Directory UMM :Data Elmu:jurnal:B:Biochemical Systematics and Ecology:Vol28.Issue8.Oct2000:
Biochemical Systematics and Ecology 28 (2000) 779}791
Discrimination and identi"cation of coastal
Douglas-"r clones using needle #avonoid
"ngerprints
Shiv Shankhar Kaundun!,",*, Philippe Lebreton", Alain Bailly#
!De& partement de Botanique, Home& opathie et Pharmacognosie, Universite& Claude Bernard-Lyon I, Faculte& de
Pharmacie, 8 avenue Rockefeller, 69373 Lyon ce& dex 08, France
"Laboratoire de Biochimie Ve& ge& tale, Universite& Claude Bernard-Lyon I, 43 Bd du 11 Novembre 1918, 69622
Villeurbanne, France
#AFOCELCentre-Ouest, Les Vaseix, 87430 Verneuil-sur-Vienne, France
Received 30 August 1999; accepted 26 October 1999
Abstract
This paper describes a method for discriminating and identifying 10 successful Douglas-"r
(Pseudotsuga menziesii var. menziesii) clones using foliar #avonoids. All the 101 individuals
analyzed by high performance liquid chromatography contained two proanthocyanidins:
prodelphinidin and procyanidin and six #avonols: myricetin, quercetin, larycitrin, kaempferol,
isorhamnetin and syringetin, but in di!erent proportions. The experimental protocol used was
very reproducible since the variation coe$cients for each #avonoid did not exceed 9%.
Submission of the #avonoid data to multivariate discriminant analysis allowed excellent
discrimination of the 10 clones with 89% of the individuals being well-grouped. Then a clonal
bank was established in which the "ngerprint of each clone is de"ned by its position in the
multidimensional space of the discriminant analysis. The clonal identity of several unknown
individuals was determined with success by projecting their #avonoid data in a subsequent
discriminant analysis. ( 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Pseudotsuga menziesii var. menziesii; Flavonoids; Genetic variability; Clonal discrimination
* Corresponding author: Present address: National Research Institute of Vegetables, Ornamental Plants
and Tea, 2769 Kanaya, Kanaya-cho, Haibara-gun, 428-8501 Shizuoka, Japan. Fax: #81-547-46-2169.
E-mail address: [email protected] (S.S. Kaundun)
0305-1978/00/$ - see front matter ( 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 3 0 5 - 1 9 7 8 ( 9 9 ) 0 0 1 1 9 - 2
780
S.S. Kaundun et al. / Biochemical Systematics and Ecology 28 (2000) 779}791
1. Introduction
Douglas-"r, Pseudotsuga menziesii (Mirb.) Franco was discovered on Vancouver
Island some 200 years ago and introduced into Europe in the middle of the 19th
century. In its natural range, it can be encountered on Northwestern America, from
the 19th parallel in Mexico to the 55th parallel in Canada and from the Paci"c Coast
to the Rockies where it reaches 3000 m in altitude (Vidakovic, 1991). Two geographically well-di!erentiated varieties, var. glauca or interior Douglas-"r and var. menziesii
or coastal Douglas-"r, separated by large morpho-anatomical (Chen et al., 1986) and
biochemical (Von Rudlo!, 1975; Li and Adams, 1989) di!erences have been fully
recognized. Because of its rapid growth, good wood quality and its resistance to major
insect pests, coastal Douglas-"r has been intensively cultivated in Europe. In France,
it has risen to the "rst place in a!orestation at the expense of numerous indigenous
conifers. In this country, improvement programs of Douglas-"r started some 30 years
ago by setting provenance trials in diverse ecological conditions with populations
representative of its natural range. From the results of several experimental trials, the
best provenances and among them the most vigorous and healthy individuals were
selected for seed orchards (Bastien et al., 1986). At the present time, local seed
production is, however, insu$cient to meet the increasing demand for Douglas-"r in
a!orestation (Michaud, 1992). Moreover, progeny tests have revealed that the heritability of such important traits as vigor and polycyclism were low in the F1 generation
(Michaud et al., 1992). For these reasons, clonal orchards were established and bulk
vegetative propagation of successful clones were undertaken. Such intensive clonal
silviculture necessitates a method to verify clonal identity and to discourage the
fraudulent commercialization of polyclonal varieties.
Needle #avonoids are robust biochemical markers that have proved their e$ciency
at the populational, speci"c and higher taxonomic levels (Niemann, 1988; Lebreton,
1995; Kaundun et al., 1997; Kaundun et al., 1998a,b). Segregation data have shown
that these secondary plant metabolites are synthesized under strict genetic control
(Yazdani and Lebreton, 1991; Forkmann, 1994). Needle #avonoid composition
was found to vary negligibly when individuals from one clone were grown under
di!erent environmental conditions, as compared to the large di!erences noted between di!erent clones originating from one site (Lebreton et al., 1990). In the
same way, no signi"cant di!erences in #avonoid metabolism were observed with the
age of a tree, or the position/orientation of a branch within a tree (Idrissi-Hassani,
1985; Lauranson, 1989). Moreover, #avonoids exist in many structural forms and
are relatively stable, easy to detect and measure (Harborne, 1975). To our knowledge,
they have not yet been applied to the clonal discrimination of conifers. The objective
of this paper is to describe a method using needle #avonoids, in an attempt to
discriminate and identify Douglas-"r clones. Special attention has been paid to
the reproducibility of the experimental protocol and to the mathematical tools
used. We have started by investigating the extent to which needle #avonoids were
capable of separating the 10 studied clones. Then we have tried to verify the identity of
several individuals after establishing a polyphenolic "ngerprint bank for di!erent
clones.
S.S. Kaundun et al. / Biochemical Systematics and Ecology 28 (2000) 779}791
781
2. Materials and methods
2.1. Plant materials
Sprigs from 10 coastal Douglas-"r clones (101 individuals in all), 13 years old, were
harvested from an experimental trial of the AFOCEL (Association Fore( t-Cellulose) in
October 1994. The stand is found at Ronno (45318@, 05319@), located in the `HautBeaujolaisa, France. Sprigs were collected from 5 to 20 individuals of each clone. The
clones originated from either Washington or Oregon State in the USA (Table 1). In
addition, one individual used to test the reproducibility of the experimental protocol
and which do not belong to any of the 10 clones, originates from the British Columbia
State (population 43 for IUFRO).
2.2. Phytochemical analyses
Two grams of pulverized air-dried needles were submitted for hot acidic hydrolysis
(160 ml HCl 2 N) in a water bath for 40 min. This acidic treatment generated
anthocyanidins from homologous proanthocyanidins, and #avonol aglycones from
corresponding #avonol glycosides. After cooling, part of the hydrolysate was kept for
relative anthocyanidin analyses by HPLC while the rest was extracted three times by
diethylether (60#60#40 ml). After extraction, the ethereal solutions containing the
#avonol aglycones were pooled and then evaporated, and the residue was redissolved
in 10 ml ethanol. After chelation with AlCl , the #avonols were measured at 425 nm
3
and expressed in quantitative units (mg/g as quercetin equivalents). In the resulting
aqueous phase, total anthocyanidins were measured at 525 nm and expressed in
arbitrary units (mg/g as procyanidin equivalents).
