6.2. Mitochondrial DNA analysis
In principle, one honey bee (worker or drone) per colony is enough to determine the maternal origin of the whole colony given the maternal inheritance of mitochondrial DNA (mtDNA), i.e. all the daughter workers and son drones from one honey bee queen share the same molecule. Due to the risk of drifting between colonies, it is ideal, and in some cases essential, to make this one collected individual of a life stage where host colony is unambiguous (e.g., a developing bee or one observed exiting from a brood cell). In cases where this is not possible, pre-flight worker bees could be substituted, although it is arguably worth sampling more than one individual to avoid mistakes in assigning colony heritage.
The most widely used mitochondrial region for population genetic studies is the intergenic region located between the tRNAleu and cox2 (subunit 2 of the cytochrome oxidase) genes. This region shows length and sequence variation that allows discrimination of honey bee evolutionary lineages (Garnery et al., 1993). It is composed of two types of sequences: P and Q. The sequence P can be absent (lineage C from east Europe) or present in four different forms: P (lineage M from west Europe), P0 (lineages A from Africa and O from Near East), P1 (Atlantic African sub-lineage) and P2 (restricted to the Y lineage from Ethiopia; De la Rúa et al., 2009). The number of Q sequences and the sequence variation developed through a RFLP test with the restriction enzyme DraI (Garnery et al., 1993) can be used to determine the haplotype within each lineage. Below is the protocol to determine the mitochondrial haplotype, modified from Garnery et al. (1993) by including a new thermal regime for PCR and optimizing the chemistry of PCR reactions. A full description of how this locus can discriminate among honey bee ecotypes is presented in the BEEBOOK paper on characterizing subspecies and ecotypes by Meixner et al. (2013).
- Honey bee samples are immediately transferred into tubes with absolute EtOH and preserved at – 20°C until DNA extraction. A single or two legs from one individual are enough to extract total DNA following the Chelex®-based (Biorad, Inc.) protocol (Walsh et al., 1991; see section 3.2.2.).
- PCR amplify the intergenic region
with the primers E2 (5’-GGCAGAATAAGTGACATTG-3’) located at the 5’ end of the
gene tRNAleu and H2 (5’-CAATATCATTGATGAACC-3’) located close to the
5’ end of the gene cox2 (Garnery et al.,
This amplification can be performed by using Ready-To-Go TM PCR Beads (product code 27-9557-01, GE Healthcare), that are pre-mixed and pre-dispensed reactions for PCR featuring, therefore reducing the pipetting steps and the chances to handling error. They contain all the necessary reagents for a 25 µl reaction volume.
- Add 20.2 µl of PCR-quality water to each tube.
- Mix by gently flicking it with the fingers.
- Add 0.4 µl of each primer (10 mM) and vortex and centrifuge the mix to get all the components at the bottom of the tubes.
- Add 4 µl of DNA extraction solution and mix.
- Place the
reaction mixtures in a thermo cycler with the following amplification program:
Denaturation at 94 °C for 5 min,
Followed by 35 cycles of:
94 °C for 45 sec,
48 °C for 45 sec,
62 °C for 2 min,
Final elongation step of 20 min at 65 °C.
- To identify successful amplicons, 2 µl of the PCR product of each sample are electrophoresed in a 1.5% agarose gel (see section 3.2.1) with ethidium bromide included and photographed over a UV light screen.
- Aliquots of
the PCR product are then digested with the endonuclease DraI
(recognition site 5’-TTTAAA-3’) by adding:
10X endonuclease buffer to a final concentration of 1X,
0.06U of DraI/,
10 µL of PCR product,
Incubate at 37 °C overnight.
- To determine RFLP patterns, the digested products of each sample are electrophoresed in a 4% agarose Nusieve® or Metaphor® (Lonza Biosciences) gel at 100 volts for ca. 1 hour 30 min and photographed over a UV light screen.
- At least one sample with a characteristic RFLP pattern should be directly sequenced using the same primers as for the amplification.
- Prior to sequencing, purify PCR products:
Either with QIAquick® PCR Purification Kit (Qiagen).
Alternatively, PCR products can be purified with isopropanol and 5 M ammonium acetate as follows:
1. To 10 µl of PCR product add:
7 µl of 5 M ammonium acetate,
17 µl of isopropanol.
2. Leave 10 min at room temperature.
3. Centrifuge 30 min to 13,500 rpm.
4. Discard the supernatant.
5. Add 500 µl of cold 70% EtOH.
6. Centrifuge 5 min at 13,500 rpm.
7. Remove supernatant and allow to dry overnight.
8. Re-suspend in 30 µl of water.