Brief Communications
J. Thors6n, P. E. Jorde, F. A.
Aravanopoulos, U. Gullberg,
and L. Zsuffa
The mode of inheritance of isozyme genes
was studied in the basket willow (Salix viminalis L), a European willow species important for biomass production. Eight enzyme systems representing 11 polymor-
phic isozyme loci (Aat-2, Acp, Gpi-2,
ldh-1, -2, -3, Mdh-1, -3, Pgd-4,
Pgm, and Skd) were examined in three
full-sib families by means of starch-gel
electrophoresis. Mendelian segregation
patterns were observed for all 11 loci.
Tests of joint segregation indicated the
presence of four weakly linked groups
(Acp-Mdh-1, Mdh-3-Skd,
ldh-2-ldh-3,
and ldh-1-Gpi-2).
These results provide
a basis for further research in willows, a
taxonomic group where information on genetics is scarce.
Biochemical genetic markers are becoming increasingly valuable tools in the
breeding of Salix species (Zsuffa and Aravanopoulos 1989). Willow species are an
important crop for short rotation intensive culture biomass plantations dedicated to feedstock and energy. In particular,
Salix eriocephala in North America and 5.
viminalis in Europe have emerged as the
most Important species for such plantations. The latter species is the focus of
this article.
Isozyme markers have been used extensively in various fields of woody plant research, such as systematics (Wheeler et
al. 1983), population genetics (Hamrick
1989; Lagercranz and Ryman 1990), characterization of breeding populations (Moran et al. 1989), monitoring of breeding
144
techniques (Adams et al. 1988; Yazdani et
al. 1989), and identification of clones
(Bergman 1987; Rajora 1988). Studies in
woody angiosperms include Citrus spp.
(Torres et al. 1985), Malus X domestica
Borkh. (Weeden and Lamb 1987), Prunus
avium L (Santi and Lemoine 1990), Juglans
spp. (Arulsekar et al. 1985), Alnus spp.
(Bousquet et al. 1988), Fagus sylvatica L.
(Kim 1979), and Quercus spp. (Guttman
and Weight 1989). Within Salicaceae most
isozyme studies have been conducted in
the genus Populus (Hyun et al. 1987a,b;
Muller-Starck 1992; Rajora 1986, 1988,
1989, 1990; Weber and Stettler 1981). In
the genus Salix, published isozyme studies are limited to the Longifolia section
(Aravanopoulos 1989, 1992; Brunsfeld et
al. 1991) and S. eriocephala (Aravanopoulos 1992).
To take full value of electrophoretically
detectable variation for genetic studies
the mode of inheritance must be known.
Inheritance studies are important for two
reasons. First, such data are needed for a
correct genetic interpretation of phenotypic patterns when these are confounded
by environmentally induced variation
(Conkle 1971; Kelley and Adams 1977), deviation from codominant gene expression
(Goodman et al. 1980; Millar 1985; Weeden
and Wendel 1989), or the presence of complex multilocus systems (Goodman et al.
1980). Second, inheritance studies are required for detection of non-Mendelian segregation patterns, such as segregation distortion (Strauss and Conkle 1986) and
linkage, which may violate assumptions
commonly made in applications of single
and multilocus systems to genetic analysis. Recently Aravanopoulos (1992) has reported the detection of linked isozyme
genes in two North American willow species, 5. eriocephala (two linkage groups)
and 5. exigua (three linkage groups).
Linkage analysis of isozymes could also
be the first step in developing a genetic
map in Salix. In Sweden we are going to
produce detailed linkage maps of Salix viminalis L and Salix dasyclados Wimm. using isozymes and DNA markers like RFLPs
and AFLPs (Vos et al. 1995). DNA markers
potentially offer much better genomic coverage than isozymes, but currently require
greater expense and effort. These genetic
maps will aid future breeding by identifying markers for important major and minor genes, allowing efficient selection at
such loci. Linkage maps will also contribute to our understanding of the evolution
of the karyotype in willows, as it has in
conifers (e.g., Muona et al. 1987)
This is the first report from an ongoing
study of the breeding structure of natural
populations of Salix viminalis L. The objective of this population study is to measure the distribution of morphological,
phenological, and genetic characters over
a large part of the natural range of this
species. These data will be utilized for the
construction of intensive breeding programs for biomass production in Sweden.
