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ELSEVIER
scrEilTtA
HORTICUTTURE
Scientia Horticulturae 85 (2000) 2I-35 www.
elsevier.com/locate/sc
ihorti
Genetic relationships
in Chaenomeles
(Rosaceae)
revealed
by isozyme analysis
Larisa P. Gark avaa'b,
Kimmo Rumpunenb,*,
Igor v. Bartishb
ulnstitute of Horticurture, Kiev-27, 252027, (Jkraine, Russia
bBalsgárd - Department of Horticultural Plant Breeding, Swedish (}niversity of Agricultural
sciences, Fjcilkestadsvcigen r23- l, s-29194 Kristianstad, sweden
Accepted 23 August 1999
Abstract
Genetic relationships were studied among four taxa in the genus Chaenomeles using isozyme
analysis' The band patterns obtained with six polymorphic isozyme systems provided 10g reliable
markers, which were scored as unordered multistate traits. A cluster analysis as well as a
multidimensional scaling analysis grouped
the taxa in agreement
with previously published results
obtained with RAPD (random amplified polymorphic DNA) analysis; C. japonica and C.
cathayensis were the most distantly related species, whereas C. speciosc took an intermediate
position together with the hybrid taxon C. x superba. Similarity mairices obtained with isozymes
and RAPDs, respectively, were closely correlated, r:0.74. The previously noted low level of
RAPD variability in C. cathayensis was indicated also with isozymes. However, the isozym e data
were less efficient than the RAPD data for intraspecific grouping of the genotypes according to the
origin of the plant material. @ 2000 Elsevier Science B.V. All rights r"r"ru"j.
Keywords: Rosaceae; chaenomeles; Genetic diversity; Isozymes; RApD; euince
1. Introduction
Domestication of the genus Chaenomeles (Maloideae, Rosaceae) as a
horticultural crop has recently been initiated in Europe and in Asia. Various
species
and species
hybrids of Chaenomeles
are
now grown for the
production
of
_-corTooondino
altfhor Tp|. J:ÁÁ ÁÁ 1<<aa. c^--. I Ar A
L.P. Garkava et al./Scientia Horticulturae 85 (2000) 2I-35
valuable
aroma
compounds and an acid
juice in Latvia and
Lithuania (Rumpunen
et al., 1998)
and China (Wang et al., 1997, 1998).
The large amount of high
quality polysaccharides
in the fruits makes this crop a promising candidate also
for the manufacture
of marÍnalades and pectins provided that well adapted, high
yielding varieties can be developed
All Chaenomeles
species are
diploid,2n : 34, and
presumed
to be outcrossing
but
the
mating system is not yet well studied. Interspecific hybridizationproduces
fertile offspring in all combinations so far tried, and several hybrid taxa have
consequently
been described
(Weber,
1964).
A strong self-incompatibility system
prevails in C. japonica (Rumpunen
et al., 1998). There is also some evidence of
self-incompatibility in C. speciosa and in various hybrids (Weber, L964).
A large collection of Chaenomeles
genotypes
has
been
gathered
at Balsgárd -
Department of Horticultural Plant Breeding, Sweden, from orchards and
botanical gardens around
the world to provide a basis for a joint North-European
plant breeding program. Three species, C. japonica (Thunb.) Lindl. (Japanese
quince), C. speciosa (Sweet) Nakai (flowering quince), and C. cathayensis
(Hemsl.)
Schneider
(Chinese
quince)
as well as the hybrid taxon C. x superba are
represented
in this collection. The fourth Chaenomeles species mentioned
in the
most recent check list of the subfamily Maloideae (Phipps et al., 1990), C.
thibetica Yi.i, has unfortunately
not yet been
lrcorporated ur this plant collection.
The present study investigates
the feasibility of using isozyme analysis for
characterizatian of genetic diversity among taxa, accessions, and individual
plants of Chaenomeles. only one minor isozyme study haŠ
previously been
reported in Chaenomeles (Ponomarenko, 1990). We also aimed to compare
isozyme data with previously published RAPD data on the same plant material
(Bartish et al., 1999),
and to estimate
the efficiency of these two marker systems.
