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13. SUPPLEMENTARY MATERIALS
The AFLP procedure was based on Vos et al. (1995) with own modifications. The concentration
of reagents in each step i.e., restriction digestion, ligation, pre-amplification and
selective amplification as well as temperature regimes are given in Tables 13.11.1-13.11.4.
After restriction digestion, ligation and pre-amplification, the efficiency of reactions was verified
on 1% agarose gel 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. Products of selective amplification were
denatured with formamide at 94 o C for 5 min. and separated on polyacrylamide gel at 45 W of
constant power up to Xylene cyanol reached two third from the beginning. The gels were
silver stained, scanned using a flatbed scanner and pictures were stored as .jpg files.
All bands that could be reliable read were scored either present (1) or absent (0). The
dominant mode of inheritance of markers generated by all primers used in this study was verified
in F 1
and F 2
samples derived from a cross between L. multiflorum cultivar Bartolini and
L. perenne ecotype from New Zealand. The presence of a band was dominant over the lack
of it. AFLP loci were named according to restriction sites flanked by adapters (MseI, EcoRI)
and three-base extensions of selective primers (e.g., McaaEact20). Numerical nomenclature
was avoided in order to improve readability without necessity to look at the standard list for
AFLP primer nomenclature.