Genotyping African Bees by PCR

I have assembled a series of excerpts from the literature in attempt to outline the current knowledge. These excerpts are snipped from my collection of more than 100 papers on this topic; no attempt has been made to cite them, except the final one.

Begin quoted material:

Genotyping by PCR is a common, cost-effective, and rapid molecular genotyping protocol that employs specific PCR primers targeting a specific DNA sequence of gene of interest in order to differentiate between varieties of genotypes. To do this, DNA is first extracted from a piece of tissue or specimen of interest. Then, a PCR reaction is run with primers targeting an area that differs between alleles of interest. The resulting reaction is loaded on an agarose gel for visualization of specific sized PCR amplicons (amplified DNA fragments).

The simplest method to detect the presence of Africanized bees in a new world population is to restrict an amplified fragment of mtDNA to determine if the African (Apis mellifera scutellata ) mitotype is present.

Identification of Africanized bees with mtDNA is relatively easy, because a single worker in any condition can represent a colony’s mtDNA lineage. Yet, since mtDNA is maternally inherited, mtDNA markers cannot determine if a European queen has mated with Africanized drones, and such colonies will remain undetected with mtDNA-based techniques.

There is only one mitochondrial genome type found in animal cells. This genome contains one circular molecule with between 11,000-28,000 base pairs. The Honey Bee Genome Consortium, describes approximately 260 million DNA base pair genome of the honey bee (Apis mellifera).

Although several regions in the honey bee mitochondrial genome exhibit size and sequence variation, the most informative has proved to be a noncoding sequence located between the genes for tRNALEU and cytochrome oxidase II (COII). Bees of the Eastern lineage have the shortest intergenic sequence, consisting of a single 192-196 bp copy of a sequence called “Q”. Bees of the Western and African lineages have longer intergenic sequences.

These mitochondrial DNA polymorphisms have been used to determine the maternal ancestry of feral African-derived honey bees in South and Central America African mtDNA haplotypes were found to be present in high frequency in feral African-derived bees from Brazil, indicating that most of them were matrilineal descendants of African bees -- most likely A. m. scutellata imported to Brazil.

Mitochondrial DNA sequencing is an unreliable test for Africanization. Mitochondria are maternally inherited without recombination and so the offspring of a European queen mated to Africanized drones will be falsely classified as European using mitochondrial DNA sequencing as will each subsequent generation arising from that mating.  

Currently, there is no reliable low-cost genetic test for detecting Africanized honeybees available. However, Whitfield et al. were able to clearly distinguish between Africanized honeybees and managed European-derived honeybees using 1136 SNPs (single nucleotide polymorphisms), suggesting that it should be possible to develop a low-cost SNP test suitable for use by industry.

Oldroyd and colleagues developed a panel of just 95 SNPs that were found to be under selection in Africanized populations (Whitfield et al. 2006) or with high pairwise population FST between three ancestral lineages (Harpur et al. 2014). 

"Differences in the proportion of African ancestry between these groups thus enable differentiation of African and Africanized honeybees from European-derived honeybees. There are a number of subspecies in Africa, and while we use 'African' ancestry to delineate Africanized honeybees from European honeybees, our current test cannot differentiate between the African subspecies." -- Oldroyd, & al. 2015, "A SNP test to identify Africanized honeybees via proportion of 'African' ancestry"

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Comment: I hope this helps. PCR technique itself is cheap and simple enough. The complicated part is choosing which genetic material to look at to answer the question at hand. The more specific the question, the more carefully you have to choose which region to look at. I would hasten to point out that genetic markers for identification purposes tell you nothing about what the genes are doing, or which genes produce characteristics such as shape, size, behavior, etc. 

PLB

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