2. Materials and methods
2.1. Plant material
A total of 42 genotypes were investigated representing four taxa in
Chaenomeles:
C. cathayensis,
C. japonica, C. speciosa and C. x superba. The
plant material used (Table 1) was the same as described in Bartish et al. (1999)
except
for genotypes
RG 4-37 and RG 4-48
(replaced
by RG 4-43 and RG 4-54),
RG 4-29 and
RG 4-30
(replaced
by RG 4-126 and
RG 4-I25).The new genotypes
were from the same accessions as the previously analysed plants which had
unfortunately
been killed by winter frost. The plant material was obtained
from
the Chaenomeles core collection maintained at Balsgárd - Department of
Horticultural Plant Breeding. One-year-old shoots were taken in the field in
January when dormancy had been terminated.
Phloem tissue was sampled from
L.P Garkava
et al-/Scientia
Horticulturae
85 (2000)
2l-35
Table 1
Plant accessions of Chaenomeles
Taxon Accession Symbol Genotype, origin
C. cathayensis
C. cathayensis
C. cathayensis
C. speciosa
C. speciosa
C. x superba
C. x superba
C. x superbo 'Pink Lady'
C. japonica
C. japonica
C. japonica
C. japonica
C. japonica
C. japonica
o
o
V
V
A
L
10
11
T2
Z
!
!
I
E
13
I4
1
4
5
6
2
a
J
A
x
RG 4-371125, RG 4-481126 Botanisk Have,
Denmark
RG 4-58, RG 4-66 Botanischer Garten,
Essen, Germany
RG 4-29143, RG 4-30154 Botanischer
Garten, Stuttgart, Germany
RG 4-79,
RG 4-80, RG 4-83, RG 4-89
Hortus Zoologius Pragensis, The Czech
Republic
RG 1-8, RG 1-10 Kyoto Takeda Herbal
Garden, Japan
RG 2-28, RG 2-30, RG 2-32, RG 2-42, RG
2-43 Botanischer Garten, Stuttgart, Germany
RG 4-119 Botanischer Garten, Stuttgart,
Germany
BG 1-l The Elite Plant Station, Sweden
RG 3-57,
RG 3-58,
RG 3-59,
RG 3-62,
RG
3-63, RG 3-64 Botanical Garden, Uppsala,
Sweden
BE 4-22, BE 7
-33 Domesticated
population,
Babtai, Lithuania
RG 5-31,
RG 5-34,
RG 5-36,
RG 5-37,
RG
5-39, RG 5-40 Domesticated
population,
Smiltene, Latvia
RG l-2I Domesticated
population,
Smiltene, Latvia
RG 1-134, RG 1-142, RG 1-145
Sendai
Botanical Garden, Japan
RG 4-21, RG 4-35, RG 4-41,
RG 4-50, RG
4-60 Salaspils Botanical Garden, Latvia
debarked
shoots whereas
young leaves were obtained
from shoots that had been
forced in a greenhouse
at l5-20oC for 3-4 weeks.
2.2. Enzyme extraction
Fresh phloem tissue (1 g) or leaves (1 g) were ground with liquid nitrogen in
pre-cooled mortars and 3 ml of extraction buffer was added. Extraction from
phloem tissue was carried out according to Boscovic et al. (1994). Extraction
from leaves
was carried out with an 0.05 M sodium phosphate
buffer (adjusted
to
pH 7.2 with HCI), containing 0.03%o Dl-dithiothreitol (DTT), 0.075Vo B-
24 L.P. Garkava et al./Scientia Horticulturae 85 (2000) 21-35
mercaptoethanol,
O.I7o ascorbic acid, 4.5Vo polyvinylpyrrolidone and 8.0Vo
sucrose.