The structure of each eluted #avonoid was determined by its HPLC-UV spectrum,
its chemical behavior with classical reagents (Mabry et al., 1970; Markham, 1982) and
co-chromatography using reference compounds.
Table 1
Origin of the ten Douglas-"r clones analyzed
Clone
State
Altitude
Latitude
Longitude
Number of
individuals
1028
1039
1321
1341
1358
1583
1566
80 324
80 377
81 455
Washington
Oregon
Washington
Washington
Washington
Washington
Washington
Oregon
Oregon
Washington
800 m
430 m
Seed Zone
Seed Zone
Seed Zone
Seed Zone
Seed Zone
900 M
900 M
450 M
463 40@ N
423 41@ N
Seed Zone
Seed Zone
Seed Zone
Seed Zone
Seed Zone
453 00@ N
453 00@ N
483 31@ N
1213 02@ W
1233 23@ W
Seed Zone 403
Seed Zone 403
Seed Zone 403
Seed Zone 403
Seed Zone 403
1223 00@ W
1223 00@ W
1213 09@ W
10
20
8
10
14
10
8
9
7
5
403
403
403
403
403
403
403
403
403
403
782
S.S. Kaundun et al. / Biochemical Systematics and Ecology 28 (2000) 779}791
The relative amount of each #avonoid was quanti"ed by HPLC on a MicroBondapak C18 column. The anthocyanidin fraction was detected at 546 nm and
eluted by a ternary mixture of H O/MeOH/AcOH (60/30/10) at a #ow rate of
2
1.0 ml/min. Flavonol aglycones, detected at 365 nm, were eluted with the same ternary
mixture of H O/MeOH/AcOH, but in the proportion of 60/40/5 and a #ow rate of
2
1.5 ml/min. On the HPLC chromatogram the peak height of each #avonoid was
expressed as a percentage of the sum of the heights of all #avonoid peaks.
2.3. Data analyses
Principal component analysis and discriminant analysis were carried out using the
STAT-ITCF version 5.0 statistical software (ITCF, Paris).
3. Results
Two proanthocyanidins and six #avonols were detected and measured in all 101
trees (Table 2). Total proanthocyanidin concentration varied from 5.2 to 8.4 mg/g and
was composed of prodelphinidin (67.3$4.2%) and procyanidin (32.7$4.2%). The
#avonol fraction ranged from 2.1 to 3.0 mg/g. It was mainly dominated by kaempferol
(35.9%) and quercetin (34.4%); myricetin averaged 13.7%, isorhamnetin 9.8% while
larycitrin and syringetin, present in small quantities, totaled less than 7%. The
chemical structures of the needle #avonoids detected in the Douglas-"r clones were
identical to those previously found in Douglas-"r trees from di!erent origins (Lebreton et al., 1980; Niemann and van Genderen, 1980; Kaundun et al., 1998a).
Before using the above #avonoid traits as markers in Douglas-"r clones, the
reproducibility of the experimental protocol was tested. One Douglas-"r tree was
analyzed 12 times on di!erent days with the complete set of analyses described in
Materials and Methods section (Table 3). The variation coe$cients for total proanthocyanidin and #avonol concentrations as well as relative proanthocyanidin and
major #avonols contents did not exceed 6%. Minor #avonols were a!ected by
variation coe$cients reaching 9%. The low variation coe$cients for all #avonoids
indicated that the protocol used was very reproducible. Thus, the #avonoid data
collected here could legitimately be used in an attempt to characterize the Douglas-"r
clones. In the "rst instance, principal component analysis (which searches for natural
a$nity between individuals) was applied on all trees and #avonoid variables. The "rst
three factor scores explained 41, 19 and 14% of total variation. On the F ]F plane,
1
2
clones 81 455, 1358 and 1341 were relatively well-grouped and separated from the
others, while plane F ]F individualized clones 80 324, 80 377 and 1039 (Fig. 1).
1
3
Three other planes, F ]F , F ]F and F ]F (not shown here) grouped clones
1
4 2
3
3
5
1563, 1566 and 1028, respectively. This suggests that individuals belonging to the same
clone possessed the same #avonoid pattern. We were therefore allowed to use the
#avonoid data in a discriminant analysis, in order to de"ne the polyphenolic "ngerprint of each clone. This multivariate analysis assumes clustering between individuals
and proceeds by allocating each tree to the highest probability clone. Fig. 2 represents
Clone
Lat mg/g
LD%
LC%
Amg mg/g
Myr%
Que%
Lar%
Kpf%
Iso%
Syr%
1028
1039
1321
1341
1358
1563
1566
80 324
80 377
81 455
Mean!
and S.D.
5.8$1.0
6.6$1.0
5.9$0.9
6.9$0.7
8.4$0.7
5.8$0.4
5.6$0.5
7.3$1.0
5.2$0.8
5.2$0.5
6.3$1.0
(15.9%)
69.3$2.4
72.2$4.0
67.1$5.9
65.0$6.8
66.7$4.3
70.2$4.4
65.2$4.4
72.7$4.5
58.1$3.6
66.1$2.0
67.3$4.2
(6.2%)
30.7$2.4
27.8$4.0
32.9$5.9
35.0$6.8
33.3$4.3
29.8$4.4
34.8$4.4
27.3$4.5
41.9$3.6
33.9$2.0
32.7$4.2
(12.8%)
2.6$0.2
2.5$0.2
2.9$0.3
2.2$0.4
2.9$0.2
2.4$0.3
2.3$0.3
2.1$0.2
3.0$0.2
2.5$0.2
2.5$0.3
(12.0%)
12.2$1.5
12.5$1.4
15.6$2.2
10.1$2.1
15.4$0.8
12.5$1.0
13.2$1.6
16.1$1.3
14.3$2.4
15.5$1.8
13.7$1.9
(13.9%)
35.3$2.1
34.4$1.3
31.6$5.0
39.5$3.3
35.1$1.6
35.4$1.2
33.8$1.3
33.3$1.7
31.6$0.9
33.8$0.7
34.4$2.3
(6.7%)
3.1$0.4
3.3$0.3
4.5$0.7
2.6$0.9
4.4$0.5
3.4$0.6
3.7$0.8
4.3$0.4
4.9$0.5
5.3$0.3
4.0$0.9
(22.5%)
38.1$3.1
39.4$1.7
37.0$3.3
37.3$2.3
31.1$1.7
36.0$1.3
37.9$1.7
33.4$1.7
34.2$2.1
35.0$1.9
35.9$2.5
(7.0%)
9.3$0.9
8.8$0.5
9.2$0.5
9.0$0.8
11.2$0.6
10.8$0.6
9.4$0.5
10.4$0.6
12.6$0.7
7.7$0.5
9.8$1.4
(14.3%)
2.0$0.4
1.6$0.2
2.3$0.3
1.6$0.3
2.8$0.3
2.0$0.3
2.0$0.4
2.6$0.4
2.5$0.3
2.7$0.2
2.2$0.4
(18.2%)
!Means and standard deviations. The percentage in brackets is the coe$cient of variation. (LAt"total proanthocyanidins, LD"prodelphinidin,
LC"procyanidin, Amg"total #avonol aglycones, Myr"myricetin, Que"quercetin, Lar"larycitrin, Kpf"kaempferol, Iso"isorhamnetin, Syr"syringetin).
