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13. SUPPLEMENTARY MATERIALS
ANNEX 13.7. AMPLIFICATION OF MITOCHONDRIAL DNA (mtDNA)
Amplification of the intron between B and C exons in nad1 gene
Total DNA of individual plants was used in PCR reaction. The intron between B and C
exons in nad1 gene of mitochondrial genome was amplified by the pair of “universal primers”
with following sequences:
• nad1-F 5’GCA TTA CGA TCT GCA GCT CA3’
• nad1-R 5’GGA GCT CGA TTA GTT TCT GC3’
At the first step, the annealing temperature between 48 o C and 58 o C was tested using
PTC200 gradient thermal cycler (BioRad). Because no single product was obtained, the temperature
with clear reproducible pattern was regarded as optimal. The maternal inheritance
of observed bands was verified in F 1
and 20 F 2
individuals derived from a cross between
L. multiflorum cultivar, Bartolini and L. perenne ecotype from New Zealand.
Each PCR reaction was performed in a total volume 20 µl. Concentrations of reagents
and thermal conditions are shown in Table 13.7.1. PCR products were loaded on 2% (w/v)
agarose gels containing 0.5 µg/ml ethidium bromide, separated in 1 x TBE buffer (Tris-
Borate-EDTA; Sambrook et al. 1989) at 100 V constant power, visualized under UV light
(312 nm), photographed and stored as .jpg files. Each fragment was scored as present (1)
and absent (0).