In the present study we analyze the pattern of inheritance and linkage in 11 out
of 13 isozyme loci so far found to be polymorphic in 5. viminalis.
Materials and Methods
Plant Material
Three full-sib families originating from unrelated female and male parental clones
were included in the investigation. These
were family I: P213 x P125; family II: F78
x P321; and family III: F66 X F85 (clone
designations In Gullberg 1989).
In selecting parental clones for the
above families, we chose individuals that
were heterozygous in many loci to gather
as much information of inheritance and
linkage as possible. All crossings were
done in a greenhouse. Branches with catkins were collected from the female and
male clones, placed in water, and kept at
20°C. Pollen was collected and the female
flowers were pollinated with the appropri-
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Inheritance and Linkage of
Isozyme Loci in the Basket
Willow (Salix viminalis L.)
Table 1. Enzyme systems assayed
Enzyme
Abbr.
Aspartate amlnotransferase
Acid phosphatase
Glucose-6-phosphate Isomerase
Isocltrate dehydrogenase
Malate dehydrogenase
Phosphogluconate dehydrogenase
Phosphoglucomutase
Shlldmate dehydrogenase
AAT
ACP
GPI
IDH
MDH
PGD
PGM
SKD
Structure
EC no.
Buffer
Dimerlc
Dlmerlc
Dimerlc
Dlmerlc
Dimerlc
Dlmerlc
Monomerlc
Monomerlc
2.6.1.1
3.13.2
5.3.1.9
1.1.1.42
1.1.U7
1.1.1.44
5.4.2.2
1.1.1.25
A
A
D
C
B
B
C
B
Nomenclature of the enzymes follows the guidelines and recommendations of the International Union of Biochemists Nomenclature Committee (IUBNC 1984). See text for electrophoretlc buffer descriptions.
Electrophoresis
The first two to three fully grown leaves
from the top of each shoot were collected
and homogenized in approximately 250 \L\
cold extraction buffer (Coulhart and Denford 1982) using a power-driven pestle.
For isocitrate dehydrogenase and phosphoglucomutase isozymes, we used the
vegetative extraction buffer II of Cheliak
and Pitel (1984). Microglass beads were
added during homogenization in order to
facilitate tissue breakage. The homogenate
was centrifuged for 10 min in a refrigerated centrifuge at 7,000 rpm and then stored
at — 70°C until electrophoretic analysis.
Electrophoretic separation techniques
followed Cheliak and Pitel (1984) and Lagercranz et al. (1988), using 12.5% starch
gels. The separation buffer systems used
for the various enzymes (Table 1) were
•Buffer A (Ashton and Braden 1961).
Stock I: 0.191 M boric acid and 0.05 M
LiOH, pH 8.1. Stock II: 0.051 M Tris and
0.008 M citric acid, pH 8.1. Gels were made
using stocks I and II in proportions 1:9;
electrode buffer was undiluted stock I.
•Buffer B (Clayton and Tretiak 1972).
Gel: 0.002 M citric acid. Electrode: 0.04 M
citric acid. Both buffers were adjusted to
pH 6.5 with N-(3-aminopropyl)-morpholine; 30 mg NAD+ per 100 ml buffer were
added to the gel and to the anodal tray
buffer.
•Buffer C (Aravanopoulos 1992; modified
from Clayton and Tretiak 1972). Gel: 0.001
M Tris and 0.003 M citric acid, pH 6.7. Electrode: 0.223 M Tris and 0.086 M citric acid,
pH 6.3. Both buffers were pH adjusted
with N—(3-amlnopropyl)-morpholine; 30
mg NADP+ per 100 ml buffer were added
to the gel and to the anodal tray buffer.