S.S. Kaundun et al. / Biochemical Systematics and Ecology 28 (2000) 779}791
Table 2
Proanthocyanidin and #avonol composition of the 10 Douglas-"r clones analyzed
783
784
Sample
Lat mg/g
LD %
LC %
Amg mg/g
Myr %
Que %
Lar %
Kpf %
Iso %
Syr %
1
2
3
4
5
6
7
8
9
10
11
12
Mean!
and S.D.
6.2
6.0
5.9
5.4
5.9
6.1
6.0
5.2
5.5
5.6
5.6
5.7
5.8$0.3
(5.2%)
65.0
65.6
66.1
65.9
66.7
66.0
66.0
65.6
65.6
65.2
65.7
64.8
65.7$0.5
(0.8%)
35.0
34.4
33.9
34.1
33.3
34.0
34.0
34.4
34.4
34.8
34.3
35.2
34.3$0.5
(1.5%)
3.1
3.4
3.1
3.0
2.9
3.0
3.0
2.9
2.9
3.0
3.1
2.8
3.0$0.2
(5.4%)
9.4
9.8
10.0
10.3
9.3
9.0
8.8
9.5
8.2
9.0
8.7
7.8
9.2$0.7
(7.6%)
32.9
31.9
31.9
33.3
31.3
29.1
32.0
31.0
30.9
30.5
30.8
30.6
31.4$1.1
(3.5%)
3.1
3.1
3.1
3.1
3.1
2.8
2.9
2.9
2.9
3.0
3.0
3.1
3.0$0.1
(3.3%)
43.1
44.0
43.9
42.1
44.0
47.2
44.6
45.3
46.9
45.9
45.6
46.2
44.9$1.5
(3.3%)
9.1
9.0
8.9
8.8
9.6
9.7
9.5
8.9
9.0
9.3
9.4
9.5
9.2$0.3
(3.3%)
2.3
2.2
2.2
2.2
2.6
2.2
2.2
2.3
2.0
2.3
2.4
2.8
2.3$0.2
(8.7%)
!The percentage in brackets is the coe$cient of variation. (LAt"total proanthocyanidins, LD"prodelphinidin, LC"procyanidin, Amg"total #avonol
aglycones, Myr"myricetin, Que"quercetin, Lar"larycitrin, Kpf"kaempferol, Iso"isorhamnetin, Syr"syringetin).
S.S. Kaundun et al. / Biochemical Systematics and Ecology 28 (2000) 779}791
Table 3
Reproducibility of the experimental protocol used: analysis of one Douglas-"r tree (originating from the state of British Columbia) 12 times
S.S. Kaundun et al. / Biochemical Systematics and Ecology 28 (2000) 779}791
785
Fig. 1. Principal planes (F ]F ) and (F ]F ) of the PCA based on 9 #avonoid variables and 101
1
2
1
3
individuals; corresponding correlation diagrams (LAt"total proanthocyanidins, LD%"prodelphinidin,
Amg"total #avonol aglycones, Myr"myricetin, Que"quercetin, Lar"larycitrin, Kpf"kaempferol,
Iso"isorhamnetin, Syr"syringetin). * For clarity, the clones which were not grouped on one plane were
indistinctly represented by an empty circle.
786
S.S. Kaundun et al. / Biochemical Systematics and Ecology 28 (2000) 779}791
Fig. 2. Principal plane (F ]F ) of the discriminant analysis based on 9 #avonoid variables and 101
1
2
individuals; corresponding correlation diagram (LAt"total proanthocyanidins, LD%" prodelphinidin,
Amg"total #avonol aglycones, Myr"myricetin, Que"quercetin, Lar"larycitrin, Kpf"kaempferol,
Iso"isorhamnetin, Syr"syringetin).
the "rst factorial plane of the discriminant analysis which explained 67% (F "48%
1
and F "19%) of the total inertia. The "rst axis was explained by kaempferol with
2
a negative sign of myricetin, larycitrin, isorhamnetin and syringetin, all four present in
small quantities. Factor 2 was mainly due to relative quercetin and prodelphinidin
contents, as well as total proanthocyanidin concentrations. On the F ]F plane,
1
2
clones 80 377 and 1358 were completely separated from each other and from the other
clones. On the other hand, clones 1341 and 1039 highly overlapped with each other,
while individuals of clone 1028 were scattered among several other clones. If the nine
axes were considered simultaneously, further clones could be discriminated in such
a way that 89% of the 101 individuals were grouped in their respective clones. The
data concerning the percentage of trees correctly classi"ed are summarized in Table 4;
the larger the proportion of trees allocated to the actual clone, the better the clone is
assumed to be discriminated. Clones 1358, 80 377 and 81 455 were the most highly
Clone
1028
1028 (n"10)
1039 (n"20)
1321 (n"8)
1341 (n"10)
1358 (n"14)
1563 (n"10)
1566 (n"8)
80 324 (n"9)
80 377 (n"7)
81 455 (n"5)
8
1039!
1321
18
1
6
1
1
1
1341
1358
1563
1566
1
1
1
9
1
7
1
80 324
80 377
81 455
1
8
14
1
8
7
5
!In clone 1039, 18 out of 20 individuals were well-classi"ed, 1 was attached to clone 1321 and the other to clone 1566.
Percentage individuals
well grouped
80
90
75
80
100
90
88
89
100
100
S.S. Kaundun et al. / Biochemical Systematics and Ecology 28 (2000) 779}791
Table 4
Percentage of well-grouped individuals for each of the 10 clones in the discriminant analysis
787
788
S.S. Kaundun et al. / Biochemical Systematics and Ecology 28 (2000) 779}791
Table 5
Paired Mahalanobis distances between the 10 clones
Clone
1028
1039
1321
1341
1358
1563
1566
80 324
80 377
1028
1039
1321
1341
1358
1563
1566
80 324
80 377
81 455
2.3181
2.3603
2.7664
2.9347
1.8474
2.2183
3.0199
3.7705
3.9725
1.9321
2.4244
2.9037
2.4605
2.1638
2.9384
3.7000
3.8168
3.2990
2.7738
2.7592
1.9906
3.0383
3.0226
3.0168
3.1210
2.6838
2.2511
3.2967
3.9564
3.7560
2.8596
3.0694
2.7239
3.4745
4.0790
1.9350
2.3871
3.0986
4.2645
2.4335
2.9488
3.1601
4.0248
4.0094
4.6019
Table 6
Mahalanobis distances (D) between the individuals to be identi"ed and the "rst three closest clones in the
clone bank
Individual to be identi"ed
(Actual clone in parentheses)
Distance to 1st closest Distance to 2nd closest Distance to 3rd closest
clone
clone
clone
1 (1039)
Clone 1039
D"1.93
Clone 1039
D"1.16
Clone 81 455
D"2.18
Clone 1039
D"3.48
Clone 1028
D"1.82
2 (1039)
3 (81 455)
4 (1321)
5 (1028)
Clone 1028
D"1.99
Clone 1321
D"2.21
Clone 1321
D"3.82
Clone 1321
D"3.60
Clone 1563
D"2.92
Clone 1321
D"2.57
Clone 1566
D"2.22
Clone 1566
D"3.91
Clone 1028
D"3.62
Clone 1039
D"3.44
discriminated whereas clone 1321 was relatively more heterogeneous. Mahalanobis
values given in Table 5 expressed the distances separating the 10 clones in the
discriminant analysis. The greatest distance was observed between clones 80 377 and
81 455, whereas 1028 and 1563 were the closest clones.
