Identifying diagnostic genetic markers for a cryptic invasive agricultural pest: a test case using the apple maggot fly, Rhagoletis pomonella (Diptera: Tephritidae)

Insect pests destroy ~15% of all USA crops, resulting in losses of $15 billion annually. Thus, developing cheap, quick and reliable methods for detecting harmful species is critical to curtail insect damage and lessen economic impact. The apple maggot fly, Rhagoletis pomonella (Diptera: Tephritidae), is a major invasive pest threatening the multibillion-dollar apple industry in the Pacific Northwest USA. The fly is also sympatric with a benign but morphologically similar and genetically closely related species, R. zephyria, which attacks non-commercial snowberry. Unambiguous species identification is essential due to a zero-infestation policy of apple maggot for fruit export. Mistaking R. zephyria for R. pomonella triggers unnecessary and costly quarantines, diverting valuable control resources. Here we develop and apply a relatively simple and cost-effective diagnostic approach using Illumina sequencing of double digest restriction-site associated DNA markers. We identified five informative single nucleotide polymorphisms (SNPs) and designed a diagnostic test based on agarose gel electrophoresis of restriction enzyme digested polymerase chain reaction amplification products (RFLPs) to distinguish fly species. We demonstrated the utility of this approach for immediate, one day species identification by scoring apple- and snowberry-infesting flies of known host plant identity, reared directly from 11 sites throughout Washington. However, if immediate diagnosis is not required, or hundreds to thousands of specimens must be assessed, then a direct Illumina-based sequencing strategy, similar to that used here for diagnostic SNP identification can be powerful and cost-effective. The genomic strategy we present is effective for R. pomonella and also transferable to many cryptic pests.Filtered vcf file with genotype probabilities: DoellmanHoodRzephSNPs.vcf.gz This is a vcf file generated using the GATK unified genotyper, then filtered as describe in the paper. The PL field provides the phred-scaled and normalized genotype probabilities produced using the GATK model. File mapping individual IDs to host associations: DoellmanHoodRzeph_popIDs.txt column 1 = sample id (sample id in the vcf file), column 2 = host association Raw Illumina sequencing data and barcodes for demultiplexing, Lane 1 DoellmanHoodRzephL1_1.fq.gz Illumina forward reads DoellmanHoodRzephL1_2.fq.gz Illumina paired reads DoellmanHoodRzephL1_barcodes.txt column 1 = inline barcode (5' end of the forward read), column 2 = sample id (sample id in the vcf file) Raw Illumina sequencing data and barcodes for demultiplexing, Lane 2 DoellmanHoodRzephL2_1.fq.gz Illumina forward reads DoellmanHoodRzephL2_2.fq.gz Illumina paired reads DoellmanHoodRzephL2_barcodes.txt column 1 = inline barcode (5' end of the forward read), column 2 = sample id (sample id in the vcf file) Raw Illumina sequencing data and barcodes for demultiplexing, Lane 3 DoellmanHoodRzephL3_1.fq.gz Illumina forward reads DoellmanHoodRzephL3_2.fq.gz Illumina paired reads DoellmanHoodRzephL3_barcodes.txt column 1 = inline barcode (5' end of the forward read), column 2 = sample id (sample id in the vcf file) Raw Illumina sequencing data and barcodes for demultiplexing, Lane 4 DoellmanHoodRzephL4_1.fq.gz Illumina forward reads DoellmanHoodRzephL4_2.fq.gz Illumina paired reads DoellmanHoodRzephL4_barcodes.txt column 1 = inline barcode (5' end of the forward read), column 2 = sample id (sample id in the vcf file) Funding provided by: U.S. Department of AgricultureCrossref Funder Registry ID: http://dx.doi.org/10.13039/100000199Award Number: NIFA-2015-67013-23289Funding provided by: National Science FoundationCrossref Funder Registry ID: http://dx.doi.org/10.13039/100000001Award Number: IIS-1560363Funding provided by: Washington Tree Fruit Research CommissionCrossref Funder Registry ID: http://dx.doi.org/10.13039/100012487Award Number: Funding provided by: Washington State Commission on Pesticide RegistrationCrossref Funder Registry ID: http://dx.doi.org/10.13039/100013663Award Number