Directory UMM :Data Elmu:jurnal:T:Tree Physiology:Vol16.1996:

Tree Physiology 16, 547--555
© 1996 Heron Publishing----Victoria, Canada

Relationship between nuclear DNA markers and physiological
parameters in Sitka × interior spruce populations
STEVEN C. GROSSNICKLE, BEN C. S. SUTTON, RAYMUND S. FOLK and
ROBERT J. GAWLEY
Forest Biotechnology Centre, B.C. Research Inc., 3650 Wesbrook Mall, Vancouver, B.C. V6S 2L2, Canada

Received March 7, 1995

Summary Eight populations of Sitka spruce (Picea sitchensis (Bong.) Carr.) and interior spruce (Picea glauca (Moench)
Voss × Picea engelmannii Parry ex. Engelm.) seedlings were
sampled from a zone of Sitka--interior spruce introgression in
British Columbia, Canada. Restriction fragment length polymorphisms of the nuclear ribosomal RNA genes (rDNA) were
used to define species-specific hybridization patterns for the
Sitka spruce and interior spruce populations. Hybridization
was estimated from an index based on the relative abundance
of polymorphic rDNA combining bands for each population.
Sitka × interior hybrid seedlings had an index value for the
relative abundance of interior spruce rDNA (Si-rDNA) ranging

from 0.07 (Lower Nass; the most westerly collected source) to
0.95 (Bulkley Valley low-elevation seed orchard). During shoot
elongation, osmotic potential at saturation (Ψsat ) and turgor loss
point (Ψtlp ) increased, whereas total turgor (Ψ PTotal ) decreased.
After bud set in the summer and throughout the fall, Ψsat and
Ψtlp decreased, whereas Ψ PTotal increased. At all times of year,
populations with a higher Si-rDNA index had lower Ψtlp and
Ψsat and higher Ψ PTotal than populations with a lower Si-rDNA
index. During the fall, Sitka × interior hybrid seedlings exhibited a seasonal decline in the temperature causing 50% needle
electrolyte leakage (LT50) and in the critical temperature indicating the initial point of freezing injury. Seedlings with a
higher Si-rDNA index had lower LT50 and critical temperature
values indicating greater freezing tolerance in the fall.
Throughout most of the year, seedling population Si-rDNA
index was related to the degree of drought and freezing tolerance.
Keywords: freezing tolerance, nuclear DNA markers, Picea
engelmannii, Picea glauca, Picea sitchensis, water relations
parameters.

Introduction
Introgression zones are areas where tree species mix genes

through repeated crossing and back crossing. The Nass Skeena
Transition in British Columbia, Canada, is a zone where introgressive hybridization occurs between Sitka spruce (Picea
sitchensis (Bong.) Carr.) and interior spruce (Picea glauca

(Moench) Voss × Picea engelmannii Parry ex. Engelm.) (Little
1953, Daubenmire 1968, Roche 1969). Sitka spruce occurs
naturally in wet, maritime climates that do not experience
extreme winter freezing conditions, whereas white spruce and
Engelmann spruce grow in continental areas that experience
summer droughts and severe winters (Burns and Honkala
1990). The exact extent of the introgression zone between
these species is unclear and there is little information on
representative genotypes. There are risks associated with using
seed from the Sitka × interior transition zone that has unknown
genetic characterization because this might result in progeny
being planted off site, thereby resulting in poor survival or
growth.
Yeh and Arnott (1986) examined spruce introgression by
comparing isozyme alleles with morphological characteristics
to calculate genetic distance between seedlots. Recently, DNA

markers have been used to identify polymorphisms within a
given taxon (Wagner 1992) and to identify distinct populations
that occur within introgression zones. Use of chloroplast DNA
to estimate species components of Sitka and interior spruce
hybrid seedlots gave good agreement with growth patterns and
seedling morphological development (Sutton et al. 1991a). In
addition, restriction fragment length polymorphisms (RFLP)
of nuclear ribosomal RNA genes (rDNA) have identified species-specific hybridization patterns for Sitka and interior
spruce populations (Sutton et al. 1994). Intermediate rDNA
patterns between these two species were found in their introgression zone and were used to generate an index that represents the contribution of each species to the nuclear genome
(Sutton et al. 1994).
Successful reforestation of the Nass Skeena Transition
would be facilitated by use of seedlings with appropriate
adaptive traits. Not only do populations need to be identified,
but information is also needed on how the rDNA index relates
to seasonal physiological patterns of Sitka × interior genotypes. There have been few attempts to use genetic markers as
indicators of physiological patterns in conifers (Mitton 1995);
however, the use of genetic markers that are related to physiological traits that respond to environmental factors could
provide an opportunity to improve the matching of genotypes

