Data from: Positional cloning of rp2 QTL associates the P450 genes CYP6Z1, CYP6Z3 and CYP6M7 with pyrethroid resistance in the malaria vector Anopheles funestus

Pyrethroid resistance in Anopheles funestus is threatening malaria control in Africa. Elucidation of underlying resistance mechanisms is crucial to improve the success of future control programs. A positional cloning approach was used to identify genes conferring resistance in the uncharacterised rp2 QTL previously detected in this vector using F6 Advanced Intercross Lines (AIL). A 113 kb BAC clone spanning rp2 was identified and sequenced revealing a cluster of fifteen P450 genes and one salivary protein gene (SG7-2). Contrary to An. gambiae, AfCYP6M1 is triplicated in An. funestus while AgCYP6Z2 ortholog is absent. 565 new SNPs were identified for genetic mapping from rp2 P450s and other genes revealing high genetic polymorphisms with 1 SNP every 36bp. A significant genotype/phenotype association was detected for rp2 P450s but not for a cluster of cuticular protein genes previously associated with resistance in An. gambiae. QTL mapping using F6 AIL confirms the rp2 QTL with an increase logarithm of odds (LOD) score of 5. Multiplex gene expression profiling of 15 P450s and other genes around rp2 followed by individual validation using qRT-PCR indicated a significant over-expression in the resistant FUMOZ-R strain of the P450s AfCYP6Z1, AfCYP6Z3, AfCYP6M7 and the glutathione-s-transferase GSTe2 with respective fold-change of 11.2, 6.3, 5.5 and 2.8. Polymorphisms analysis of AfCYP6Z1 and AfCYP6Z3 identified amino acid changes potentially associated with resistance further indicating that these genes are controlling the pyrethroid resistance explained by the rp2 QTL. The characterisation of this rp2 QTL significantly improves our understanding of resistance mechanisms in An. funestus.

富氏按蚊（Anopheles funestus）对拟除虫菊酯的抗性正威胁着非洲的疟疾防控工作。阐明其抗性潜在机制，对提升未来疟疾防控项目的实施成效至关重要。本研究采用位置克隆（positional cloning）技术，针对此前通过F6代高级互交系（Advanced Intercross Lines, AIL）在该传疟媒介中检测到的未被表征的rp2数量性状位点（Quantitative Trait Locus, QTL），开展抗性相关基因的鉴定工作。
研究人员成功鉴定并测序了一段长度为113 kb、覆盖rp2位点的细菌人工染色体（Bacterial Artificial Chromosome, BAC）克隆，该克隆包含15个细胞色素P450基因（cytochrome P450 genes）与1个唾液蛋白基因（SG7-2）的基因簇。与冈比亚按蚊（An. gambiae）不同，富氏按蚊中的AfCYP6M1基因存在三倍化现象，而冈比亚按蚊AgCYP6Z2的直系同源基因在富氏按蚊中缺失。
研究从rp2位点的P450基因及其他基因中鉴定出565个新的单核苷酸多态性（Single Nucleotide Polymorphism, SNP）位点用于遗传作图，结果显示该区域遗传多态性较高，平均每36 bp即存在1个SNP。研究检测到rp2位点的P450基因与抗性表型存在显著的基因型/表型关联，但此前在冈比亚按蚊中被报道与抗性相关的表皮蛋白基因簇则未发现该关联。
利用F6代高级互交系开展的QTL定位实验证实了rp2 QTL的存在，其对数似然比（logarithm of odds, LOD）评分提升了5分。对rp2位点周边的15个P450基因及其他基因进行多重基因表达谱分析，并通过实时定量反转录聚合酶链式反应（quantitative real-time polymerase chain reaction, qRT-PCR）开展单个基因验证，结果显示：抗性株系FUMOZ-R中，P450基因AfCYP6Z1、AfCYP6Z3、AfCYP6M7以及谷胱甘肽S-转移酶GSTe2（glutathione S-transferase GSTe2）的表达量显著上调，其折叠变化分别为11.2、6.3、5.5与2.8。
对AfCYP6Z1与AfCYP6Z3的多态性分析发现了可能与抗性相关的氨基酸改变，进一步证实这些基因正是rp2 QTL所调控的拟除虫菊酯抗性的关键介导因子。本次对rp2 QTL的表征工作，显著加深了我们对富氏按蚊抗性机制的理解。