For both
kinds of tissue,
samples were
centrifuged
(7000
rpm) for 20 min
at Z"C and the supernatant
was collected and kept at -20"C until used.
2.3. Electrophoresis
Isozyme analysis was caffied out by electrophoresis
in polyacrylamide gels
(16cm x 18cm, 15 wells,0.75mm thick).
Density
gradient
gels,
5.8-1I.8Vo,
were prepared
according to Boscovic et al. (1994) and 1.57o
gels were prepared
according
to Davis (1964).
Electrophoretic separation
was carried
out at 4"C with
a running
time of 3.51h at 30mA on LKB 200I vertical electrophoresis
unit
with LKB 2003 power supply.
All samples
were electrophoresed
twice.
2.4. Gel staining , i
The enzyme assay procedures for acid phosphatase
(ACP, EC 3.I.3.2),
superoxide
dismutase
(SOD,
EC 1.15.1.1),
malic enzyme
(ME,EC 1.1.1.40),
and
esterases
@ST, EC 3.1.1.-)
were as described
by Wendel and Weeden (1989).
Alcohol dehydrogenase
(ADH, EC I1.I.1), alkaline phosphatase
(AKP' EC
3.I.3.1),
glucose-6-phosphate
dehydrolenase
(G-6-PDH,
EC 7.1.L49),
glucose-
6-phosphate
isomerase
(GPI, EC 5.3.1.9),
glutamate dehydrogenase
(GDH' EC
I.4.I.2), glutamate oxaloacetate
transaminase
(GOT, EC 2.6.1.D, isocitrate
dehydrogenase
(IDH, EC 1 .7.I.42),
malate dehydrogenase
(MDH, EC 1
J.1.37),
shikimate
dehydrogenase
(SKDH, EC I :.1.25), phosphogluconate
dehydrogen-
ase (PGD, EC 11.I.44) and phosphoglucomutase
(PGM, EC 5.4.2.2)
were
stained
according
to Vallejos
(1983).
Peroxidase
(PRX, EC 1.11.1.7)
was stained
with 0.0917o
benzidine in 0.32
M sodium acetate
buffer adjusted
to pH 5.4, and
bands
were revealed
with 0.0IVo
hydrogen
peroxide. A11 isozyme systems
except
SOD were stained in the dark at 31-38"C. SOD were stained
under light after
dark treatment
at the same temperature
as the other systems.
Gels stained for
PRX, ACP, EST and GOT were fixed tn 957o
ethanol and
30Vo
glycerol, whereas
gels stained for the other enzyme systems were fixed in 407o glycerol when a
suitable
band intensity had been attained.
2.5. Data analysis
Data were collected immediately when gels had been fixed. Only clear and
consistent
isozyme bands were scored.
Band patterns from each zone of activity
were numbered in ascending order starting
with the fastest
migrating band for
each enzyme
system
and scored
as unordered
multistate
traits (Beer et al., 1993;
Strefeler
et al.. 1996a,b). Thus a matrix for genotypes
and isozyme systems
was
L.P. Garkava et al./Scientia Horticulturae 85 (2000) 21-35 25
SPSS data analysis
package
(Norusis, 1990) and NTSYS-pc (Rohlf, 1991)
were
used for the statistical analyses. Cronbach's a (procedure
reliability, SPSS) was
used to investigate the raw data and remove unreliable bands before further
statistical treatment.
Jaccard's coefficient of similarity was calculated for all pairwise comparisons
between
individual samples to provide a distance
matrix. A cluster
analysis based
on average linkage between groups (unweighted
pair group method algorithm,
UPGMA) was constructed
based on this matrix, and a dendrogram
was produced.
A multidimensional scaling analysis (MDS) was conducted
(procedure
alscal,
SPSS) using a genetic distance matrix obtained from the binary data set with
negated and rescaled
(0-1) values for Jaccard's
coefficient of similarity. A scaling
solution with three dimensions was used in our study.