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GROSSNICKLE, SUTTON, FOLK AND GAWLEY

to site conditions. Although tightly linked RFLP markers are
used in marker-aided selection (Tauer et al. 1992) in advanced
breeding programs, these markers are not suitable for discriminating among a wide range of populations. For this purpose,
genetic markers are needed that provide a rapid measure of
population differences in physiological traits.
We have assessed the ability of DNA analysis to characterize
seasonal physiological profiles of Sitka × interior spruce hybrids collected from sites in the Nass Skeena Transition zone.
The specific objectives of the study were to: (1) characterize a
range of Sitka × interior spruce hybrid populations by DNA
analysis, (2) characterize the seasonal drought and freezing
tolerance profiles of these populations, and (3) examine the
relationship between DNA analysis and the seasonal drought
and freezing tolerance profiles.
Materials and methods
Plant material
Eight seed sources were obtained from locations across the

Nass Skeena Transition in British Columbia, Canada (Table 1;
a map identifying collection sites is given in Sutton et al.
1994). Four seed sources were from wild seed collections
(1004, 4075, 4132 and 14657) in three seed zones. One seed
source was an operational seedlot pooled from the entire Bulkley Valley seed orchard (Seedlot 6308). Two seed sources were
from open-pollinated families in the Bulkley Valley seed orchard (each seedlot was pooled in groups of five families based
on the locations of original parent trees). One seed source came
from a pool of six crosses between interior and Sitka spruce
sources. One seed source comprised full-sib F1 crosses derived
from crosses between selected interior spruce parent trees from
Prince George (PG) combined with Sitka spruce from the
Queen Charlotte Islands (QCI) or Vancouver Island (VI) (i.e.,
PG4 × VI187, PG21 × VI130, PG135 × QCI266, PG4 ×
QCI242, PG135 × VI161, PG42 × VI58).
Sitka × interior spruce seedlings were grown in 615 Styroblock containers under an intensive nursery culture from
March until September 1992 at the Saanich Nursery Facility,
B.C. Ministry of Forests, British Columbia (48°28′ N,
124°10′ W). All seedlings had set bud by mid-September and
populations ranged in height from 47.9 ± 1.2 to 36.9 ± 1.3 cm,
and diameter from 7.8 ± 0.2 to 6.9 ± 0.2 mm. After bud set,

seedlings were maintained outdoors to acclimate to fall environmental conditions.
Field site
Seedlings were planted in mid-November 1992 at the Plant
Sciences Field Reseach Facility, University of British Columbia (49°15′ N, 123°15′ W, 25 m elev.). The field site has a
homogeneous cultivated sandy loam soil. Seedlings were
planted at a 1.0 × 1.0 m spacing with ten rows per seed source
randomly located across the site, with 15 seedlings per row.
Within each row, seedlings used for drought tolerance measurements were randomly selected from each seed source population and planted in 11-liter plastic containers that were then
buried within each row. Seedlings used for freezing tolerance

measurements were randomly selected from each seed source
population and tagged. Site environmental conditions were
monitored daily throughout the study. Fall mean daily air
temperatures were calculated for periods between freezing
tolerance tests. Precipitation was not recorded on site, though
a nearby (5 km) Environment Canada weather station reported
typically high amounts of precipitation from January through
June, moderate amounts from July through September, and
high amounts from October through December. Vegetation
around the seedlings was cut on a regular basis to remove

aboveground competition.
Sampling procedure
Bud break, shoot elongation and bud set patterns were monitored during the growing season. Because of seedling size,
physiological measurements and DNA analysis were taken
from two separate samplings of the same population. Eighteen
seedlings from each seed source were repeatedly measured for
seasonal physiological patterns. At each drought tolerance
sampling, measurements alternated between two sample
groups, each of six seedlings per seed source. Fall--early winter
freezing tolerance patterns were measured on a third group of
six seedlings from each seed source. From the test population
used for physiological measurements, DNA analysis was conducted on 12 randomly selected seedlings from each seed
source.
DNA Analysis
Each DNA analysis was made on three buds from each selected
seedling as described by Sutton et al. (1994). The DNA was
extracted by digestion with restriction enzyme, HindIII, followed by electrophoresis and Southern blotting. Hybridization
was carried out with yeast 18S rRNA gene. Autoradiograms
obtained with the 18S rRNA gene probe were quantified by
image analysis. For each individual, the total optical density

