126.96.36.199. Microsatellite analyses
Microsatellites are regions containing tandem repeats of 1-six-nucleotide repeats that are widespread in eukaryotic genomes including that of the SHB (please refer to Evans et al., 2013 for details). For example a CA12 microsatellite has 12 adjoining ‘CA’ nucleotides in the middle of non-repetitive DNA. These regions are inherently unstable, adding and clipping repeat units during cell replication and recombination. As a result, individuals often differ from each other in their genotypes at these loci, and they offer a powerful tool for describing populations and gene flow. Several microsatellite loci have been described for the SHB (Evans et al., 2008) and these loci have proven useful in mapping the movement of SHB in the Americas (Lounsberry et al., 2010). Below is a protocol for genotyping using dinucleotide (CA) microsatellites.
- Prepare 5 μl reaction mixes for each sample and
- 1 Unit Taq DNA polymerase with appropriate 1X buffer (Invitrogen),
- 1 mM dNTP mix,
- 2 mM added MgCl2,
- 0.2 μM of each forward and reverse primer,
- 0.08 μM of the Forward primer end-labelled on the 5’ end with FAM or HEX fluorophores (ordered from Invitrogen, or another supplier).
- Carry out the polymerase chain reaction (PCR) with a cycling program of
- 96°C for 2 min., then
- 3 cycles of 96°C for 30 sec.,
- 60°C for 30 sec. (-1° C/Cycle),
- 65°C for 1 min.,
- followed by 35 cycles of
- 96°C for 30 sec.,
- 56°C for 30 sec.,
- 65°C for 1 min, and
- a final extension at 65°C for 2 min.
- Add 1 μl each PCR product (diluted 1:20) to 10 μl Formamide containing 1X LIZ size standard (Applied Biosystems, ABI).
- Determine labelled product size via one capillary gel run using an ABI 3730 DNA machine and GeneMapper software (e.g. version 3.7, ABI)
- Carry out population-genetic analyses with the programs GenAlex (Peakall and Smouse, 2006), STRUCTURE (Falush et al., 2007) or other software programs designed to assess multi-allelic data, see section 3.3.2. on DNA microsatellites of the BEEBOOK paper on methods for characterising subspecies and ecotypes of Apis mellifera (Meixner et al., 2013).