Correspondence between the two distance matrices obtained here with
isozymes and previously with RAPDs (Bartish et al., 1999), respectively,
was
analysed with the MXCOMP procedure (NTSYS) which produces the Pearson
product-moment
correlation,
r, and
the Mantel test statistic,
Z.If the two matrices
show similar relationships, Z is large in comparison to chance expectation.
The
estimated Z was compared with its permutational
distribution obtained
from 9999
random samples of all possible permutations
of the matrices.
A combined matrix was also produced,
including the present
isozyme data as
well as the previously obtained RAPD data. The MXCOMP procedure
was used
on each data set separately as well as on the combined data set to obtain the
cophenetic correlation between the UPGMA matrix and the distance matrix as a
measure
of goodness
of fit for the cluster analysis.
2.6. Experimental error
The fact that four C. cathayensis
genotypes
had to be replaced in the present
isozyme study due to death of plants used in the RAPD study,
may have biased
some of the results. However, this bias is probably insignificant since C.
cathayensis
appears
to be the least variable species, and since the new genotypes
were selected from the same accessions as the original plants.
3. Results
Initially, starch gel electrophoresis
was used for some systems but sufficient
resolution was only obtained
for MDH and ACP. For other
systems the resolution
was poor or there was no activity. Moreover, the isozyme patterns
obtained for
MDH and ACP with starch gel electrophoresis were less polymorphic than the
patterns
obtained with polyacrylamide gels. Therefore, only data derived from
L.P. Garkava et al./Scientia Horticulturae 85 (2000) 21-35
3.1. Polymorphism of isozyme
systems
Thirteen (ACP, ADH, AKP, EŠr GDH, GoT G-6PDH' IDH, MDH, PGM,
PRX, SKDH and SOD) of the 16
isozyme systems
initially tested
showed
enzyme
activity. Based on these preliminary investigations,
four systems (ACP, EST,
MDH and PRX) were selected
which consistently
yielded good resolution,
clear
bands and
reproducible
profiles
both for phloem and
leaf tissue.
In addition,
PGM
was selected
for analysis of leaf extracts
and GOT was selected
for analysis of
phloem extracts (Table 2). Other isozyme systems did not result in sufficient
resolution or produced inconsistent band patterns. For PRX, a discontinuous
electrophoretic
system was used while for ACP, EST GOT, MDH and PGM a
gradient system
resulted
in the best resolution.
More than one zone of activity was observed for all the selected isozyme
systems.
These zones of activity were probably the result of different isozyme
loci but this was not analysed furthel. i
Acid phosphatase. Four monomeric zones and one dimeric zone of activity
were observed
for ACP in the phloem extracts.
Twelve bands
were scored
in the
monoÍTlpric
zones but no sufficiently clear bands could bb scored
in the dimeric
zone.
All C. cathayensls
genotypes had the same
ACP profile,
but
this system
was
polymorphic for the other taxa. { '
Leaf extracts
yielded four monomeric zones that seemed
homologous to the
zones produced by phloem extracts.
However, with leaf extracts
one additional
fast migrating zone was obtained. Leaf extract-derived
bands in the dimeric zone
stained
faintly. Altogether 14 polymorphic bands were selected
for scoring from
leaf extracts.
Esterases. u-esterase
activity was detected
in both leaf and phloem extracts
whereas B-esterase
activity was detected
only in the phloem extracts.
Table 2
Number of total bands and number of polymorphic bands for each of the six isozyme systems
studied
Isozyme system Total bands Polymorphic bands
Leaves Phloem Leaves Phloem
ACP (EC 3.r.3.2)
EST
(EC
3.1.1.-)
GOT
(EC
2.6.r.r)
MDH (EC 1.1.1.37)
PER
(EC 1.11.1.7)
PGM (EC 5.4.2.2)
15
I7
8
1'.7
6
LZ
12
10
9
19
14
I4
7
15
6
12
tz
10
9
I6
<o
L.P. Garkava et al./Scientia Horticulturae 85 (2000) 2I-35 27
Two cathodal zones and one anodal zone of o-esterase activity were observed
in the phloem extracts.