•Buffer D (Cheliak and Pitel 1984). Gel:
0.01 M histidine-HCl and 0.28 mM EDTA,
adjusted to pH 7.0 with 1 M Tris. Electrode: 0.125 M Tris adjusted to pH 7.0 with
1 M citric acid.
Enzyme staining recipes were from Cheliak and Pitel (1984) with some modifications, mainly in relation to the adoption of
the "agar-overlay" technique (Harris and
HopMnson 1976). Tissue extractions and
enzyme electrophoresis of the parental
clones were repeated at least twice for
each clone. This procedure was also done
for a random subset of progenies from all
three crossings. In every case the zymograms obtained proved to be stable and
repeatable.
Genotypes of all individuals were inferred from the interpretation of the observed banding patterns based on the
quaternary structure of proteins (Table 1;
Harris and Hopkinson 1976; Weeden and
Wendel 1989), progeny phenotype distribution, and comparisons with related species. For multilocus enzyme systems, the
most anodal (i.e., the fastest migrating)
isozyme was designated as controlled by
locus 1 and additional loci were numbered
(2, 3, etc.) in the cathodal direction. Similarly, within each locus, the allozymes
were lettered in the anodal to cathodal direction with lowercase letters (a, b, c,
etc.).
Subcellular Location of Isozymes
Isolation and purification of mitochondria
was carried out after Boutry et al. (1984),
with some modifications as described in
Hikansson et al. (1988). Isolation of chloroplast was carried out as a step during
the mitochondria isolation: after the first
slow centrifuging the supernatant was decanted into fresh 50 ml centrifuge tubes
and spun at 4,500 rpm (2,600 g) to sediment the chloroplasts. Chloroplast or mitochondrial pellets were suspended in ex-
Genetic Analysis
The segregation of allozymes among offspring of controlled crosses was analyzed
by testing its conformity with a Mendelian
mode of inheritance using the chi-square
test for goodness-of-fit. Differences in segregation ratios over families was tested,
whenever possible, using the heterogeneity chi-square test (cf., Sokal and Rohlf
1981).
Independent assortment of pairs of loci
was tested by using the contingency chisquare test (Sokal and Rohlf 1981). Linkage can only be detected in families where
at least one of the parents is a double heterozygote. In cases where one of the parents was a double heterozygote and the
other a single heterozygote involving a
third allele (e.g., ab/ab x aa/ac), this third
allele gives no further information about
linkage and was therefore ignored. Contingency chi-square tests were thus performed on the resulting 2 x 2 tables.
The recombination fraction (r) was estimated from two-locus genotype counts
in progenies as the fraction of putative recomblnants over total number of analyzed
Individuals. In the absence of knowledge
of parental gametic phase, these putative
recombinants were defined as the two
complimentary genotypes with least observed numbers. For all crosses reported
here, the resulting estimate is also the
maximum likelihood estimate of r (Mather
1938:47-48). The standard error (SE) of r
was computed from the binomial variance,
that is, where n is the number of progenies.
Results and Discussion
Inheritance Analysis
The results of the single-locus inheritance
analysis are presented in Table 2. Comparisons with inheritance studies in other
species concentrate on woody angiosperms of the family Salicaceae.
Aspartate aminotransferase (AAT). Two
well-defined zones of activity were observed on gels stained for this enzyme
(Figure 1A). An additional weak band was
sometimes observed between the two major zones. In the slowest migrating (i.e.,
least anodal) zone, three distinct phenotypes were observed: two single-banded
phenotypes (one of which overlapped
Brief Communications 1 4 5
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ate male clone. Seeds were sown in a
greenhouse, and seedlings from families II
and III were sampled after 6 weeks. The
crossing of family I was made a year earlier and their offspring planted outdoors.
For electrophoresis, cuttings were taken
from dormant branches, planted in a
greenhouse and sampled after 2 weeks.
