Rapid identification of Africanized honey bee <i>Apis mellifera</i> (hymenoptera: apidae) using high-resolution melting analysis DNA from honey

Genetic characterization of honey bee populations, particularly the identification of Africanized honey bees, has traditionally been based on different molecular methods that involve the amplification and analysis of different regions of the mitochondrial or nuclear genome which often take a long time. We introduce high-resolution melting analysis (HRM) as a novel approach for rapid and cost-effective identification of the African lineage in <i>Apis mellifera</i> populations from mitochondrial DNA extracted from honey. Samples were collected from 111 apiaries in 13 Argentinean provinces between 2012 and 2018. Total DNA was extracted from 10 g honey sediment and subjected to quantitative PCR (qPCR) using specific primers (APIS-F/Afr207R) amplifying a 207 bp product. Positive controls (Africanized/Non-Africanized) were used for mitotype validation and a subset of qPCR products were sequenced to compare different haplotypes. HRM analysis enabled the quick detection of sequence polymorphisms in short amplicons generated by qPCR. Among the analyzed apiaries, 26 located above 35° S in Misiones, La Rioja, Catamarca, Formosa, Corrientes, Córdoba, and Santa Fé exhibited the Africanized mitotypes while the remaining apiaries below this latitude, mainly in Buenos Aires, Rio Negro, Neuquén, and Entre Ríos, showed the European mitotypes. These results align with previous characterizations in the region. HRM analysis demonstrated high sensitivity and effectiveness, requiring minimal DNA input, and reducing costs and sampling efforts. This approach provides a rapid and reliable method for identifying the African lineage, facilitating honey bee breeding programs, conservation efforts, and management strategies in regions with Africanized honey bee populations.蜜蜂种群的遗传鉴定，尤其是非洲化蜜蜂的识别，传统上依赖于各类分子生物学方法，这类方法需对线粒体或核基因组的不同区域进行扩增与分析，且往往耗时较长。本研究引入高分辨率熔解曲线分析（high-resolution melting analysis, HRM）作为一种全新方法，可快速且经济高效地从蜂蜜提取的线粒体DNA中，识别西方蜜蜂（Apis mellifera）种群中的非洲血统。 2012年至2018年间，研究团队从阿根廷13个省份的111个蜂场采集了样本。从10克蜂蜜沉淀物中提取总DNA，随后使用特异性引物APIS-F/Afr207R进行定量聚合酶链式反应（quantitative PCR, qPCR），扩增得到207 bp的目的片段。以非洲化/非非洲化样本作为阳性对照，用于线粒体单倍型验证；同时对部分qPCR产物进行测序，以比对不同的单倍型。HRM分析可快速检测qPCR扩增得到的短扩增片段中的序列多态性。在所分析的蜂场中，有26个蜂场位于南纬35°以南的米西奥内斯、拉里奥哈、卡塔马卡、福尔摩沙、科连特斯、科尔多瓦及圣菲省，其样本呈现非洲化线粒体单倍型；剩余蜂场均位于该纬度以北，主要分布于布宜诺斯艾利斯、内格罗河、内乌肯及恩特雷里奥斯省，样本则表现为欧洲线粒体单倍型。该研究结果与该区域此前的相关鉴定结果一致。HRM分析展现出极高的灵敏度与有效性，仅需极少量的DNA模板，同时降低了实验成本与采样工作量。该方法为非洲血统识别提供了一种快速可靠的技术手段，有助于在存在非洲化蜜蜂种群的区域开展蜜蜂育种计划、资源保护工作及种群管理策略制定。