- A simple PCR method was used to amplify pubically avaliable and in-house SSRs with parental DNA (Mar34 & GD33) and DNA from a subset of the mapping population, to ensure that the markers produced polymorphic products.
- In the initial screen size differences were visualised on agarose gels stained with ethidium bromide.
- To genotype the population in a more high through put way, the SSRs were flourescently labelled at the 5' end and run through the ABI DNA sequencer in the genomics centre. The PCR products could then be visualised as peaks in Genemarker.
- The parental peaks sizes for all SSRs were recorded and the mapping population were genotyped according to which parental peak was present for each individual marker.
- A data matrix was constructed which used a genotpye code of A (Mar34) and B (GD33) to distinguish between the parental contributions.
Amplified Fragment Length Polymorphims (AFLPs)
- Thhe AFLP technique allows the visulaisation of hundreds of amplified DNA restriction fragments simultaneously. The polymorpisms found in the AFLP banding patterns can by used to genotype individuals based on the alleles they carry.
- There are 4 mains steps to this technique:
- Digestion: Target DNA is digested with two enzymes (EcoRI & MseI) simultaneously to provide a template for the AFLP reaction.
- Ligation: Adapter sequences are attached to provide primer binding sites for amplification of fragments using PCR.
- Pre-amplification: DNA fragments are amplified using EcoRI & Mse I primers with one selective nucleotide (A & C, repectively), to provide cleaner AFLP banding patterns.
- Touchdown PCR: Selective amplification is carried out with EcoRI and MseI primers containing 2/3 selective nucleotides, this give an average of 50 amplified fragments per sample.
- The touchdown PCR products are fluorecently labelled with the 5' dye FAM and run through an ABI DNA sequencer.
- The AFLP banding pattern can then be visualised in Genemarker, where polymorphisms can be identified and used to genotype the mapping population.
- A total of 8 AFLP combinations were used to genotype the mapping population, individuals were assigned the letter A or B to distingusih between the parental contributions
- The AFLP genotype information was added to the data matrix which was used in the construction of a framework linkage map.