We analyzed 68 offspring from family I and
80 offspring each from families U and III.
traction buffer before electrophoresis was
carried out. Putative subcellular localization of isozymes was based on the relative
enzyme activity detected in mitochondria,
chloroplast, and cytosolic extracts.
Table 2. Parental clones and progeny genotype distribution for all loci and families
Family
Cross
Aat-2
I
II
III
1
1!
Ill
in
ab X bb
bbx bb
bbx bb
be x ab
bbx cc
bexbb
bbx bb
ab x aa
ab x aa
aa x aa
ab x bb
ab x bb
aa x aa
ac x ab
ac x aa
bbxbb
bbx be
bbx bb
aa X ac
aa x aa
aa X aa
ab x bb
aa X aa
aa x aa
i
cc X ce
n
in
cc x cc
be x cc
aa x aa
bcx cc
Acp
Gpi-2
I
n
in
Idh-l
i
II
ui
ldh-2
i
II
in
ldh-3
i
II
in
Mdh-1
i
n
in
Mdh-3
PgcH
Pgm
i
II
i
II
m
Skd
i
II
in
be
be
cc
cc
Progeny genotypes
aa
ab
ac
bb
32
34
13
22
80
19
32
68
36
be
cc
0.06
df
P
1
ns
—
13
3.63
3
ns
39
0.69
1
ns
0.46
1
ns
1.02
0.06
1
1
ns
5.20
2.45
3
1
ns
ns
0.45
1
ns
1
ns
1
ns
1
ns
3
1
1
ns
ns
ns
42
—
68
35
37
24
33
14
44
35
26
16
47
68
43
80
38
37
—
0.94
30
—
—
80
37
0.53
31
—
—
—
—
80
X ab
X cc
X ac
X cc
ns
41
38
0.11
—
9
14
37
23
19
29
37
42
6.81
0.97
0.32
—
Single-locus segregation In the progenies of controlled crosses was tested against expected numbers with x1 goodness-of-fit test (df = degrees of freedom; ns = not significant at the 556 level). A dash (u—") Indicates no data for
this particular cross.
with the weak band) and a three-banded
phenotype displaying both these bands
and an additional band centered between
them.
We conservatively interpreted AAT to
be coded for by two loci in the leaves of
S. viminalis. The first locus (Aat-f) was
invariant, whereas the second locus
(Aat—2) segregated for two codominant alleles (a and 6). The three-banded phenotype of the presumed heterozygote individuals is in accordance with the pattern
expected for this dimeric protein.
We made two different crosses involving
Aat-2 (Table 2). In family I we crossed a
heterozygote (ab') with a homozygote
(bb), obtaining progeny of the parental
types only, in numbers not significantly
different from the 1:1 ratio expected under
Mendellan segregation (x2, = 0.06 with P
> .05). In family III two identical homozygotes produced progeny of the parental
type only. These results are in accordance
with Mendelian inheritance at the Aat-2
locus.
One AAT locus has been reported for 5.
eriocephala and 5. exigua (Aravanopoulos
1 4 6 The Journal of Heredity 1997.88(2)
1992). One to four AAT loci were observed
in Populus spp. (Cheliak and Dancik 1982;
Farmer et al. 1988; Hyun et al. 1987b; Rajora 1990).
Acid phosphatase (ACP). One zone of activity was observed in gels stained for
ACP. The observed zymogram pattern suggests that this dimeric protein is coded for
by a single locus (Acp) segregating for
three alleles—a, b, and c (Figure IB).
We made two crosses involving Acp. In
family I two heterozygote parents (6c and
ab) produced progeny of both parental
types and two recombinant types in numbers not significantly different from the expected 1:1:1:1 ratio (x23 = 3.63 with P >
.05; Table 2). In family III a heterozygote
(be) was crossed with a homozygote (bb)
and the progeny contained the parental
types in a ratio that was not significantly
different from 1:1 (x2, = 0.69 with P> .05).
