BACKGROUND Chromosomal inversion polymorphisms play a role in adaptation to heterogeneous environments. Inversion polymorphisms are implicated in the very high ecological flexibility of the three main malaria vector species of the Afrotropical Anopheles gambiae complex, facilitating the exploitation of anthropogenic environmental modifications and promoting a strong association with humans. In addition to extending the species' spatial and temporal distribution, inversions are associated with epidemiologically relevant mosquito behavior and physiology, underscoring their medical importance. We here present novel PCR-RFLP based assays strongly predictive of genotype for the cosmopolitan 2Rb inversion in An. coluzzii and An. gambiae, a development which overcomes the numerous constraints inherent to traditional cytological karyotyping. METHODS We designed PCR-RFLP genotyping assays based on tag SNPs previously computationally identified as strongly predictive (> 95%) of 2Rb genotype. We targeted those tags whose alternative allelic states destroyed or created the recognition site of a commercially available restriction enzyme, and designed assays with distinctive cleavage profiles for each inversion genotype. The assays were validated on 251 An. coluzzii and 451 An. gambiae cytologically karyotyped specimens from nine countries across Africa and one An. coluzzii laboratory colony. RESULTS For three tag SNPs, PCR-RFLP assays (denoted DraIII, MspAI, and TatI) reliably produced robust amplicons and clearly distinguishable electrophoretic profiles for all three inversion genotypes. Results obtained with the DraIII assay are ≥ 95% concordant with cytogenetic assignments in both species, while MspAI and TatI assays produce patterns highly concordant with cytogenetic assignments only in An. coluzzii or An. gambiae, respectively. Joint application of species-appropriate pairs of assays increased the concordance levels to > 99% in An. coluzzii and 98% in An. gambiae. Potential sources of discordance (e.g. imperfect association between tag and inversion, allelic dropout, additional polymorphisms in the restriction target site, incomplete or failed restriction digestion) are discussed. CONCLUSIONS The availability of highly specific, cost effective and accessible molecular assays for genotyping 2Rb in An. gambiae and An. coluzzii allows karyotyping of both sexes and all developmental stages. These novel tools will accelerate deeper investigations into the role of this ecologically and epidemiologically important chromosomal inversion in vector biology.

中文翻译：

---

用于对冈比亚按蚊复合体的疟疾载体中广泛的2Rb倒置进行核型分析的高度特异性PCR-RFLP分析。

背景技术染色体倒位多态性在适应异构环境中起作用。倒序多态性与非洲嗜热按蚊复杂的三种主要疟疾媒介物种的极高生态灵活性有关，从而促进了对人为环境的利用，并促进了与人类的牢固联系。除了扩大物种的空间和时间分布外，倒置还与流行病学上与蚊子的行为和生理有关，这突出了它们的医学重要性。我们在这里提出了新颖的基于PCR-RFLP的检测方法，可强有力地预测An。中大都会2Rb转化的基因型。coluzzii和An。冈比亚的发展，克服了传统细胞学核型分型固有的众多限制。方法我们基于标签SNP设计了PCR-RFLP基因分型检测方法，该标签SNPs先前已被计算为2Rb基因型的强预测（> 95％）。我们针对那些标签的替代等位基因状态破坏或创建了市售限制酶的识别位点，并针对每种转化基因型设计了具有独特切割谱的分析方法。该测定法在251 An上进行了验证。coluzzii和451 An。来自非洲9个国家和1个非洲国家的冈比亚细胞学核型分析标本。coluzzii实验室菌落。结果对于三个标签SNP，PCR-RFLP分析（分别表示为DraIII，MspAI和TatI）可靠地产生了稳健的扩增子，并为所有三种反转基因型提供了清晰可辨的电泳图谱。通过DraIII分析获得的结果在两个物种中均≥95％与细胞遗传学分配一致，而MspAI和TatI分析仅在An中产生与细胞遗传学分配高度一致的模式。coluzzii或An。冈比亚 联合应用适当种类的检测对可将An中的一致性水平提高到> 99％。coluzzii和98％的An 冈比亚 讨论了潜在的不一致性来源（例如，标签与倒位之间的关联不完善，等位基因缺失，限制性内切酶靶位点的其他多态性，限制性内切酶消化不完全或失败）。结论高特异性，高性价比和可访问的分子分析技术可用于对An。2Rb进行基因分型。冈比亚和安。coluzzii可以进行性别和所有发育阶段的核型分析。