The space occupied by the individuals of a clone in the nine dimensions of the
discriminant analysis represented the polyphenolic "ngerprint of that particular
clone. From the clonal bank thus established, the determination of the clonal origin of
an individual was carried out by projecting its #avonoid data in a subsequent
discriminant analysis. As an example, "ve known individuals were randomly withdrawn from the 101 individuals forming the clonal bank. Two of them belonged to
clones 1039 and the three others to clones 81 455, 1321 and 1028 (Table 6). They were
then projected on a new discriminant analysis composed of the 10 same clones, but
consisting of 96 individuals only. The distances separating the individuals to be
recognized to the center of gravity of the clones in the clonal bank is given in Table 6.
S.S. Kaundun et al. / Biochemical Systematics and Ecology 28 (2000) 779}791
789
The individuals were attached to the clone to which they were closest. With the
exception of individual 4, all the other trees were successfully identi"ed (Table 6).
4. Discussion
The study was based on needle #avonoids in view of proposing a method to verify
the identity of Douglas-"r clones. The experimental protocol used was quite reproducible. Indeed, the variation coe$cients associated with the #avonoid traits did not
exceed 9% in any case. Two proanthocyanidins and six #avonols were detected and
measured in all 101 samples analyzed. The #avonoid patterns were relatively constant
among individuals of the same clone. At the same time, interclonal variability was
su$ciently high to allow good discrimination of the ten clones.
Multivariate discriminant analysis was used to de"ne #avonoid "ngerprints of
each clone and for identifying unknown individuals. Allocation of an individual to
a clone in a discriminant analysis is based on the multidimensional distance separating the individual to the center of gravity of the clones in the clone bank. Thus, the
greater the number of individuals used to de"ne the clonal "ngerprints, the higher are
the chances of allocating an individual to its proper clone. However, for practical
reasons, an optimum number of individuals should be determined for each clone; the
higher the intraclonal variability the larger the number of individuals. In addition,
when an individual to be identi"ed is attached to a clone, this does not necessarily
imply that it belongs to that clone, but rather that it does not belong to the other
clones.
The geographical variability of coastal Douglas-"r has recently been investigated
with needle #avonoids (Kaundun et al., 1998a). The amplitudes of variations for all
#avonoid traits in the latter study were much higher than that observed here. This
suggests that the 10 studied clones were not representative of the #avonoid patternings of the species and that further clones could be discriminated and introduced
simultaneously in the clone bank.
Clones 80 377 and 80 324, which originated from the same Oregon population, were
completely distinguished from one another (Fig. 2). This is an interesting "nding since
the most promising individuals selected for a!orestation in a particular region were
found to originate from a limited zone of the species range (Michaud et al., 1988). On
the other hand, two geographically distant clones, for instance clones 1039 and 1341,
were relatively close to each other. This observation supports several morphoanatomical (Chen et al., 1986) and biochemical (Zavarin and Snagberg, 1975; Yeh and
O'Malley, 1980; Li and Adams, 1989) studies which showed that the intrapopulational
variability of coastal Douglas-"r as much more important than its interpopulational
variability.
As shown on the correlation diagram of Fig. 2, interclonal variability in Douglas-"r
was biogenetically structured. Indeed, axis 1 explained the metabolic balance: B-ring
monohydroxylation (kaempferol) against B-ring trihydroxylation and/or methylation
(myricetin, larycitrin, syringetin and isorhamnetin), whereas axis 2 was negatively
de"ned by B-ring dihydroxylation (quercetin), as well as tannin concentration (total
790
S.S. Kaundun et al. / Biochemical Systematics and Ecology 28 (2000) 779}791
proanthocyanidin and prodelphinidin contents). In this respect, the #avonol metabolism in clones 1358 and 80 377 was more directed towards B-ring trihydroxylation
and/or methylation and less so regarding B-ring monohydroxylation compared to
clones 1028 and 1039. The two former clones were distinguishable upon their degree
of tannin synthesis; clones 1358 and 80 377 being, respectively, characterized by high
and low levels of tannin.
The method of clonal identi"cation has been established upon 13- year old trees. To
become a more interesting tool it should be tested on two-year old nursery samples, in
order to avoid errors in a!orestation. This cheap, rapid and reproducible method
could most probably be extended to other conifers and may constitute an important
tool in monitoring clonal bank management activities.
Acknowledgements
The authors would like to express their gratitude to Mr. T. Fauconnier and Mr. C.
Julien (AFOCEL, Sud-Est) for their help in sample collection and to Mr. J. Reynaud
(University of Lyon), Mr. J.C. Bastien (INRA, Orleans) and Mr. B. Fady (INRA,
Bormes-les-Mimosas) for reviewing the manuscript. The "rst author was supported
by a stipend from the `ReH gion Rho( ne-Alpesa, France.
References
Bastien, J.C., Roman-Amat, B., Michaud, D., 1986. Douglas. Rev. For. Fr. 38, 113}120.
Chen, Z.Y., Scagel, R.K., Maze, J., 1986. A study of the morphological variation in Pseudotsuga menziesii in
southwestern British Columbia. Can. J. Bot. 64, 1654}1663.
Forkmann, G., 1994. Genetics of #avonoids. In: Harborne, J.B. (Ed.), The Flavonoids. Chapman & Hall,
London, pp. 537}558.
Harborne, J.B., 1975. The biochemical systematics of #avonoids. In: Harborne, J.B., Mabry, T.J., Mabry, H.
(Eds.), The Flavonoids. Chapman & Hall, London, pp. 1056}1095.
Idrissi-Hassani, M., 1985. Etude de la variabiliteH #avonique chez deux conife`res meH diterraneH ens: le pin
maritime: Pinus pinaster Ait. et le geneH vrier thurife`res: Juniperus thurifera L., Ph.D. Thesis. Faculty of
Sciences, University of Lyon, France.
Kaundun, S.S., Fady, B., Lebreton, P., 1997. Genetic di!erences between Pinus halepensis, Pinus brutia and
Pinus eldarica based on needle #avonoids. Biochem. Syst. Ecol. 25, 553}562.
Kaundun, S.S., Lebreton, P., Bailly, A., 1998a. Needle #avonoid variation in coastal Douglas-"r (Pseudotsuga menziesii var. menziesii) populations. Can. J. Bot. 76, 2076}2083.
Kaundun, S.S., Lebreton, P., Fady, B., 1998b. Geographical variability of Pinus halepensis Mill. as revealed
by foliar #avonoids. Biochem. Syst. Ecol. 26, 83}96.
Lauranson, J., 1989. Exploration de la diversiteH biochimique chez les conife`res: contribution a` l'eH tude de
l'hybridation Pinus uncinata Ram. X Pinus sylvestris L., et a` la connaissance du complexe speH ci"que
Pinus nigra Arn. Ph.D. Thesis. Faculty of Sciences, University of Lyon, France.
Lebreton, P., 1995. Genetics and biodiversity of #avonoids in conifers. In: Baradat, P., Adams, W.T.,
MuK ller-Stark, G. (Eds.), Population Genetics and Genetic Conservation of Forest Trees. SPB Academic
Publishing, Amsterdam, pp. 41}54.
