High sample throughput genotyping for estimating C-lineage introgression in the dark honeybee: an accurate and cost-effective SNP-based tool

Dora Henriques,Per Kryger,M Alice Pinto,Melanie Parejo,Grace P Mccormack,Fiona Highet,Tom C Freeman,Irene Muñoz,Keith A Browne,Mark W Barnett,Lionel Garnery,J Spencer Jonhston

doi:10.1038/s41598-018-26932-1

Abstract

The natural distribution of the honeybee (Apis mellifera L.) has been changed by humans in recent decades to such an extent that the formerly widest-spread European subspecies, Apis mellifera mellifera, is threatened by extinction through introgression from highly divergent commercial strains in large tracts of its range. Conservation efforts for A. m. mellifera are underway in multiple European countries requiring reliable and cost-efficient molecular tools to identify purebred colonies. Here, we developed four ancestry-informative SNP assays for high sample throughput genotyping using the iPLEX Mass Array system. Our customized assays were tested on DNA from individual and pooled, haploid and diploid honeybee samples extracted from different tissues using a diverse range of protocols. The assays had a high genotyping success rate and yielded accurate genotypes. Performance assessed against whole-genome data showed that individual assays behaved well, although the most accurate introgression estimates were obtained for the four assays combined (117 SNPs). The best compromise between accuracy and genotyping costs was achieved when combining two assays (62 SNPs). We provide a ready-to-use cost-effective tool for accurate molecular identification and estimation of introgression levels to more effectively monitor and manage A. m. mellifera conservatories.

Highlights

Pollination by the honeybee (Apis mellifera L.) is a blended ecosystem service of managed and unmanaged colonies that is under threat from human-mediated environmental changes including climate change, habitat loss, habitat fragmentation, pesticides, and introduced parasites and pathogens[1,2]
Of the 144 highly-informative SNPs selected by Muñoz, et al.[20], the Assay Design software was able to multiplex 127 into four assays, each containing a variable number of SNPs ranging from 38 in M1 to 24 in M4 (Supplementary Table S1)
While adoption of microsatellites represented a major step in conservation management of A. m. mellifera[12], it has been shown that a reduced number of high-graded SNPs20, outperform the multiallelic marker in estimating introgression[19,26]

Summary

Introduction

Pollination by the honeybee (Apis mellifera L.) is a blended ecosystem service of managed and unmanaged (feral or wild) colonies that is under threat from human-mediated environmental changes including climate change, habitat loss, habitat fragmentation, pesticides, and introduced parasites and pathogens[1,2]. Lineage C occurs in a smaller geographical area composed of the Apennine and Balkan peninsulas and includes the most widely kept honeybee subspecies: the Italian Apis mellifera ligustica and the Carniolan Apis mellifera carnica. It has been argued that, unlike with other domesticated stock organisms, management and selective breeding in honeybees increase genetic diversity through introgression[14]. This form of admixture reduces the frequency of locally adapted gene complexes, leading to an increased likelihood of reduced survival rates of colonies[15]. Mellifera are gathering momentum in multiple European countries (www.sicamm.org) and with the knowledge that reduced adapted genetic diversity threatens both managed and unmanaged populations, the interests of commercial beekeeping and honeybee conservationists should be aligning, in A. m. These properties make SNPs a powerful tool for testing the breeding stock in A. m. mellifera conservatories and SNP data can be readily incorporated in shared genetic databases, facilitating implementation of a conservation strategy at the European scale

Methods

Results

Conclusion