In total, seven clear and consistent bands were scored, of
these six were in the cathodal zone.
All C. cathayensis
genotypes
had the same
cathodal u-esterase
profile, which differed from profiles of other species. All C.
speciosa
and C. x superba
genotypes
had one intensely stained
s-esterase band in
the
anodal
zone. The C. cathayensis
genotypes
also had this band,
but stained less
intensely.
Only three
out of 23 genotypes
of C. japonica had this band.
B-esterase
showed two zones of activity in the phloem extracts, one fast
migrating and one slowly migrating. The slowly migrating zone had poor
resolution and was not taken into account for this study. In total five polymorphic
bands were scored. C. cathayensis had one monomorphic band in the fast
migrating zone, whereas C. x superba and C. japonica genotypes
had another
band. C. speciosa genotypes
each had two or three
bands in this zone.
At least seven zones of a-esterase activity were observed in the leaf extracts.
Six of these zones together
yielded 17 well separated
bands, but the three fastest
migrating bands were monomorphic. Thus, fourteen polymorphic bands were
scored.
Glutamate oxaloacetate
transaminase. Ten bands representing
four zones were
found for GOT in the
phloem extracts. Leaf extracts
yielded only one active zone.
Malate dehydrogenase.
Up to four zones
of MDH activity were observed in the
phloem extracts and nine bands were scored, all of them
polymorphic. In the leaf
extracts,
eight bands were well separated and seven
of these were polymorphic.
Peroxidase.
Seven zones (PRX-1-PRX-7) and 19 bands of peroxidase
activity
were observed
in the phloem extracts. The same seven zones were found also in
the leaf extracts but here the activity in PRX- 1 and PRX -2 zones was very low.
PRX-5 and PRX-6 zones were monomorphic.
C. cathayensis had one major band
with very high staining intensity in the PRX-4 zone. This band, however, less
intensely stained, was also found in the C. speciosa and C. x superba genotypes
but not in any of the C. japonica genotypes.
Phosphoglucomutase.
Two zones
of activity were observed for PGM in the leaf
extracts,
but only one of them was well resolved.
Six bands were consistent and,
therefore, selected for scoring. PGM activity was found also in the phloem
extracts,
but resolution was not sufficient to allow reliable scoring.
Based on the calculations
of Cronbach's il,, seven out of 115 initially scored
bands (6Vo)
were removed from our data set. The remaining bands resulted
in a
matrix of 42 genotypes
and 108 bands,
representing
six isozyme systems, which
were then used for the further analvses.
3.2. Multivariate analyses
The cophenetic correlation for the cluster analvsis was hish. r: 0.90.
28 L.P. Garkava et al./Scientia Horticulturae 85 (2000) 21-35
major clusters (Fig. 1). C. cathayensis
isozyme phenotypes
appear
to be rather
similar to each other and formed,the
most distant
cluster.
Another major cluster
was formed by C. iaponica. Genoiyp"t of C. speciosa and C. x superba
clustered
together.
Inside th! major clusters,
genotypes
generally grouped
according to the
origin of the plant material.
The three-dimensional
MDS yielded a.plot of dimension 1 vs 2 (Fig. 2) with
three main groups,
which were in good agreement
with the cluster analysis' C'
35 45 55 65 75 85 95 SimilaritY (%)
o RG 4-58
o RG 4-126
o RG 4-43
o BG 4-54
o RG 4-66
O RG 4-12s
V RG 1-8
V RG 1.10
A BG 2-32
n ie z-qz
A BG 1.1
Y BG 4.79
V RG 4.83
v nc +.gÓ
Y RG 4.80
A RG 2.28
A RG 2.30
A RG 2-43
a RG 4-119
v BÉ 4.22
0a BE 7-33
I RG 3-64
x RG 3-59
a BG 3-63
B HG 3.62
B RG 3.58
tr RG 5-31
I RG 3-57
E RG 4.41
E RG 4-50
$ RG 4-21
ts RG 4-35
I RG 1-142
I RG 1-145
I BG 1-134
tr RG 5-40
D BG 5.34
! RG 5-36
tr RG s-37
EB RG 4.60
tr RG s-39
E RG 7-21
Fig. 1. Dendrogram based on upGMA analysis of genetic similarity estimates (Jaccard's
coefficient) for isozyme data, showing relationships among individual plants' The 42 genotypes are
classified
into three
major clusters.