Mendelian inheritance in ACP coding
loci has also been verified in 5. eriocephala
and S. exigua (Aravanopoulos 1992). One
ACP coding locus was also reported in the
Salix species of the Longifoliae section
(Brunsfeld et al. 1991). On the other hand,
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Locus
two ACP loci were reported in 5. eriocephala and S. exigua (Aravanopoulos 1992)
and P. trichocarpa (Weber and Stettler
1981).
Glucose-6-phosphate isomerase (GPl).
Two zones of activity were observed on
gels stained for this dimeric enzyme (Figure 1C). Both zones were variable, but the
faster (i.e., the most anodal) zone was
poorly resolved and therefore excluded
from further considerations. We interpreted GPI to be coded for by at least two loci,
of which the second locus (Gpi-2) is polymorphic for two codominant alleles (a and
b) in the present material. A third allele, c,
was found in material not used in our genetic analysis but is included in the figure.
We made two crosses involving Gpi—2.
In family I the parental clones were both
homozygote 66 and the progeny revealed
the parental type only. In family II a heterozygote (ab) was crossed with a homozygote (aa). The progeny showed both the
parental types in a ratio not significantly
different from the expected 1:1 ratio (x2,
= 0.46 with P > .05; Table 2).
Two Mendelian loci coding for GPI were
also found in the willows of the Longifolia
section (Aravanopoulos 1992; Brunsfeld et
al. 1991), in 5. exigua (Aravanopoulos
1992), and in Populus spp. (Cheliak and Pitel 1984; Hyun et al. 1987b; Muller-Starck
1992; Rajora 1990).
Isocitrate dehydrogenase (IDH). Two major zones of activity were observed on gels
stained for this dimeric protein (Figure
ID). The slower zone displayed a complex
variability pattern with a total of five distinct bands distributed over eight different
phenotypes (Figure 2). Four phenotypes
displayed all five bands, differing only in
the relative staining intensities between
them. Two phenotypes displayed the
three most anodal bands only, whereas
two other phenotypes displayed the three
least anodal bands only. In all cases, phenotypes with the same set of bands differed only in the relative staining intensities among bands.
The simplest possible model to account
for this banding pattern seems to be that
of a single locus with two alleles in which
additional (artifact) bands are formed (cf.,
Harris and Hopkinson 1976). However, this
hypothesis was rejected on the basis of
progeny phenotype distribution in both
families II and III (x22 = 11.475 with P <
.01, and x2i = 5.128 with P < .05, respectively; data not shown).
With this simple model refuted, we postulate that the slower IDH zone is coded
for by two loci (ldh-2 and Idh-J) segre-
B
— Aat-1
— Acp (a)
— Acp jb)
— Acp (c)
— Aat-2 (a)
_ Aat-2 (b)
bcbbacbbccbbbbbbbccc
Genotypes
a£>
aa
bo
Acp
Aat-2
Genotypes
— ldh-1 (a)
— ldh-1 (b)
— Gpi-1
— Gpi-2 (a)
— Gpi-2 (b)
— Gpi-2 (c)
bbbcbbbbbb abtftabDcoc
Genotypes
Gp-2
bbabbbabbbabbbab
abaaacabtcacbcaa
b e b b b b b b b e t c bbbc
Genotvoes
ktt-1
Mh-2
kJh-3
Figure 1. Starch-gel zymograms of eight enzyme systems In the leaves of Salix viminalis. "O" designates the
origin of migration of Isozymes. Designation of loci and alleles are shown. (Some of the genotypes shown are from
a population screening study.)
cates the scoring and interpretation of
phenotypes, but it also provides a tool of
high discriminating power for identification of S. viminalis clones. Both the dimeric structure of IDH and the formation of
intergenic heterodimers has been observed and verified in a series of other
taxa (Gottlieb 1982; Weeden and Wendel
1989).
Three IDH coding loci were also observed in 5. eriocephata (Aravanopoulos
1992), compared to two to four loci in Populus spp. (Rajora 1986; Muller-Starck 1992;
Viquez-Lopez 1988). Mendelian inheritance in IDH coding loci has been reported
for S. eriocephala and Populus (see references above).
