A PCR-RFLP method for genotyping of inversion 2Rc in Anopheles coluzzii.
A PCR-RFLP method for genotyping of inversion 2Rc in Anopheles coluzzii.
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DOI：
10.1186/s13071-021-04657-x
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发表时间：
2021-03-22
影响因子：
3.2
通讯作者：
Besansky NJ
中科院分区：
文献类型：
--
作者：
Montanez-Gonzalez R;Vallera AC;Calzetta M;Pichler V;Love RR;Guelbeogo MW;Dabire RK;Pombi M;Costantini C;Simard F;Della Torre A;Besansky NJ
疟疾病媒（如按蚊）中多态性染色体倒位的基因分型coluzzii Coetzee & Wilkerson是重要的，因为它们会导致神秘的种群结构，可能会误导媒介分析和控制，因为它们会影响流行病学相关的生态表型。传统的细胞遗传学基因分型方法是一个障碍，因为它是劳动密集型的，需要专门的培训，只能应用于一个性别和发育阶段。在这里，我们绕过这些限制，开发一个简单而快速的分子分型方法，在AN倒位2 Rc。coluzzii既经济又友好。这种反转强烈暗示在时间和空间适应气候和生态变化，特别是干旱。使用一组标签的单核苷酸多态性（SNP）与反向方向密切相关，我们确定了那些重叠的限制性内切酶识别位点，并开发了四个聚合酶链反应（PCR）限制性片段长度多态性（RFLP）分析，区分替代等位基因状态的标签SNP。我们使用布基纳法索的蚊子种群样本进行了细胞遗传学核型分析和基因分型，使用两种基于标签SNP的互补高通量分子方法评估了这些检测方法的性能。使用来自其他西非（贝宁、马里、塞内加尔）和中非（喀麦隆）国家的蚊子种群样本进行了进一步验证。在测试的四种测定中，两种在所有样品中与2 Rc细胞遗传学核型> 90%的时间一致。我们建议这两种检测方法串联使用，以获得可靠的基因分型。通过仅接受两种测定法一致的基因型分配，预期> 99%的分配是准确的。我们已经开发了串联PCR-RFLP分析的准确基因分型的倒位2 Rc在AN。猫猴。由于这种方法简单、便宜，而且只需要基本的分子生物学设备，因此它是广泛可用的。这些为探索与疟疾流行病学和病媒控制相关的生态表型的分子基础提供了重要工具。 在线版本包含补充材料，可通过10.1186/s13071-021-04657-x获得。
Genotyping of polymorphic chromosomal inversions in malaria vectors such as An. coluzzii Coetzee & Wilkerson is important, both because they cause cryptic population structure that can mislead vector analysis and control and because they influence epidemiologically relevant eco-phenotypes. The conventional cytogenetic method of genotyping is an impediment because it is labor intensive, requires specialized training, and can be applied only to one gender and developmental stage. Here, we circumvent these limitations by developing a simple and rapid molecular method of genotyping inversion 2Rc in An. coluzzii that is both economical and field-friendly. This inversion is strongly implicated in temporal and spatial adaptations to climatic and ecological variation, particularly aridity. Using a set of tag single-nucleotide polymorphisms (SNPs) strongly correlated with inversion orientation, we identified those that overlapped restriction enzyme recognition sites and developed four polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) assays that distinguish alternative allelic states at the tag SNPs. We assessed the performance of these assays using mosquito population samples from Burkina Faso that had been cytogenetically karyotyped as well as genotyped, using two complementary high-throughput molecular methods based on tag SNPs. Further validation was performed using mosquito population samples from additional West African (Benin, Mali, Senegal) and Central African (Cameroon) countries. Of four assays tested, two were concordant with the 2Rc cytogenetic karyotype > 90% of the time in all samples. We recommend that these two assays be employed in tandem for reliable genotyping. By accepting only those genotypic assignments where both assays agree, > 99% of assignments are expected to be accurate. We have developed tandem PCR-RFLP assays for the accurate genotyping of inversion 2Rc in An. coluzzii. Because this approach is simple, inexpensive, and requires only basic molecular biology equipment, it is widely accessible. These provide a crucial tool for probing the molecular basis of eco-phenotypes relevant to malaria epidemiology and vector control. The online version contains supplementary material available at 10.1186/s13071-021-04657-x.
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影响因子：
2.6
作者：
Love, R. Rebecca;Pombi, Marco;Besansky, Nora J.
通讯作者：
Besansky, Nora J.
影响因子：
1.9
作者：
PETRARCA, V;VERCRUYSSE, J;COLUZZI, M
通讯作者：
COLUZZI, M
影响因子：
3.7
作者：
Ma, Jianzhong;Amos, Christopher I.
通讯作者：
Amos, Christopher I.
影响因子：
3.2
作者：
Montanez-Gonzalez, Raquel;Pichler, Verena;Besansky, Nora J.
通讯作者：
Besansky, Nora J.
影响因子：
56.9
作者：
Coluzzi, M;Sabatini, A;Petrarca, V
通讯作者：
Petrarca, V