Highly specific PCR-RFLP assays for karyotyping the widespread 2Rb inversion in malaria vectors of the Anopheles gambiae complex
Highly specific PCR-RFLP assays for karyotyping the widespread 2Rb inversion in malaria vectors of the Anopheles gambiae complex
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DOI：
10.1186/s13071-019-3877-x
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发表时间：
2020-01-10
影响因子：
3.2
通讯作者：
Besansky, Nora J.
中科院分区：
文献类型：
--
作者：
Montanez-Gonzalez, Raquel;Pichler, Verena;Besansky, Nora J.
染色体倒位多态性在适应异质环境中起着重要作用。倒置多态性与非洲热带冈比亚按蚊（Anopheles gambiae）复合体的三种主要疟疾媒介物种具有非常高的生态灵活性有关，有助于利用人为环境改变并促进与人类的密切联系。除了扩大物种的空间和时间分布外，倒挂还与流行病学相关的蚊子行为和生理有关，强调了它们的医学重要性。我们在此提出了一种新的基于PCR-RFLP的检测方法，该方法可以强烈预测An中普遍存在的2Rb基因型。coluzzi和An。冈比亚，这一发展克服了传统细胞学核型固有的众多限制。方法我们设计了PCR-RFLP基因分型方法，该方法基于先前计算确定的标记snp对2Rb基因型具有很强的预测作用（约95%）。我们针对那些替代等位基因状态破坏或创建商业上可用的限制性内切酶识别位点的标签，并设计了针对每种反转基因型的独特切割谱的测定方法。该方法在251 An上得到了验证。coluzzi and 451 An。对非洲9个国家和非洲1个国家的冈比亚菌细胞学核型标本进行了研究。科鲁兹实验室菌落。对于三个标签snp， PCR-RFLP分析（分别为DraIII、MspAI和TatI）可靠地产生了稳健的扩增子，并为所有三种倒置基因型提供了清晰可区分的电泳图谱。DraIII测定的结果与两种物种的细胞遗传学分配具有b> = 95%的一致性，而MspAI和TatI测定的结果仅与An的细胞遗传学分配高度一致。coluzzi或An。分别冈比亚按蚊。联合应用适合种属的分析对，使安省的一致性水平提高到约99%。coluzzi的比例为98%。冈比亚按蚊。讨论了不一致的潜在来源（如标签和反转之间的不完全关联，等位基因缺失，限制性酶切靶位点的额外多态性，不完整或失败的限制性酶切）。结论高特异性、低成本、易获取的分子检测方法可用于安家鼠2Rb基因分型。冈比亚和安哥拉。Coluzzii允许两性和所有发育阶段的染色体组型。这些新工具将加速深入研究这种在生态学和流行病学上重要的染色体反转在媒介生物学中的作用。
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.