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.农业害虫年均损毁美国约15%的农作物，造成的经济损失高达150亿美元。因此，研发廉价、快速且可靠的有害物种检测方法，对于减少虫害损失、降低经济影响至关重要。苹果实蝇（Rhagoletis pomonella，双翅目：实蝇科）是一种极具破坏性的入侵害虫，严重威胁着美国太平洋西北地区市值数十亿的苹果产业。该实蝇还与一种形态相似、亲缘关系紧密的无害物种——雪果实蝇（R. zephyria）同域分布，后者仅取食非商业化的雪果属植物。由于苹果实蝇针对果品出口实施零侵染政策，因此对其进行准确的物种鉴定至关重要。若将雪果实蝇误判为苹果实蝇，将引发不必要且成本高昂的检疫措施，挤占宝贵的虫害防控资源。本研究开发并应用了一种基于双酶切限制性酶切位点相关DNA标记（double digest restriction-site associated DNA markers）的Illumina测序（Illumina sequencing）诊断方法，该方法简便易行且成本效益优异。本研究鉴定出5个具有区分价值的单核苷酸多态性（single nucleotide polymorphisms, SNPs），并设计了基于限制性酶切聚合酶链反应扩增产物琼脂糖凝胶电泳的限制性片段长度多态性（restriction fragment length polymorphisms, RFLPs）诊断检测方案，用于区分两种实蝇物种。本研究通过对从华盛顿州全境11个采样点直接采集的、已知寄主植物来源的苹果寄主与雪果寄主实蝇样本进行分型验证，证实该方法可在1天内完成快速物种鉴定。但若无需快速诊断，或需要对数百至数千份样本进行鉴定，则采用本研究中用于诊断性SNP分型的直接Illumina测序策略，同样具备高效性与成本效益优势。本研究提出的基因组学策略不仅适用于苹果实蝇的鉴定，还可推广应用于多种隐蔽性害虫的物种识别。