The multiple data sets from the five procedures of the three methods were then separately analyzed
with the
PHYLIP
program
CONSENSE
to obtain bootstrap values reflecting the consistency of tree
branching patterns, and dendrograms were pro- duced with the
PHYLIP
program
DRAWGRAM
. The chalcone synthase-like protein sequence from
Streptomyces griseus, a prokaryote, was used to root all trees.
3. Results
Primers were designed which would amplify the full length of a stilbene synthase gene. Amplifica-
tion of genomic DNA from V. 6inifera cv. Optima and V. riparia cv. Gloire de Montpellier both
yielded a single band of 1550 bp data not shown. Cloning and sequencing of the stilbene
synthase gene from V. riparia Genbank accession no. AF128861 revealed that it had 99 nucleotide
identity to the stilbene synthases of V. 6inifera but only 63 identity to the chalcone synthases of V.
6 inifera Fig. 1. Nucleotide identity of the V.
riparia stilbene synthase gene was 65, 63 and 62, respectively, with the stilbene synthases of A. hy-
pogaea, P. strobus and P. syl6estris. An examina- tion of the predicted protein sequence of the V.
riparia gene showed that the sequence motif, GVLFGFGPGLT, which is the family signature
sequence for stilbene and chalcone synthases [9], was present Fig. 1. Furthermore, the sequence
motif, IPNSAGAIAGN, which is specific for stil- bene synthases [15], was found also in the pre-
dicted protein sequence of the V. riparia gene Fig. 1. The size of the predicted protein was 42716
daltons with an isoelectric point of 5.81. One intron was present which interrupted the codon
for cysteine at the same location as reported in all other stilbene and chalcone synthase genes. The
intron sequence was compared with that of two stilbene synthase genes of V. 6inifera cv. Optima
Table 1 Stilbene and chalcone synthase gene sequences obtained from Genbank except for S-Vitis3 and used for phylogenetic analyses
Species Gene
Common Genbank
Abbreviation accession no.
name 58314
Peanut S-Arachis
Arachis hypogaea Stilbene synthase
Pine S-Pinus1
Pinus strobus 762992
S-Pinus2 260926
P. syl6estris Pine
S-Pinus3 Pine
20656 P. syl6estris
S-Vitis1 Vitis 6inifera cv. Optima
Grape 235943
V. 6inifera cv. Labrusco a Foglia Frastagliata Grape
499024 S-Vitis2
V. riparia cv. Gloire de Montpellier Grape
AF128861 S-Vitis3
Bromheadia finlaysoniana Orchid
Chalcone synthase 2253119
C-Bromhead C-Betula
Birch 1834387
Betula pendula Chrysosplenium americanum
Saxifrage 2078561
C-Chrysosplen Callistephus chinensis
Aster 3059083
C-Callistephus C-Glycine
116377 Soybean
Glycine max Ipomoea nil
Morning glory 2329835
C-Ipomoea1 I. purpurea
Morning glory 2189961
C-Ipomoea2 C-Juglans
Walnut 1296816
Juglans sp. Oryza sati6a
Rice 1816444
C-Oryza Perilla frutescens
Perilla 1944195
C-Perilla P. strobus
Pine 3114911
C-Pinus1 P. syl6estris
Pine 20689
C-Pinus2 2642606
Radish Raphanus sati6us
C-Raphanus Scutellaria baicalensis
C-Scuttellaria 2618488
Skullcap Solanum tuberosum
Potato 1292924
C-Solanum Grape
V. 6inifera cv. Cabernet Sauvingnon 3288721
C-Vitis1 V. 6inifera
Grape 2465406
C-Vitis2 3702261
Actinomycete Streptomyces griseus
C-Streptomyces Chalcone synthase-
like
Fig. 1. Nucleotide sequence alignment of the coding regions of a stilbene synthase of V. riparia NS-Vitis3, a stilbene synthase of V. 6inifera NS-Vitis1 and a chalcone synthase of V. 6inifera NC-Vitis2. Nucleotides corresponding to the amino acid
signature sequences for stilbene synthases are shaded in reverse, and the stars below each line of alignment indicate fully conserved sites.
Fig. 2 The homology was very high 86.2 iden- tity with the similarly-sized intron of the Vst
1
gene of V. 6inifera cv. Optima, but there was only 53.7 identity with the intron of the Vst
2
stilbene synthase gene. There were four differences in the
21-bp region of the intron in the V. riparia gene that had been found to be identical between the
Vst
1
and Vst
2
genes [15]. Alignment of the amino acid sequence of the
stilbene synthase of V. riparia with that of other stilbene and chalcone synthases was done with five
different procedures Fig. 3 involving distance, parsimony and maximum likelihood methods. Us-
ing a parsimony method, Tropf et al. [11] reported that stilbene synthases did not cluster separately
from chalcone synthases but both grouped to- gether and could be subdivided based on the sys-
tematics of the plant families. Our analyses with all five procedures also found that stilbene syn-
thases did not form a separate cluster from chal-
Fig. 2. The intron sequence of the stilbene synthase of V. riparia NS-Vitis3 aligned with those of the Vst
1
and Vst
2
stilbene synthase genes of V. 6inifera [15]. The stars below each line of alignment indicate fully conserved sites.
cone synthases Fig. 3. However, UPGMA analy- sis placed all the chalcone synthases, including
those from Pinus spp., in one cluster, and all the stilbene synthases of different genera as succes-
sively more distant from chalcone synthases Fig. 3A. In the other types of analyses Fig. 3B – E,
stilbene and chalcone synthases of Pinus spp. clus- tered together, but for the Vitis spp., the stilbene
and chalcone synthases did not cluster together. Parsimony Fig. 3D and Maximum Likelihood
Fig. 3E analyses were most supportive of this distinction with bootstrap values above 68 at the
branching points of these two groups. The chal- cone synthase of soybean C-Glycine and the
stilbene synthase from peanut S-Arachis, both members of the Fabaceae, did not cluster together
in any of the analyses.
In some instances branching patterns reflected the taxonomic position of the species sequenced.
For instance, except for UPGMA analysis, the chalcone synthase sequences of P. frutescens C-
Perilla and S. baicalensis C-Scutellaria clustered
Fig. 3. Consensus trees from phylogenetic analyses of aligned amino acid sequences of 25 chalcone synthase or stilbene synthase genes listed in Table 1. The alignments were analyzed either by distance methods A, UPGMA; B, neighbor-joining; C, fitch
found within the software program
PHYLIP
, or by maximum parsimony D, using
PHYLIP PROTPARS
or maximum likelihood E, using
MOLPHY PROTML
. These procedures are described in the text. Bootstrap percentages greater than 50 are shown above or below each branch. In the distance-based trees, branch lengths reflect the distance between taxa except for the outgroup
C-Streptomyces, which is much more distant indicated by squiggle. For D and E, absolute branch lengths do not reflect differences between taxa.
together bootstrap values ranged from 82 to 98 and both are members of the Lamiaceae. How-
ever, the chalcone synthases of I. purpurea clus- tered closer to S. tuberosum than to I. nil for all
the types of analysis other than UPGMA, even though I. purpurea and I. nil are in the same genus
in the Convolvulaceae, whereas S. tuberosum is a member of the Solanaceae.
4. Discussion