mtDNA melting analysis for quick characterization of Africanized honeybee Apis mellifera populations using honey samples

Abstract

Honey contains mitochondrial DNA (mtDNA) from bees that produce it and can be used to identify their evolutionary origin, maternal lineage, or even subspecies. The mtDNA variation in Apis mellifera has been detected by different molecular methods (RFLPs, PCR-RFLPs, sequencing) and was previously used to characterize Africanized populations. High-resolution melting (HRM) analysis is a new technique that allows rapid identification of sequence polymorphisms in short amplicons generated by qPCR. The aim of this work is to identify the African mitotype in Apis mellifera populations by applying HRM. Honey samples were collected from 111 apiaries in 13 Argentine provinces between 2012 and 2018. Total DNA was extracted from sediment obtained by centrifugation of 10 g of honey and then subjected to qPCR in a final volume of 20 µl using a fluorescent intercalator and primers (APIS -F/Afr207R) that amplify a 207Pb product that allows discrimination between European and African origin. Positive controls (European/African) were used to corroborate qPCR product sizes and fusion curves. In addition, some products were purified and sequenced to confirm mitochondrial haplotypes. Only 26 honey samples from apiaries above 35° S in the provinces of Misiones, La Rioja, Catamarca, Formosa, Corrientes, Córdoba and Santa Fé showed an Africanized mitotype. The remaining apiaries below this border, mainly in the provinces of Buenos Aires, Rio Negro, Neuquén, and Entre Ríos, showed a European mitotype. The results are consistent with previous characterizations and with the distribution limit of Africanized populations in Argentina. Likewise, HRM analysis showed high sensitivity and effectiveness in mitotype characterization, as it requires very little DNA and requires only a single amplification reaction, reducing the cost and effort of sampling. In addition, it can be readily used in genetic selection programs in transition regions between European and Africanized populations, as it allows the identification of genetic resources of honey bee colonies based on the honey they produce.