Revision of Passiflora Subgenus Decaloba ... - Passion Flowers

APPENDIX
CLADISTIC ANALYSIS OF GRANULE-BOUND STARCH SYNTHASE (WAXY)
GENE SEQUENCES
Introduction
Granule-bound starch synthase (sometimes referred to as “waxy” because of
its mutant phenotype in maize) was investigated in Passiflora supersection Cieca
because this gene has demonstrated phylogenetic utility in other groups of plants
and appears to be a single- or low-copy gene (Mason-Gamer et al., 1998; Miller
& Rausher, 1999; Evans et al., 2000; Mason-Gamer, 2001; Evans & Campbell,
2002; Mathews et al., 2002; Smedmark et al., 2003). In addition, the preliminary
data presented here indicates that GBSSI displays as many, if not more,
informative sites than the ITS region in supersection Cieca and has the potential
to answer difficult questions about evolution within this complex group of plants.
Materials and Methods
Total genomic DNA was extracted from fresh, heat dried or silica dried leaves
or flowers utilizing the CTAB method of Doyle and Doyle, scaled down to 1.0 ml
extraction volumes (Doyle & Doyle, 1987). DNA was precipitated for
approximately one to eight hours at -20ºC with 0.65 volumes of isopropanol,
centrifuged, washed twice with 70% ethyl alcohol, and dried. The pellet was
resuspended in 75µl of Tris-EDTA buffer and stored at -20ºC.
Amplification of the granule-bound starch synthase region (GBSSI) of nuclear
ribosomal DNA (nrDNA) was performed using 50 µl reactions containing Taq
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