Malate dehydrogenase (MDH). One extensive zone of activity was observed on
gels stained for this dimeric enzyme (Fig-
ure IE). Six different phenotypes were easily distinguished regarding band numbers
and band intensities; in particular, two
five-banded, two six-banded, and two seven-banded phenotypes were observed
(five of which are shown in the figure).
We interpreted MDH to be coded for by
three loci in the leaves of S. uiminalis. The
first locus (Mdh-f) is polymorphic for
two alleles (a and c), the second locus
(Mdh -2) is invariant, and the third locus
(Mdh-3) is polymorphic for two codominant alleles (a and b) in the present study.
An additional allele (6) in Mdh-] has been
found elsewhere. The c allozyme in Mdh-]
partially overlaps with the Mdh-2 band.
The three-banded phenotypes of the presumed heterozygote individuals in both
Mdh—] and Mdh —3 are in accordance with
the dimeric structure of this protein. Het-
Brief Communications
147
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gating for a total of three alleles. Two of
these alleles (6 and c) are apparently
shared between both loci. The three alleles a, b, and c produce homodimeric
products, identified as the most anodal,
the median, and the least anodal band, respectively. Heterodimeric products form
within (with the apparent 1:2:1 staining ratio) as well as between loci. These latter
(intergenic) heterodlmers apparently stain
less intensely than either the intragenic or
the homodimeric products, forming the
basis for assigning single-locus genotypes.
Some bands overlap in electrophoretic
mobility, thereby intensifying the staining
at such positions.
At locus Idh -2 we crossed two different
heterozygote parents (ac and ab), obtaining progenies of both parental types as
well as two recombinant types, as expected (Table 2; family II). The observed proportion of progeny genotypes did not significantly deviate from the 1:1:1:1 ratio expected under Mendelian segregation (x23
= 5.20, P > .05), although a slight excess
of nonparental types was present in our
sample. A second cross (Table 2, family
III) was between a heterozygote (ac) and
a homozygote (ad) parent resulting in
progeny of parental types only in proportions not significantly different from the
expected 1:1 ratio (x2, = 2.45 with P >
.05).
At locus Idh—3 we crossed a homozygote with a heterozygote (family II), obtaining parental types only, in the expected 1:1 ratio (x2, = 0.45 with P > .05). In
two additional crosses, between two identical homozygotes (family I and III), only
the parental type was observed in the
progeny.
Unlike the complexity of the slow IDH
zone, the fast migrating zone revealed two
phenotypes only, one single-banded and
one triple-banded with the middle band
more intensely stained. This zone was assumed to be coded for by a single locus
(Idh-1) segregating for two alleles. We
crossed a single-banded individual with a
triple-banded individual (in both family U
and III) obtaining the parental types in the
expected 1:1 proportion (x2, = 1.02 and x2,
= 0.06, respectively, with P > .05 in both
crosses). No significant heterogeneity was
observed for ldh-\ among the two families (x2, = 0.76, P > .05; data not shown).
These results support the Mendelian inheritance at IDH coding loci in 5. uiminalis.
The presence of two different loci that
share allelic positions in the lower zone of
IDH activity and the occurrence of overlapping intergenic heterodimers compli-
1 4 8 The Journal of Heredity 1997:88(2)
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Pgd—2). No variation was found in this
zone. The slower migrating zone also consisted of three bands and was presumed
to be coded for by loci Pgd-3 and Pgd-4.
At locus Pgd—4, a total of four alleles have
been found during a population screening.
— Mdh-1 (a)
The present investigation involves only
— Mdh-2+Mdh-1 (c)
two of these (alleles b and c; the three ge— Mdh-3 (a)
notypes shown in Figure IF).
— Mdh-3 (b)
We made one cross involving Pgd-4,
namely be x cc (family III; Table 2). The
progeny of this cross contained the parenbb ab ab ab aa aa ab aa aa aa
tal types only in numbers not significantly
different from the expected 1:1 ratio (x2,
_ Pgd-4 (b)
= 0.11 with/>> .05).