C. cathayensls
(C) form the
most distant
cluster.
Another major
.,.^-*L^l/A\clrrctcr
L.P. Garkava et al./Scientia Horticulturae 85 (2000) 21-35
#
ďr#
It
D
!
!
29
ol
tr
.9
0
tr
o
tr
i5
N
AnV Ý
VV
&
o
Dimension
1
Fig. 2. Plot of MDS analysis of genetic similarity estimates
(Jaccard's coefficient) for isozyme
data, showing groups among individual plants. C. cathayensis
(C) and C. japonica (l) form two
well-separated
groups, whereas C. speciosa (V) and C. x superba (l\) together form a third
intermediate group.
cathayensis and C. japonica fonrred two well-separated groups, whereas C.
speciosa and C. x superba together
formed a third group.
3.3. Comparing isozyme and RAPD data
The Pearson product-moment
conelation coefficient calculated between the
elements of the distance matrices for the isozyme data here presented and
the RAPD data previously reported (Bartish et aL., 1999) was r - 0.74. In
addition,
no estimate of Z from 9999 random permutations of the matrices was
equal
to or larger than the observed
Z (16.9).In conclusion,
the two data
sets
thus
provided highly coffesponding estimates
of genetic relationships among the 42
genotypes.
3.4. Combining isozyme
and RAPD data
When the isozyme and RAPD data sets were combined in one matrix, the
cophenetic
correlation coefficient reached
r - O.9I indicating a very good fit of
30 L.P. Garkava et al./Scientia Horticulturae 85 (2000)
21-35
Similarity (%)
o RG 4-66
o RG 4-30/54
o RG4-29143
o RG 4-58
o RG4.37lí25
o RG4-48/126
V RG 4.83
Y RG 4-79
a RG4-119
A RG 2.28
A RG 2.30
A HG
2-43
V BG 4-89
V RG 4-so
V RG1.í0
V RG1.8
A RG 2.32
L Rcz-42
A BG1-1
N RG7.2í
fl RG 5-34
tr RG s-37
Ú HG
5-36
fl BG 5-40
tr RG 5-39
Ú| BE 4.22
n BE7-33
tr RG 5-3í
B RG 3-59
tr RG 3-64
El RG 3-63
B RG 3-57
I RG 3-58
El RG 3-62
E RG 4.60
E RG 4-50
E RG 4-41
E BG 4.35
E RG4-21
I RGl-142
I RG1.í45
I BG1-134
Fig. 3. Dendrogram based on UPGMA analysis of genetic similarity estimates (Jaccard's
coefficient) for the combined data set (isozymes and RAPDs), showing relationships among
individual plants. The dendrogram is in good agreement with the taxonomy, and the genotypes in
most cases cluster according to accession or postulated
relatedness.
C. cathayensis
(C) form the
most distant cluster. Another major cluster is formed by C. japonica (J). Genotypes of. C. speciosa
(V) and C. x superbc (A) cluster together
except for one accession of C. speciosa (Y) which is
split in two groups of which genotype RG 4-83 and RG 4-79 form an intermediate group to C'
cathayensis.
agreement with the taxonomy, and the genotypes in most cases clustered
according to accession or postulated relatedness. However, one accession
(number 10) of C. speciosa was split in two different groups in the combined
data set (Fis. 3) whereas in the isozvme data
set (Fig. 1) the four genotypes
(RG
L.P. Garkava et al./Scientia Horticulturae 85 (2000) 21-35
4. Discussion
Six isozyme systems were found that are useful for characterization
of genetic
resources
and to investigate
genetic relationships among and within groups of
closely related Chaenomeles taxa. In addition, some of the initially tested
isozyme systems could probably also yield useful data with further improvement
of the methodology. Some other isozyme systems, that have been used
successfully in apples (Chevreau et al., L999), pears (Chevreau et al., 1997)
and different Prun s species (Mowrey et al., 1990, Mowrey and Werner, 1990;
Granger, 1996), could also be tried on Chaenomeles.
