 sequencing followed by Sanger sequencing to confirm the detection of known and candidate resistance alleles to Cry1F corn in field-collected S. frugiperda from continental USA, Puerto Rico, Africa (Ghana, Togo, and South Africa), and Southeast Asia (Myanmar). Results confirm that the distribution of a previously characterized resistance allele (SfABCC2mut) is limited to Puerto Rico and identify 2 new candidate SfABCC2 alleles for resistance to Cry1F, one of them potentially spreading along the S. frugiperda migratory route in North America. No candidate resistance alleles were found in samples from the invasive S. frugiperda range. These results provide support for the potential use of targeted sequencing in Bt resistance monitoring programs. [ABSTRACT FROM AUTHOR]

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. 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. [ABSTRACT FROM AUTHOR]