A novel tetra-primer ARMS-PCR approach for the molecular karyotyping of chromosomal inversion 2Ru in the main malaria vectors Anopheles gambiae and Anopheles coluzzii.
A novel tetra-primer ARMS-PCR approach for the molecular karyotyping of chromosomal inversion 2Ru in the main malaria vectors Anopheles gambiae and Anopheles coluzzii.
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一种新型的TETRA-PRIMER ARMS-PCR方法，用于在主要疟疾载体Anopheles Gambiae和Anopheles Coluzzii中染色体反转2RU的分子核分型。
DOI：
10.1186/s13071-023-06014-6
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发表时间：
2023-10-27
影响因子：
3.2
通讯作者：
中科院分区：
文献类型：
--
作者：
染色体倒位多态性与两种主要的非洲热带疟疾媒介--哥卢兹按蚊和冈比亚按蚊的适应性行为、生理、形态和生活史特征相关。对染色体倒位系统适应性价值的理解受到细胞学核型分析可行性的限制。近年来，在计算机和分子方法已经开发了最广泛的倒位（2La，2 Rb和2 Rc）的基因分型。2 Ru倒位，跨越约8%的染色体2 R，通常是多态性的西非人口的安。coluzzii和安氏隐翅虫An.在冈比亚，随着季节性和地理上降雨量的增加，发生频率明显增加。这项工作的目的是克服目前可用的细胞学和高通量分子检测的限制，通过开发一种简单的PCR检测方法对两种蚊子物种的个体标本中的2 Ru倒位进行基因分型。我们设计了四引物扩增难治性突变系统（ARMS）-PCR检测的基础上，5个标签的单核苷酸多态性（SNP），以前被证明是强烈相关的2 Ru倒位方向。最有前途的检测方法进行了验证，对实验室和现场样品的安。coluzzii和安氏隐翅虫An.使用测序的千分之一基因分型（GT-seq）高通量方法对冈比亚的染色体进行细胞遗传学或分子核型分析，该方法采用多重PCR扩增子的靶向测序。基于位置2 R 31710303处的标签SNP设计了成功的测定，其高度预测2 Ru基因型。该检测方法仅需一次PCR，除电泳外无需额外的PCR后处理，在检测的454份标本中，98.5%的标本产生了清晰的带型，与已建立的核型分析方法一致率为96.7%。获得了9个AN的序列。coluzzii标本与GT-seq显示2 Ru基因型差异。这些不一致的可能来源进行了讨论。四引物ARMS-PCR检测代表了一种准确，简化和成本效益的方法，用于2 Ru倒位的分子核型分析在An。coluzzii和安氏隐翅虫An.冈比亚。与其他常见的多态性反转，2La，2 Rb和2 Rc的方法已经可用，该试验将允许调查的适应价值的复杂的反转系统中观察到的两个主要的疟疾病媒在非洲热带地区。在线版本包含补充材料，可通过10.1186/s13071-023-06014-6获得。
Chromosomal inversion polymorphisms have been associated with adaptive behavioral, physiological, morphological and life history traits in the two main Afrotropical malaria vectors, Anopheles coluzzii and Anopheles gambiae. The understanding of the adaptive value of chromosomal inversion systems is constrained by the feasibility of cytological karyotyping. In recent years in silico and molecular approaches have been developed for the genotyping of most widespread inversions (2La, 2Rb and 2Rc). The 2Ru inversion, spanning roughly 8% of chromosome 2R, is commonly polymorphic in West African populations of An. coluzzii and An. gambiae and shows clear increases in frequency with increasing rainfall seasonally and geographically. The aim of this work was to overcome the constraints of currently available cytological and high-throughput molecular assays by developing a simple PCR assay for genotyping the 2Ru inversion in individual specimens of both mosquito species. We designed tetra-primer amplification refractory mutation system (ARMS)-PCR assays based on five tag single-nucleotide polymorphisms (SNPs) previously shown to be strongly correlated with 2Ru inversion orientation. The most promising assay was validated against laboratory and field samples of An. coluzzii and An. gambiae karyotyped either cytogenetically or molecularly using a genotyping-in-thousands by sequencing (GT-seq) high-throughput approach that employs targeted sequencing of multiplexed PCR amplicons. A successful assay was designed based on the tag SNP at position 2R, 31710303, which is highly predictive of the 2Ru genotype. The assay, which requires only one PCR, and no additional post-PCR processing other than electrophoresis, produced a clear banding pattern for 98.5% of the 454 specimens tested, which is a 96.7% agreement with established karyotyping methods. Sequences were obtained for nine of the An. coluzzii specimens manifesting 2Ru genotype discrepancies with GT-seq. Possible sources of these discordances are discussed. The tetra-primer ARMS-PCR assay represents an accurate, streamlined and cost-effective method for the molecular karyotyping of the 2Ru inversion in An. coluzzii and An. gambiae. Together with approaches already available for the other common polymorphic inversions, 2La, 2Rb and 2Rc, this assay will allow investigations of the adaptive value of the complex set of inversion systems observed in the two major malaria vectors in the Afrotropical region. The online version contains supplementary material available at 10.1186/s13071-023-06014-6.
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影响因子：
64.8
作者：
Joron, Mathieu;Frezal, Lise;Jones, Robert T.;Chamberlain, Nicola L.;Lee, Siu F.;Haag, Christoph R.;Whibley, Annabel;Becuwe, Michel;Baxter, Simon W.;Ferguson, Laura;Wilkinson, Paul A.;Salazar, Camilo;Davidson, Claire;Clark, Richard;Quail, Michael A.;Beasley, Helen;Glithero, Rebecca;Lloyd, Christine;Sims, Sarah;Jones, Matthew C.;Rogers, Jane;Jiggins, Chris D.;Ffrench-Constant, Richard H.
通讯作者：
Ffrench-Constant, Richard H.
影响因子：
4.9
作者：
Coughlan, Jennifer M.;Willis, John H.
通讯作者：
Willis, John H.
影响因子：
4.9
作者：
Cassone, Bryan J.;Molloy, Matthew J.;Besansky, Nora J.
通讯作者：
Besansky, Nora J.
影响因子：
64.8
作者：
Jones, Felicity C.;Grabherr, Manfred G.;Chan, Yingguang Frank;Russell, Pamela;Mauceli, Evan;Johnson, Jeremy;Swofford, Ross;Pirun, Mono;Zody, Michael C.;White, Simon;Birney, Ewan;Searle, Stephen;Schmutz, Jeremy;Grimwood, Jane;Dickson, Mark C.;Myers, Richard M.;Miller, Craig T.;Summers, Brian R.;Knecht, Anne K.;Brady, Shannon D.;Zhang, Haili;Pollen, Alex A.;Howes, Timothy;Amemiya, Chris;Lander, Eric S.;Di Palma, Federica;Lindblad-Toh, Kerstin;Kingsley, David M.
通讯作者：
Kingsley, David M.
影响因子：
2.6
作者：
Love, R. Rebecca;Pombi, Marco;Besansky, Nora J.
通讯作者：
Besansky, Nora J.