Lebreton, P., Laracine-Pittet, C., Bayet, C., Lauranson, J., 1990. VariabiliteH pheH nolique et systeH matique du
Pin sylvestre. Pinus sylvestris L. Ann. Sci. For. 47, 117}130.
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Lebreton, P., Thivend, S., Boutard, B., 1980. Distribution des proanthocyanidines chez les Gymnospermes.
Plant. MeH d. PhytotheH r. 15, 105}129.
Li, P., Adams, W.T., 1989. Range-wide patterns of allozyme variation in Douglas-"r (Pseudotsuga menziesii).
Can. J. For. Res. 19, 149}161.
Mabry, T.J., Markham, K.R., Thomas, M.S., 1970. The Systematic Identi"cation of Flavonoids. Springer,
Berlin.
Markham, K.R., 1982. Techniques of Flavonoid Identi"cation. Academic Press, New York.
Michaud, D., 1992. Comportement de Douglas issus de peuplements franc7 ais classeH s. Afocel, InformationsFore( t, Fasc. 438, 237}252.
Michaud, D., Bouvet, A., Gousseau, C., Besse, A., 1992. Analyse d'une expeH rimentation multistationelle de
descendances maternelles de Douglas issues de peuplements franc7 ais classeH s. Annales de Recherches
Sylvicoles, Afocel, pp. 89}124.
Michaud, D., Guinaudeau, F., Bouvet, A., 1988. Aspects de la variabiliteH des provenances de Douglas de
l'ouest de l'Etat de Washington. Annales de Recherches Sylvicoles, Afocel, 151}191.
Niemann, G.J., 1988. Distribution and evolution of the #avonoids in gymnosperms. In: Harborne, J.B. (Ed.),
The Flavonoids. Advances in Research since 1980. Chapman & Hall, London, pp. 469}478.
Niemann, G.J., van Genderen, H.H., 1980. Chemical relationships between Pinaceae. Biochem. Syst. Ecol.
8, 237}240.
Vidakovic, M., 1991. Conifers Morphology and Variation. Gra"cki Zavod Hrvatske, Zagreb.
Von Rudlo!, E., 1975. Volatile Leaf oil analysis in chemosystematic studies of North American conifers.
Biochem. Syst. Ecol. 2, 131}167.
Yazdani, R., Lebreton, P., 1991. Inheritance pattern of the #avonic compounds in Scots Pine (Pinus
sylvestris L.). Silvae Genet 40, 57}59.
Yeh, F.C., O'Malley, D., 1980. Enzyme variations in natural populations of Douglas-"r (Pseudotsuga
menziesii (Mirb.) Franco from British Columbia. I. Genetic variation patterns in coastal populations.
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Zavarin, E., Snagberg, K., 1975. Pseudosuga menziesii chemical races of California and Oregon. Biochem.
Syst. Ecol. 4, 121}129.
Discrimination and identi"cation of coastal
Douglas-"r clones using needle #avonoid
"ngerprints
Shiv Shankhar Kaundun!,",*, Philippe Lebreton", Alain Bailly#
!De& partement de Botanique, Home& opathie et Pharmacognosie, Universite& Claude Bernard-Lyon I, Faculte& de
Pharmacie, 8 avenue Rockefeller, 69373 Lyon ce& dex 08, France
"Laboratoire de Biochimie Ve& ge& tale, Universite& Claude Bernard-Lyon I, 43 Bd du 11 Novembre 1918, 69622
Villeurbanne, France
#AFOCELCentre-Ouest, Les Vaseix, 87430 Verneuil-sur-Vienne, France
Received 30 August 1999; accepted 26 October 1999
Abstract
This paper describes a method for discriminating and identifying 10 successful Douglas-"r
(Pseudotsuga menziesii var. menziesii) clones using foliar #avonoids. All the 101 individuals
analyzed by high performance liquid chromatography contained two proanthocyanidins:
prodelphinidin and procyanidin and six #avonols: myricetin, quercetin, larycitrin, kaempferol,
isorhamnetin and syringetin, but in di!erent proportions. The experimental protocol used was
very reproducible since the variation coe$cients for each #avonoid did not exceed 9%.
Submission of the #avonoid data to multivariate discriminant analysis allowed excellent
discrimination of the 10 clones with 89% of the individuals being well-grouped. Then a clonal
bank was established in which the "ngerprint of each clone is de"ned by its position in the
multidimensional space of the discriminant analysis. The clonal identity of several unknown
individuals was determined with success by projecting their #avonoid data in a subsequent
discriminant analysis. ( 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Pseudotsuga menziesii var. menziesii; Flavonoids; Genetic variability; Clonal discrimination
* Corresponding author: Present address: National Research Institute of Vegetables, Ornamental Plants
and Tea, 2769 Kanaya, Kanaya-cho, Haibara-gun, 428-8501 Shizuoka, Japan. Fax: #81-547-46-2169.
E-mail address: [email protected] (S.S. Kaundun)
0305-1978/00/$ - see front matter ( 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 3 0 5 - 1 9 7 8 ( 9 9 ) 0 0 1 1 9 - 2
780
S.S. Kaundun et al. / Biochemical Systematics and Ecology 28 (2000) 779}791
1. Introduction
Douglas-"r, Pseudotsuga menziesii (Mirb.) Franco was discovered on Vancouver
Island some 200 years ago and introduced into Europe in the middle of the 19th
century. In its natural range, it can be encountered on Northwestern America, from
the 19th parallel in Mexico to the 55th parallel in Canada and from the Paci"c Coast
to the Rockies where it reaches 3000 m in altitude (Vidakovic, 1991). Two geographically well-di!erentiated varieties, var. glauca or interior Douglas-"r and var. menziesii
or coastal Douglas-"r, separated by large morpho-anatomical (Chen et al., 1986) and
biochemical (Von Rudlo!, 1975; Li and Adams, 1989) di!erences have been fully
recognized. Because of its rapid growth, good wood quality and its resistance to major
insect pests, coastal Douglas-"r has been intensively cultivated in Europe. In France,
it has risen to the "rst place in a!orestation at the expense of numerous indigenous
conifers. In this country, improvement programs of Douglas-"r started some 30 years
ago by setting provenance trials in diverse ecological conditions with populations
representative of its natural range. From the results of several experimental trials, the
best provenances and among them the most vigorous and healthy individuals were
selected for seed orchards (Bastien et al., 1986). At the present time, local seed
production is, however, insu$cient to meet the increasing demand for Douglas-"r in
a!orestation (Michaud, 1992). Moreover, progeny tests have revealed that the heritability of such important traits as vigor and polycyclism were low in the F1 generation
(Michaud et al., 1992). For these reasons, clonal orchards were established and bulk
vegetative propagation of successful clones were undertaken. Such intensive clonal
silviculture necessitates a method to verify clonal identity and to discourage the
fraudulent commercialization of polyclonal varieties.