When integrating isozyme band patterns for the same isozyme system for
different tissues (such as leaves and phloem) in the same data set there is a
possibility that results may be biased. Some loci may be expressed in both tissues
and thus could be scored twice. However, the profiles obtained for the different
tissues were clearly different within the isozyme systems studied except for
peroxidase. To completely avoid such bias, only the tissue yielding the most
polymorphic isozyme pattern
should be selected but at the same time valuable
information from additional tissue specific loci may be lost. A better strategy
would thus be to score the tissue specific loci from each tissue together
with the
common tissue
unspecific loci. To perform such an investigation
unambiguously
it is, however,
necessary to have complete knowledge on the inheritance of the
loci. Such information can only be obtained from an analysis of plant material
derived through controlled crosses
(Manganaris and Alston, I992c).
The bands were not scored as loci and alleles since the genetic
background
of
the different isozyme systems employed is not yet known in Chaenomeles.
However, scoring of band patterns
as unordered multistate traits has previously
proved to be useful for studies where loci cannot be unambiguously identified
(Beer et al., 1993, Strefeler et al., I996a,b). Consequently, this approach was
considered appropriate also for the present investigation. In addition, highly
variable
multilocus systems such
as esterases
and acid phosphatase
are difficult to
score
in any other
way since homologous
loci often cannot be identified
between
taxa (Weeden
and Lamb, 1987).
Nevertheless,
these systems are quite useful for
providing numerous,
highly polymorphic bands.
4.
I. Multivariate analysis
Cluster analysis and MDS of the isozyme data yielded rather
similar results.
C.
cathayensis and C. japonica appear
to be the most distantly related species,
whereas
C. speciosa clustered
together
with C. x superba. However,
despite the
large number of isozyme markers
employed in this study,
only some association
was found between origin of the plant material and the intraspecific variabilitv
31
32 L.P. Garkava et al./Scientia Horticulturae 85 (2000) 2l-35
4.2. Comparing isozyme and RAPD data
Our isozyme data partitione&
genetic diversity in Chaenomeles
in almost the
same way as previously reported with RAPD markers (Bartish et a1., 1999).
However,
some differences
were observed.
Isozyme data
yielded a more compact
clustering of C. cathayensis genotypes, thus suggesting even less genetic
variability than indicated with RAPD data. By contrast,
C. japonica genotypes
clustered less densely when isozyme data was used compared to RAPD data.
Both data sets placed C. speciosa and C. x superba close together, and in an
intermediate
position between
C. cathayensis
and C. japonica. However, a more
compact
cluster was obtained for C. speciosa and C. x superba with isozyme data
than with RAPD data. Intraspecific
variation was more closely associated
with
origin of the analysed plant material when based on RAPD data compared to
when based on isozyme data. These findings are in agreement with studies on
several other species
(Heun et al,, 1994: Maass and Klaas, 1995; Staub et al.,
r9e1).
Rather few investigations
have been
published
on thg correlation
between
gene
diversity estimates obtained
with isozymes and RAPDs on woody plants.
RAPDs
are considered to be selectively neutral and to sample
the total genome
randomly .
(Dawson
et al., 1995) but there a.e ďia*backs due,}o..^u-ple, to their dominant
nature.
The distance matrices based on isozyme and RAPD data, respectivelyi
were well correlated (r - 0.74) in our investigation. Complete congruence in
diversity estimates
was found in black spruce
(Picea mariana) when enzyme loci
and RAPD loci were studied in haploid plant tissue, whereas biased estimates
were obtained with dominant RAPD phenotypes
(Isabel
et a1., 1995).