(OD) for each band was recorded relative to total OD in the
lane (to correct for differences in DNA loading) and then
compared to the relative intensities of the bands in pure interior
or pure Sitka spruce populations as determined by Sutton et al.
(1994). A maximum of five bands was observed in the interior
spruce species, two of which were absent in Sitka spruce. For
pure interior spruce, optical densities (i.e., %OD per band) of
bands one through five were: 2.70 ± 1.72, 55.87 ± 5.30, 13.78
± 2.10, 5.82 ± 1.30, and 21.92 ± 1.99, respectively. For pure
Sitka spruce, optical densities of bands one through five were:
39.82 ± 2.46, 16.54 ± 2.87, 43.66 ± 1.97, 0, and 0, respectively.
The average of the ratios for all five bands per tree was used to
calculate an index of interior spruce (Si) rDNA in the nuclear
genome from the equation: Si-rDNA Index = Σ((%ODx −
%ODSs)/(%ODSi − %ODSs))/5, where %OD is the relative band
intensity for tree x, interior spruce (Si), or Sitka spruce (Ss).
Although the Si-rDNA index gives an overall estimate of
hybrid nature, the index does not account for possible segregation of the observed bands in more advanced hybrid populations. In practice we found that variation in the Si-rDNA index
among mature trees from one location gave rise to standard
errors of about 0.05 to 0.10 ( Sutton et al. 1994).

RELATIONSHIP OF DNA MARKERS TO PHYSIOLOGICAL PARAMETERS

Drought tolerance
Pressure-volume data were collected as described by Grossnickle (1989). Seedlings in 11-liter containers were removed
from the field and rehydrated in the dark at 22 °C in a controlled environment. After 14 h in the dark, lateral shoots were
removed from the potted seedlings and their saturated weight
measured. The seedlings were returned to the field, within 24 h
of being sampled, and placed in their original location. Pressure-volume curves were determined by periodic measurements of shoot mass and water potential. Between
measurements, shoots transpired in a controlled environment
room (air temperature = 22 °C, relative humidity = 50% and
PFD = 300 µmol m −2 s −1). Dry weights of shoots were measured after oven drying at 65 °C for 48 h. Shoots consisted of
one-year-old needles for pressure-volume analysis in February, one-year-old and current-year needles in March and April,
and current-year needles from May through November. Data
were collected at 4- to 8-week intervals from February through
November.
Pressure-volume curves were used to determine osmotic
potential at saturation (Ψ sat ) and turgor loss point (Ψtlp ) and
relative water content at turgor loss point (RWCtlp) as outlined
by Schulte and Hinckley (1985). Höfler diagrams were used to

determine total turgor (Ψ PTotal , MPa) (Roberts and Knoerr
1977, Grossnickle 1988, Colombo and Teng 1992).
Freezing tolerance
Freezing tolerance of needles was monitored every two weeks
from September until mid-December. At each sampling, needles were removed from three branches on the middle third of
the stem and subjected to a freeze-induced electrolyte leakage
(FIEL) procedure (Burr et al. 1990). Needles from each seedling were cut at both ends into 0.5 cm lengths, washed in
deionized water and transferred, in random groups of 24, to
glass culture tubes containing 0.5 ml of deionized water. One
tube from each seedling was stoppered and placed in ice water
as a control at 1 °C. Four tubes from each seedling were placed
in an ethanol bath at −2 °C (Forma Scientific MC-8-80). Water
in all tubes in the ethanol bath was nucleated simultaneously
with ice crystals after 0.5 h, and tubes were stoppered. The
ethanol bath was then cooled at 5 °C h −1.
Four temperatures were selected to bracket the anticipated
50% tissue electrolyte leakage value. When one of the selected
temperatures was reached, a tube for each seedling was removed and the contents were allowed to thaw in ice water.
After the contents of all tubes had thawed, 5.5 ml of deionized
water was added to each tube. Tubes were then stoppered and

placed on a 100-rpm shaker at 24 °C for 20--24 h. Conductivity
of the solution in each tube was measured after incubation.
Tubes were then placed in a 90 °C water bath for 15 min to
induce maximum tissue injury and conductivity was remeasured after an additional 20 h on a 100-rpm shaker at 24 °C.
Measured FIEL values were converted to an index of injury
(II) (Dexter et al. 1930, Flint et al. 1967, Burr et al. 1990):
II =

1 − ( T1 /T2)
100,
1 − ( C1 /C2 )

549

where T1 and T2 are the conductivity of treatment tubes after
freezing and after boiling, respectively, and C1 and C2 are the
conductivity of control tubes before and after boiling, respectively. Temperatures at which 50% needle electrolyte leakage
occurred (i.e., LT50), were calculated from a linear regression
model for each seedling.
For each measurement period, a critical temperature, defined as the highest temperature at which freezing injury can
be detected, was determined for each Sitka × interior spruce
population by a modification of the procedures described by
DeHayes and Williams (1989). On each measurement date, a
one-way analysis of variance was conducted for each Sitka ×
interior spruce population (n = 6); the ANOVA table included
a relative conductivity value for each temperature and an error
term with 4 and 25 degrees of freedom, respectively. For each
population, the mean square error (MSE) and error degrees of
freedom (df) were used in the Dunnett’s equation to determine
a critical value, d′:
d′ = t√