— Pgd-4 (c)
Three PGD coding loci were found in the
willows of the Longifoliae section (Aravanbcbbbobcbcccccccccbc
Pgd-4
opoulos 1992; Brunsfeld et al. 1991) and in
Genotypes
S. eriocephala (Aravanopoulos 1992). Mendelian inheritance has been reported in S.
exigua and 5. eriocephala (Aravanopoulos
— Pgm (a)
1992). Two to five PGD loci were reported
— Pgm (b)
j ,
in different poplar species, and Mendelian
— Pgm (c)
"
inheritance was verified for some of them
(Hyun et al. 1987b; Muller-Starck 1992; Rajora 1990).
Phosphoglucomutase (PGM). One zone of
activity was observed in gels stained for
this enzyme. The observed zymogram pat— Skd (a)
tern
suggests the presence of a single lo— Skd (b)
cus (Pgm) segregating for three alleles—a,
— Skd (c)
b, and c (Figure 1G). The two-banded patterns of the presumed heterozygote individuals are in accordance with the monomeric structure of this protein.
We made one cross involving Pgm (famoc be ab be
Pgm
ily III; Table 2). In this cross, two different
Genotypes
heterozygote parents (be and ab), produced four progeny types (ab, ac, bb, and
Figure 1. Continued.
6c) in numbers not significantly different
from the 1:1:1:1 ratio expected under Mendelian segregation (x23 = 6.81 with P >
erodimers are formed between the pri- cal homozygotes, resulting in progeny of
.05).
mary products of all three loci.
the parental type only. Thus the results of
all crosses are in accordance with MenWe made two different crosses involving
One Mendelian locus coding for PGM
Mdh-1 (Table 2). In family I an aa homo- delian Inheritance of MDH coding loci in 5. was also reported in S. eriocephala and 5.
oiminalis.
zygote was crossed with a heterozygote
exigua (Aravanopoulos 1992) as well as in
(ac). The progeny showed both parental
Three MDH coding loci were also re- Populus spp. and hybrids (Muller-Starck
types in a ratio not significantly different
1992). Up to three PGM coding loci have
ported in S. eriocephala (Aravanopoulos
from the expected 1:1 (x2, = 0.94 with P >
been described in various Populus species
1992), while four loci were found in the
.05). In family III both parental clones were
willows of the Longifoliae section (Aravan- (Cheliak and Dancik 1982; Farmer et al.
homozygote aa, and so were all their prog- opoulos 1992; Brunsfeld et al. 1991), and 1988; Rajora 1990).
enies, as expected. At Mdh—2 the proge- four to six loci were described in Populus
Shikimate dehydrogenase (SKD). One
nies of all crosses revealed the parental
spp. (Farmer et al. 1988; Rajora 1990). zone of activity was observed on gels
(homozygote) type only. We made two dif- Mendelian inheritance of MDH coding loci
stained for this monomeric enzyme. The
ferent crosses involving Mdh —3 (Table 2). has been reported in Populus (Rajora
observed zymogram pattern suggests the
In family I we crossed a heterozygote (ab)
1986; Muller-Starck 1992).
presence of a single locus (Skd) segregatwith a homozygote (bb) and the progeny
Phosphogluconate dehydrvgenase (PGD). ing for three alleles—a, b, and c (Figure
revealed the parental types in numbers
1H).
Two major zones of activity were obnot significantly different from the 1:1 ra- served on gels stained for this dimeric enWe made two crosses involving Skd. In
tio expected under Mendelian segregation
zyme (Figure IF). The fastest migrating
family I and II a homozygote individual
2
(X , = 0.53 with P > .05). A second cross
zone consisted of three distinct bands, in- (cc) was crossed with a heterozygote in(family ID) was made between two identi- ferred to be coded by two loci (Pgd-1 and
dividual (different in each family; Table 2).