Needle #avonoids are robust biochemical markers that have proved their e$ciency
at the populational, speci"c and higher taxonomic levels (Niemann, 1988; Lebreton,
1995; Kaundun et al., 1997; Kaundun et al., 1998a,b). Segregation data have shown
that these secondary plant metabolites are synthesized under strict genetic control
(Yazdani and Lebreton, 1991; Forkmann, 1994). Needle #avonoid composition
was found to vary negligibly when individuals from one clone were grown under
di!erent environmental conditions, as compared to the large di!erences noted between di!erent clones originating from one site (Lebreton et al., 1990). In the
same way, no signi"cant di!erences in #avonoid metabolism were observed with the
age of a tree, or the position/orientation of a branch within a tree (Idrissi-Hassani,
1985; Lauranson, 1989). Moreover, #avonoids exist in many structural forms and
are relatively stable, easy to detect and measure (Harborne, 1975). To our knowledge,
they have not yet been applied to the clonal discrimination of conifers. The objective
of this paper is to describe a method using needle #avonoids, in an attempt to
discriminate and identify Douglas-"r clones. Special attention has been paid to
the reproducibility of the experimental protocol and to the mathematical tools
used. We have started by investigating the extent to which needle #avonoids were
capable of separating the 10 studied clones. Then we have tried to verify the identity of
several individuals after establishing a polyphenolic "ngerprint bank for di!erent
clones.
S.S. Kaundun et al. / Biochemical Systematics and Ecology 28 (2000) 779}791
781
2. Materials and methods
2.1. Plant materials
Sprigs from 10 coastal Douglas-"r clones (101 individuals in all), 13 years old, were
harvested from an experimental trial of the AFOCEL (Association Fore( t-Cellulose) in
October 1994. The stand is found at Ronno (45318@, 05319@), located in the `HautBeaujolaisa, France. Sprigs were collected from 5 to 20 individuals of each clone. The
clones originated from either Washington or Oregon State in the USA (Table 1). In
addition, one individual used to test the reproducibility of the experimental protocol
and which do not belong to any of the 10 clones, originates from the British Columbia
State (population 43 for IUFRO).
2.2. Phytochemical analyses
Two grams of pulverized air-dried needles were submitted for hot acidic hydrolysis
(160 ml HCl 2 N) in a water bath for 40 min. This acidic treatment generated
anthocyanidins from homologous proanthocyanidins, and #avonol aglycones from
corresponding #avonol glycosides. After cooling, part of the hydrolysate was kept for
relative anthocyanidin analyses by HPLC while the rest was extracted three times by
diethylether (60#60#40 ml). After extraction, the ethereal solutions containing the
#avonol aglycones were pooled and then evaporated, and the residue was redissolved
in 10 ml ethanol. After chelation with AlCl , the #avonols were measured at 425 nm
3
and expressed in quantitative units (mg/g as quercetin equivalents). In the resulting
aqueous phase, total anthocyanidins were measured at 525 nm and expressed in
arbitrary units (mg/g as procyanidin equivalents).
The structure of each eluted #avonoid was determined by its HPLC-UV spectrum,
its chemical behavior with classical reagents (Mabry et al., 1970; Markham, 1982) and
co-chromatography using reference compounds.
Table 1
Origin of the ten Douglas-"r clones analyzed
Clone
State
Altitude
Latitude
Longitude
Number of
individuals
1028
1039
1321
1341
1358
1583
1566
80 324
80 377
81 455
Washington
Oregon
Washington
Washington
Washington
Washington
Washington
Oregon
Oregon
Washington
800 m
430 m
Seed Zone
Seed Zone
Seed Zone
Seed Zone
Seed Zone
900 M
900 M
450 M
463 40@ N
423 41@ N
Seed Zone
Seed Zone
Seed Zone
Seed Zone
Seed Zone
453 00@ N
453 00@ N
483 31@ N
1213 02@ W
1233 23@ W
Seed Zone 403
Seed Zone 403
Seed Zone 403
Seed Zone 403
Seed Zone 403
1223 00@ W
1223 00@ W
1213 09@ W
10
20
8
10
14
10
8
9
7
5
403
403
403
403
403
403
403
403
403
403
782
S.S. Kaundun et al. / Biochemical Systematics and Ecology 28 (2000) 779}791
The relative amount of each #avonoid was quanti"ed by HPLC on a MicroBondapak C18 column. The anthocyanidin fraction was detected at 546 nm and
eluted by a ternary mixture of H O/MeOH/AcOH (60/30/10) at a #ow rate of
2
1.0 ml/min. Flavonol aglycones, detected at 365 nm, were eluted with the same ternary
mixture of H O/MeOH/AcOH, but in the proportion of 60/40/5 and a #ow rate of
2
1.5 ml/min. On the HPLC chromatogram the peak height of each #avonoid was
expressed as a percentage of the sum of the heights of all #avonoid peaks.
2.3. Data analyses
Principal component analysis and discriminant analysis were carried out using the
STAT-ITCF version 5.0 statistical software (ITCF, Paris).
3. Results
Two proanthocyanidins and six #avonols were detected and measured in all 101
trees (Table 2). Total proanthocyanidin concentration varied from 5.2 to 8.4 mg/g and
was composed of prodelphinidin (67.3$4.2%) and procyanidin (32.7$4.2%). The
#avonol fraction ranged from 2.1 to 3.0 mg/g. It was mainly dominated by kaempferol
(35.9%) and quercetin (34.4%); myricetin averaged 13.7%, isorhamnetin 9.8% while
larycitrin and syringetin, present in small quantities, totaled less than 7%. The
chemical structures of the needle #avonoids detected in the Douglas-"r clones were
identical to those previously found in Douglas-"r trees from di!erent origins (Lebreton et al., 1980; Niemann and van Genderen, 1980; Kaundun et al., 1998a).
Before using the above #avonoid traits as markers in Douglas-"r clones, the
reproducibility of the experimental protocol was tested. One Douglas-"r tree was
analyzed 12 times on di!erent days with the complete set of analyses described in
Materials and Methods section (Table 3). The variation coe$cients for total proanthocyanidin and #avonol concentrations as well as relative proanthocyanidin and
major #avonols contents did not exceed 6%. Minor #avonols were a!ected by
variation coe$cients reaching 9%. The low variation coe$cients for all #avonoids
indicated that the protocol used was very reproducible. Thus, the #avonoid data
collected here could legitimately be used in an attempt to characterize the Douglas-"r
clones. In the "rst instance, principal component analysis (which searches for natural
a$nity between individuals) was applied on all trees and #avonoid variables. The "rst
three factor scores explained 41, 19 and 14% of total variation. On the F ]F plane,
1
2
clones 81 455, 1358 and 1341 were relatively well-grouped and separated from the
others, while plane F ]F individualized clones 80 324, 80 377 and 1039 (Fig. 1).
1
3
Three other planes, F ]F , F ]F and F ]F (not shown here) grouped clones
1
4 2
3
3
5
1563, 1566 and 1028, respectively. This suggests that individuals belonging to the same
clone possessed the same #avonoid pattern. We were therefore allowed to use the
#avonoid data in a discriminant analysis, in order to de"ne the polyphenolic "ngerprint of each clone. This multivariate analysis assumes clustering between individuals
and proceeds by allocating each tree to the highest probability clone. Fig. 2 represents
Clone
Lat mg/g
LD%
LC%
Amg mg/g
Myr%
Que%
Lar%
Kpf%
Iso%
Syr%
1028
1039
1321
1341
1358
1563
1566
80 324
80 377
81 455
Mean!
and S.D.
