PCR analyses were determined through real-time quantitative PCR, on a LightCycler 2.0 instrument (Roche) using the LightCycler FastStart DNA MasterPLUS SYBR Green I kit (Roche) essentially as described in Terol et al. (2015) (link). Each individual PCR reaction contained 2 ng of genomic DNA extract from leaves using the DNeasy Plant Mini Kit (Qiagen) according to the manufacturer’s instructions. Cycling protocol consisted of 10 min at 95 °C for preincubation followed for 45 cycles of 10 s for denaturation, 10 s at 60 °C for annealing, and 20 s at 72 °C for extension. Specificity of the PCR reaction was assessed by the presence of a single peak in the dissociation curve after amplification and through size estimation of the amplified product. Specificity of PCR reaction was confirmed by direct Sanger sequencing of the PCR product. Primers used are listed in table 5 .
Specific Primers for the Determination of Heteroplasmic Positions.
| Name | Frame | SNP Position | Sequence |
|---|---|---|---|
| C21826-F1 | + | 21826 | 5′-GCATCTTGGACTAGCCATCG-3′ |
| C21826-R1 | − | 5′-ACCGTGGGCCATATTTCTCT-3′ | |
| C20848-F2 | + | 20848 | 5′-CCCGAATCTCAGCAATCACT-3′ |
| C20848-R2 | − | 5′-TAACCCTTCGAACACAAGCA-3′ | |
| C69792-F1 | + | 69792 | 5′-GTTGCCCACTCAATCTGTTG-3′ |
| C69792-R2 | − | 5′-AATCTGCCTTGCCTAGGAATC-3′ |
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