IDH
ldh-1 (a)
ldh-1 (b)
ldh-2 (a)
ldh-2,3 (b)
ldh-2,3 (c)
ab
ab
be
bb
aa
bb
ab
ac
bb
bb
ab
bb
ab
be
be
bb
ac
be
ab
be
bb
bb
aa
be
ldh-1
ldh-2
ldh-3
Genotypes
Figure 2. Schematic representation of banding pattern and assignment of loci and alleles In the IDH enzyme
system of Salix viminalis. "O" designates the origin of migration of Isozymes.
The progeny of both crosses revealed the
respective parental types In numbers that
did not significantly differ from the expected 1:1 ratio (x2, = 0.97 with P > .05
and x2i = 0.32 with P > .05, respectively).
Two SKD coding loci were reported in 5.
eriocephala and S. exigua (Aravanopoulos
1992; Mendellan segregation observed in
one locus) and in Populus (Rajora 1990).
On the other hand, Muller-Starck (1992)
reports only one locus, Mendelian inherited, in Populus spp. and hybrids.
Preliminary Results for Subcellular
Localization of Isozymes
Sometimes very weak activity was detected in chloroplast extract for the enzymes.
However, that activity may have resulted
from contamination. On the other hand,
the weak activity may have occurred because the number of chloroplasts was too
small to create significant activity. In the
mitochondrial extract some of the enzyme
loci showed activity, but we could not exclude the possibility that this could have
resulted from some contamination. No
data for subcellular location of loci in Salix
has been reported previously (cf., Aravanopoulos 1989, 1992; Brunsfeld et al. 1991),
precluding the comparison across species
at the present time.
Linkage Analysis
Among the 11 variable loci reported in this
study there were 23 pairwise tests of in-
dependent assortment possible (where
one pair of loci was tested twice). For family I we tested Aat-2 against Acp, Mdh-3,
and Skd; Acp against Mdh—1, Mdh—3, and
Skd; and Mdh-3 against Skd. For family II
we tested Idh -1 against Gpi -2 and Idh -2;
ldh-2 against Gpi-2, Idh-3, and Skd; and
ldh-3 against Skd. For family III we tested
Acp against ldh-1, ldh-2, Pgd-4, and
Pgm; ldh-1 against ldh-2, Pgd-4, and
Pgm; ldh-2 against Pgd-4 and Pgm; and
Pgd-4 against Pgm.
We found four cases with significant deviations from independent assortment
among the 22 different locus pairs tested.
These were between Acp and Mdh —1 (family I: r = .313; X2, = 9.689 with P < .01),
Mdh-3 and Skd (family I: r = .364; x\ =
4.735 with P < .05), Idh -2 and Idh -3 (family II: r = .388; x2, = 4.073 with P < .05; ),
and between ldh-1 and Gpi—2 (family II:
r = .364; x 2 , = 6.053 with P < .05).
In conclusion, four possible linked locus
pairs were found in 5. viminalis: AcpMdh-1, Mdh-3-Skd, ldh-2-ldh-3, and
ldh-l-Gpi-2.
These pairs presumably
mark four different physical linkage
groups (chromosome segments), but the
possibility of statistical type I errors must
be recognized, especially since three tests
were only weakly significant. Also, the
complex phenotypic expression of the loci
Idh -2 and Idh -3 are very difficult to score
(cf., Figure ID), and this may have confounded the linkage analysis for this locus
From the Department of Plant Breeding Research, Box
7003, Swedish University of Agricultural Science, S-750
07 Uppsala, Sweden (Thorsen and Gullberg), the Division of Population Genetics, Stockholm University,
Stockholm, Sweden (Jorde), and the Faculty of Forestry, University of Toronto, Toronto, Canada (Aravanopoulos and Zsuffa). Address correspondence to Dr.
Thorsen at the address above. We acknowledge the financial assistance provided from the National Board
for Industrial and Technical Development (NUTEK),
Sweden (to U.G.), and from the International Energy
Agency's Bloenergy Agreement, Canada (to LZ). During the course of this study FAA. was further supported with an Ontario Graduate Scholarship.
The Journal of Heredity 1997:88(2)
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