5.8$1.0
6.6$1.0
5.9$0.9
6.9$0.7
8.4$0.7
5.8$0.4
5.6$0.5
7.3$1.0
5.2$0.8
5.2$0.5
6.3$1.0
(15.9%)
69.3$2.4
72.2$4.0
67.1$5.9
65.0$6.8
66.7$4.3
70.2$4.4
65.2$4.4
72.7$4.5
58.1$3.6
66.1$2.0
67.3$4.2
(6.2%)
30.7$2.4
27.8$4.0
32.9$5.9
35.0$6.8
33.3$4.3
29.8$4.4
34.8$4.4
27.3$4.5
41.9$3.6
33.9$2.0
32.7$4.2
(12.8%)
2.6$0.2
2.5$0.2
2.9$0.3
2.2$0.4
2.9$0.2
2.4$0.3
2.3$0.3
2.1$0.2
3.0$0.2
2.5$0.2
2.5$0.3
(12.0%)
12.2$1.5
12.5$1.4
15.6$2.2
10.1$2.1
15.4$0.8
12.5$1.0
13.2$1.6
16.1$1.3
14.3$2.4
15.5$1.8
13.7$1.9
(13.9%)
35.3$2.1
34.4$1.3
31.6$5.0
39.5$3.3
35.1$1.6
35.4$1.2
33.8$1.3
33.3$1.7
31.6$0.9
33.8$0.7
34.4$2.3
(6.7%)
3.1$0.4
3.3$0.3
4.5$0.7
2.6$0.9
4.4$0.5
3.4$0.6
3.7$0.8
4.3$0.4
4.9$0.5
5.3$0.3
4.0$0.9
(22.5%)
38.1$3.1
39.4$1.7
37.0$3.3
37.3$2.3
31.1$1.7
36.0$1.3
37.9$1.7
33.4$1.7
34.2$2.1
35.0$1.9
35.9$2.5
(7.0%)
9.3$0.9
8.8$0.5
9.2$0.5
9.0$0.8
11.2$0.6
10.8$0.6
9.4$0.5
10.4$0.6
12.6$0.7
7.7$0.5
9.8$1.4
(14.3%)
2.0$0.4
1.6$0.2
2.3$0.3
1.6$0.3
2.8$0.3
2.0$0.3
2.0$0.4
2.6$0.4
2.5$0.3
2.7$0.2
2.2$0.4
(18.2%)
!Means and standard deviations. The percentage in brackets is the coe$cient of variation. (LAt"total proanthocyanidins, LD"prodelphinidin,
LC"procyanidin, Amg"total #avonol aglycones, Myr"myricetin, Que"quercetin, Lar"larycitrin, Kpf"kaempferol, Iso"isorhamnetin, Syr"syringetin).
S.S. Kaundun et al. / Biochemical Systematics and Ecology 28 (2000) 779}791
Table 2
Proanthocyanidin and #avonol composition of the 10 Douglas-"r clones analyzed
783
784
Sample
Lat mg/g
LD %
LC %
Amg mg/g
Myr %
Que %
Lar %
Kpf %
Iso %
Syr %
1
2
3
4
5
6
7
8
9
10
11
12
Mean!
and S.D.
6.2
6.0
5.9
5.4
5.9
6.1
6.0
5.2
5.5
5.6
5.6
5.7
5.8$0.3
(5.2%)
65.0
65.6
66.1
65.9
66.7
66.0
66.0
65.6
65.6
65.2
65.7
64.8
65.7$0.5
(0.8%)
35.0
34.4
33.9
34.1
33.3
34.0
34.0
34.4
34.4
34.8
34.3
35.2
34.3$0.5
(1.5%)
3.1
3.4
3.1
3.0
2.9
3.0
3.0
2.9
2.9
3.0
3.1
2.8
3.0$0.2
(5.4%)
9.4
9.8
10.0
10.3
9.3
9.0
8.8
9.5
8.2
9.0
8.7
7.8
9.2$0.7
(7.6%)
32.9
31.9
31.9
33.3
31.3
29.1
32.0
31.0
30.9
30.5
30.8
30.6
31.4$1.1
(3.5%)
3.1
3.1
3.1
3.1
3.1
2.8
2.9
2.9
2.9
3.0
3.0
3.1
3.0$0.1
(3.3%)
43.1
44.0
43.9
42.1
44.0
47.2
44.6
45.3
46.9
45.9
45.6
46.2
44.9$1.5
(3.3%)
9.1
9.0
8.9
8.8
9.6
9.7
9.5
8.9
9.0
9.3
9.4
9.5
9.2$0.3
(3.3%)
2.3
2.2
2.2
2.2
2.6
2.2
2.2
2.3
2.0
2.3
2.4
2.8
2.3$0.2
(8.7%)
!The percentage in brackets is the coe$cient of variation. (LAt"total proanthocyanidins, LD"prodelphinidin, LC"procyanidin, Amg"total #avonol
aglycones, Myr"myricetin, Que"quercetin, Lar"larycitrin, Kpf"kaempferol, Iso"isorhamnetin, Syr"syringetin).
S.S. Kaundun et al. / Biochemical Systematics and Ecology 28 (2000) 779}791
Table 3
Reproducibility of the experimental protocol used: analysis of one Douglas-"r tree (originating from the state of British Columbia) 12 times
S.S. Kaundun et al. / Biochemical Systematics and Ecology 28 (2000) 779}791
785
Fig. 1. Principal planes (F ]F ) and (F ]F ) of the PCA based on 9 #avonoid variables and 101
1
2
1
3
individuals; corresponding correlation diagrams (LAt"total proanthocyanidins, LD%"prodelphinidin,
Amg"total #avonol aglycones, Myr"myricetin, Que"quercetin, Lar"larycitrin, Kpf"kaempferol,
Iso"isorhamnetin, Syr"syringetin). * For clarity, the clones which were not grouped on one plane were
indistinctly represented by an empty circle.
786
S.S. Kaundun et al. / Biochemical Systematics and Ecology 28 (2000) 779}791
Fig. 2. Principal plane (F ]F ) of the discriminant analysis based on 9 #avonoid variables and 101
1
2
individuals; corresponding correlation diagram (LAt"total proanthocyanidins, LD%" prodelphinidin,
Amg"total #avonol aglycones, Myr"myricetin, Que"quercetin, Lar"larycitrin, Kpf"kaempferol,
Iso"isorhamnetin, Syr"syringetin).
the "rst factorial plane of the discriminant analysis which explained 67% (F "48%
1
and F "19%) of the total inertia. The "rst axis was explained by kaempferol with
2
a negative sign of myricetin, larycitrin, isorhamnetin and syringetin, all four present in
small quantities. Factor 2 was mainly due to relative quercetin and prodelphinidin
contents, as well as total proanthocyanidin concentrations. On the F ]F plane,
1
2
clones 80 377 and 1358 were completely separated from each other and from the other
clones. On the other hand, clones 1341 and 1039 highly overlapped with each other,
while individuals of clone 1028 were scattered among several other clones. If the nine
axes were considered simultaneously, further clones could be discriminated in such
a way that 89% of the 101 individuals were grouped in their respective clones. The
data concerning the percentage of trees correctly classi"ed are summarized in Table 4;
the larger the proportion of trees allocated to the actual clone, the better the clone is
assumed to be discriminated. Clones 1358, 80 377 and 81 455 were the most highly
Clone
1028
1028 (n"10)
1039 (n"20)
1321 (n"8)
1341 (n"10)
1358 (n"14)
1563 (n"10)
1566 (n"8)
80 324 (n"9)
80 377 (n"7)
81 455 (n"5)
8
1039!
