An economical and effective high-throughput DNA extraction protocol for molecular marker analysis in honeybees

Abstract

The honeybee is becoming an increasingly important organism in the realm of laboratory-based genetics. As honey bee health around the world is declining, measures are being taken to understand the genetic basis of a multitude of traits including behavior, disease resistance, survivability, honey production, and pollination efficiency. Quantitative Trait Loci (QTL) studies and mapping projects are elucidating the complex interactions of genetic loci that dictate the important traits being bred for by beekeepers, queen producers, and researchers alike. Quantitative Trait Loci for foraging behavior and aggression were identified nearly 15 years ago (Hunt et al., 1995, 1998), long before the advancements of the current and ongoing honey bee genome project (Honey Bee Genome Sequencing Consortium, Baylor College of Medicine, Houston, TX, USA). More recent studies have identified a single QTL for chalkbrood disease resistance (Holloway et al., 2012) and have utilized the genome data to finemap the interval to contain just two genes of potential interest (Holloway et al., 2013). As QTL studies are becoming more commonplace in the understanding of honey bee genetics, DNA extractionmethods are needing to be faster, more effective, andmore economical to keep pace with the analyses of the populations being studied. Honey bee QTL studies are generating information on potential gene or allele functionality relevant to mapping populations. Yet, narrowing the intervals and pinpointing the genes of interest for eventual marker-assisted selection requires hundreds or even thousands of phenotyped bees to be processed for DNA extraction and genotyping. Typically, the extraction processes yield good qualities and quantities of DNA per individual sample, yet are costly in time and materials. Commercial DNA extraction kits allow for consistent and reproducible yields, yet can cost several dollars per sample. Chelating agents purify DNA with high quality but may require long incubations and preparations with additional proteinase K (Giraffa et al., 2000; Casquet et al., 2012). Traditional phenol-chloroform extractions require handling, storage, and disposal of hazardous organic solvents. Honey bee DNA has recently been effectively extracted and purified by homogenization and utilizing proteinase K and ‘salting-out’ methods to remove proteins (Bourgeois et al., 2008, 2010; Bourgeois & Rinderer, 2009). However, we developed a simple, costeffective, and fast method using only sodium chloride and sodiumdodecylsulfate (SDS) to extract clean DNAdirectly useable for PCR without additional dilution as is typically required. This method is particularly useful for highthroughput extraction of DNA from large numbers of individual bees while minimizing labor, plastic consumables, and reagent requirements. The resulting DNA is of a high enough quality and quantity to directly amplify bands for molecular marker analysis.