Estimates
on intraspecific
variability should, therefore, be considered as preliminary when
based on unordered isozyme data or on RAPD phenotypes. Moreover, the
sampling effor may be rather large because of unknown genetic relationships
among our genotypes
and because of unknown linkage between
isozyme loci.
4.3. Combining isozyme and RAPD data
Combining isozyme data and RAPD data may not always be appropriate
for
analysis at the interspecific level since co-migrating
RAPD bands from different
species could be non-homologous (Rieseberg, 1996). In our investigation,
the
same argument can be applied
to the
isozymes since
we scored
these
as unordered
multistate traits. However, goodness
of fit for the cluster analyses,
measured
as
the cophenetic correlation coefficient, was even slightly higher when based on
combined data
(r: 0.91)
as compared
to only isozyme data
(r: 0.90)
or only
RAPD data
(r - 0.85). Moreover, the genotypes
of the most studied taxon, C.
innonirn.
clrrstererl
more
closelv according
to accession
when
the combined
data
L.P. Garkava et al./Scientia Horticulturae 85 (2000) 21-35 33
134) formed one cluster, genotypes from Uppsala Botanical Garden, Sweden
(RG4-2I, RG 4-35, RG 4-41, RG 4-50 and RG 4-60) formed a second cluster,
genotypes
from Salaspils Botanical
Garden,
Latvia (RG 3-57, RG 3-58,
RG 3-59,
RG 3-62,
RG 3-63 and RG 3-64)
formed a third cluster, and partly domesticated
plants originating from three fields in Latvia and Lithuania formed a fourth
cluster.
Obviously the mass selection applied in open pollinated populations in
farmers' fields has had an impact on the
plant
material
which, it was claimed,
had
originated
from European botanical gardens
about
three to four generations
ago.
Morphologically the domesticated plant material differed from wild plants by
having almost thornless
twigs.
Based on the combined data set two genotypes
of C. speciosa (accession
number 10) were
clustered in an intermediate
position between
the C. cathayensis
cluster
and the C. speciosa
I C. x superba main cluster whereas
based
on isozyme
data
all four genotypes
in the accession were clustered into one group within the
C. speciosa I C. x superba main cluster. This result demonstrates
the higher
discriminating
power of RAPDs compared
to isozymes since the same separation
was obtained also when only RAPD data were used (Bartish et al., 1999).
The
result indicates
a hybrid origin of the accession which is a possible
consequence
of spontaneous
cross pollination taking place in the botanical garden
from which
the seeds
were obtained.
5. Conclusion
The isozyme analysis
resulted
in almost the same
estimates of genetic
diversity
as previously reported
for RAPD markers. Taking into account the comparatively
low cost of chemicals and the high efficiency and reliability, isozyme analysis
appears to be useful for genetic and taxonomic investigations of the genus
Chaenomeles.
A drawback
is the relatively low number of useful systems,
which
limit the number of polymorphic loci to be studied.
Further investigations
are needed
to clarify the genetic control of the isozyme
systems in Chaenomeles, as previously conducted in several closely related
genera
like Pyrus (Chevreau et al., 1991,1999)
and Malu.s (Weeden
and Lamb,
1987;
Manganaris, 1989;
Manganaris and Alston, r992a,b,c; Manganaris and
Alston, 1997). This would allow the interpretation
of zymograms as alleles and
loci, thus raising the information content considerably.
Acknowledgements
This study was the carried out with financial support
from the Commission of
34 L.P. Garkava et al./Scientia Horticulturae 85 (2000) 21-35
programme,
CT 97-3894, Japanese
quince (C.
japonica) - a new European
fruit
crop for production of juice, flayour and fibre. It does not necessarily reflect its
views and in no way anticipates the Commission's future policy in this area.
Financial support was also received from The Swedish Institute. Valuable
comments on the manuscript were received from H. Nybom.
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