1321
18
1
6
1
1
1
1341
1358
1563
1566
1
1
1
9
1
7
1
80 324
80 377
81 455
1
8
14
1
8
7
5
!In clone 1039, 18 out of 20 individuals were well-classi"ed, 1 was attached to clone 1321 and the other to clone 1566.
Percentage individuals
well grouped
80
90
75
80
100
90
88
89
100
100
S.S. Kaundun et al. / Biochemical Systematics and Ecology 28 (2000) 779}791
Table 4
Percentage of well-grouped individuals for each of the 10 clones in the discriminant analysis
787
788
S.S. Kaundun et al. / Biochemical Systematics and Ecology 28 (2000) 779}791
Table 5
Paired Mahalanobis distances between the 10 clones
Clone
1028
1039
1321
1341
1358
1563
1566
80 324
80 377
1028
1039
1321
1341
1358
1563
1566
80 324
80 377
81 455
2.3181
2.3603
2.7664
2.9347
1.8474
2.2183
3.0199
3.7705
3.9725
1.9321
2.4244
2.9037
2.4605
2.1638
2.9384
3.7000
3.8168
3.2990
2.7738
2.7592
1.9906
3.0383
3.0226
3.0168
3.1210
2.6838
2.2511
3.2967
3.9564
3.7560
2.8596
3.0694
2.7239
3.4745
4.0790
1.9350
2.3871
3.0986
4.2645
2.4335
2.9488
3.1601
4.0248
4.0094
4.6019
Table 6
Mahalanobis distances (D) between the individuals to be identi"ed and the "rst three closest clones in the
clone bank
Individual to be identi"ed
(Actual clone in parentheses)
Distance to 1st closest Distance to 2nd closest Distance to 3rd closest
clone
clone
clone
1 (1039)
Clone 1039
D"1.93
Clone 1039
D"1.16
Clone 81 455
D"2.18
Clone 1039
D"3.48
Clone 1028
D"1.82
2 (1039)
3 (81 455)
4 (1321)
5 (1028)
Clone 1028
D"1.99
Clone 1321
D"2.21
Clone 1321
D"3.82
Clone 1321
D"3.60
Clone 1563
D"2.92
Clone 1321
D"2.57
Clone 1566
D"2.22
Clone 1566
D"3.91
Clone 1028
D"3.62
Clone 1039
D"3.44
discriminated whereas clone 1321 was relatively more heterogeneous. Mahalanobis
values given in Table 5 expressed the distances separating the 10 clones in the
discriminant analysis. The greatest distance was observed between clones 80 377 and
81 455, whereas 1028 and 1563 were the closest clones.
The space occupied by the individuals of a clone in the nine dimensions of the
discriminant analysis represented the polyphenolic "ngerprint of that particular
clone. From the clonal bank thus established, the determination of the clonal origin of
an individual was carried out by projecting its #avonoid data in a subsequent
discriminant analysis. As an example, "ve known individuals were randomly withdrawn from the 101 individuals forming the clonal bank. Two of them belonged to
clones 1039 and the three others to clones 81 455, 1321 and 1028 (Table 6). They were
then projected on a new discriminant analysis composed of the 10 same clones, but
consisting of 96 individuals only. The distances separating the individuals to be
recognized to the center of gravity of the clones in the clonal bank is given in Table 6.
S.S. Kaundun et al. / Biochemical Systematics and Ecology 28 (2000) 779}791
789
The individuals were attached to the clone to which they were closest. With the
exception of individual 4, all the other trees were successfully identi"ed (Table 6).
4. Discussion
The study was based on needle #avonoids in view of proposing a method to verify
the identity of Douglas-"r clones. The experimental protocol used was quite reproducible. Indeed, the variation coe$cients associated with the #avonoid traits did not
exceed 9% in any case. Two proanthocyanidins and six #avonols were detected and
measured in all 101 samples analyzed. The #avonoid patterns were relatively constant
among individuals of the same clone. At the same time, interclonal variability was
su$ciently high to allow good discrimination of the ten clones.
Multivariate discriminant analysis was used to de"ne #avonoid "ngerprints of
each clone and for identifying unknown individuals. Allocation of an individual to
a clone in a discriminant analysis is based on the multidimensional distance separating the individual to the center of gravity of the clones in the clone bank. Thus, the
greater the number of individuals used to de"ne the clonal "ngerprints, the higher are
the chances of allocating an individual to its proper clone. However, for practical
reasons, an optimum number of individuals should be determined for each clone; the
higher the intraclonal variability the larger the number of individuals. In addition,
when an individual to be identi"ed is attached to a clone, this does not necessarily
imply that it belongs to that clone, but rather that it does not belong to the other
clones.
The geographical variability of coastal Douglas-"r has recently been investigated
with needle #avonoids (Kaundun et al., 1998a). The amplitudes of variations for all
#avonoid traits in the latter study were much higher than that observed here. This
suggests that the 10 studied clones were not representative of the #avonoid patternings of the species and that further clones could be discriminated and introduced
simultaneously in the clone bank.
Clones 80 377 and 80 324, which originated from the same Oregon population, were
completely distinguished from one another (Fig. 2). This is an interesting "nding since
the most promising individuals selected for a!orestation in a particular region were
found to originate from a limited zone of the species range (Michaud et al., 1988). On
the other hand, two geographically distant clones, for instance clones 1039 and 1341,
were relatively close to each other. This observation supports several morphoanatomical (Chen et al., 1986) and biochemical (Zavarin and Snagberg, 1975; Yeh and
O'Malley, 1980; Li and Adams, 1989) studies which showed that the intrapopulational
variability of coastal Douglas-"r as much more important than its interpopulational
variability.
As shown on the correlation diagram of Fig. 2, interclonal variability in Douglas-"r
was biogenetically structured. Indeed, axis 1 explained the metabolic balance: B-ring
monohydroxylation (kaempferol) against B-ring trihydroxylation and/or methylation
(myricetin, larycitrin, syringetin and isorhamnetin), whereas axis 2 was negatively
de"ned by B-ring dihydroxylation (quercetin), as well as tannin concentration (total
790
S.S. Kaundun et al. / Biochemical Systematics and Ecology 28 (2000) 779}791
proanthocyanidin and prodelphinidin contents). In this respect, the #avonol metabolism in clones 1358 and 80 377 was more directed towards B-ring trihydroxylation
and/or methylation and less so regarding B-ring monohydroxylation compared to
clones 1028 and 1039. The two former clones were distinguishable upon their degree
of tannin synthesis; clones 1358 and 80 377 being, respectively, characterized by high
and low levels of tannin.
The method of clonal identi"cation has been established upon 13- year old trees. To
become a more interesting tool it should be tested on two-year old nursery samples, in
order to avoid errors in a!orestation. This cheap, rapid and reproducible method
could most probably be extended to other conifers and may constitute an important
tool in monitoring clonal bank management activities.
Acknowledgements
The authors would like to express their gratitude to Mr. T. Fauconnier and Mr. C.
Julien (AFOCEL, Sud-Est) for their help in sample collection and to Mr. J. Reynaud
(University of Lyon), Mr. J.C. Bastien (INRA, Orleans) and Mr. B. Fady (INRA,
Bormes-les-Mimosas) for reviewing the manuscript. The "rst author was supported
by a stipend from the `ReH gion Rho( ne-Alpesa, France.
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