2MSE/df,

where t is a statistical value (2.65) der ived from a t-table.The
calculatedd′ value was added to the needle relative conductivity value for the control sample of each seedling within each
Sitka ×inter iorsprucepopulation.Individualseedlingcritical
temperatures were then calculated by interpolating between
twotesttemperaturesthathad relative conductivitiesthatwere
bracketingthecriticalrelativeconductivity.
Statistical analysis
For all tests we used a randomized experimental design with n
= 6 for each seed source. For each test time, a one-way analysis
of var iance was conducted to compare the responses of the
eight seed sources: for clar ity, only four seed sources, showing
the full range of sources, are presented in the figures. When
there were significant treatment differences (based on the
one-way analysis of variance), a Pearson product moment
coefficient of correlation was conducted using all eight seed
sources to measure linear relationships between an ecophysiologicalparameter(y var iable) and the rate of Sitka ×interior
sprucehybridization,Si-rDNA index,(x var iable). Because of
atypical freezing tolerance patterns in the full-sib F1 crosses,
data are shown but not included in the correlation analysis for
freezing tolerance parameters with other spruce hybrid populations. Regression analysis of freezing tolerance in relation to
mean daily air temperature in the fall was conducted on data
from four seed sources.

Results and discussion
DNA Analysis
Sitka × interior spruce seed sources had an interior spruce (Si)
rDNA index ranging from 0.07 (Lower Nass) for the most
westerly seed source to 0.95 for the low-elevation Source No. 5
from the Bulkley Valley (Table 1). Among the eight sources
tested, only seedlings from the Kitwanga location (Source

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GROSSNICKLE, SUTTON, FOLK AND GAWLEY

Table 1. Summary of seed source information and the interior spruce rDNA index (mean± SE; n = 12) of each Sitka × interior spruce seed source.
Source

1
2
3
4
5
6
7
8
1
2

Seedlot

1004
4075
4132
14657
6308
Five OP families
Five OP families
Full-sib F1 Crosses

Seed zone1

NST
BLK
SM
NST

Location

Elevation (m)

Kitwanga, 55°20′ N, 128°05′ W
Morice West, 54°10′ N, 127°15′ W
Lower Nass, 54°58′ N, 129°38′ W
Meziadan Lake, 56°06′ N, 129°19′ W
Bulkley Valley, 53°30′ N, 55°40′ N,127° W
Morice Lake, 54°20′ N, 127°30′ W
North Babine, 55°35′ N, 126°40′ W

305
760
10
290
840
840
840

Si (rDNA) index
MT2

Seedlings

0.35 + 0.06
0.80 + 0.08
0.01 + 0.04
0.44 + 0.05
-0.80 + 0.08
0.96 + 0.10
--

0.43 ± 0.05
0.94 ± 0.04
0.07 ± 0.02
0.36 ± 0.04
0.95 ± 0.05
0.79 ± 0.08
0.84 ± 0.02
0.63 ± 0.05

NST = Nass Skeena Transition, BLK = Bulkley Valley, and SM = Spruce Maritime.
MT = Mature trees. Surveyed by Sutton et al. (1994).

No. 1) exhibited a large variation in Si-rDNA index between
the drought tolerant (0.51 ± 0.08) and freezing tolerant (0.35 ±
0.07) populations (Grossnickle and Sutton, unpublished data).
The spruce seedling populations had similar Si-rDNA index
values to the mature trees that were surveyed previously from
the same approximate locations (Sutton et al. 1994, and Table 1).
Seed from the full-sib F1 crosses had an Si-rDNA index of
0.63, indicating that the contribution from each of the parental
species to the nuclear genome was not equal. Based on estimates of rDNA copy number from hybridization intensities,
Sitka spruce has a copy number of approximately 80% that of
interior spruce. The Si-rDNA index averaged over all F1 seedlings measured was 0.58 ± 0.16 (n = 22), which may reflect the
difference in copy number. Another possibility is that some
individuals may not be hybrids, as a result of errors in pollination. However, when Sutton et al. (1991) analyzed representatives of these crosses using chloroplast probes the

expected inheritance pattern was observed in every case, indicating that it is unlikely that errors have occurred in the generation of F1 hybrids.
Drought tolerance
Before bud break (Julian Day (JD) 47), seedlings from all Sitka
× interior spruce populations had low osmotic potentials at
saturation (Ψ sat ) (Figure 1) and turgor loss point (Ψtlp ) (Figure 2). During bud break and at the start of shoot elongation
(JD 90 to 140), Ψ sat and Ψtlp increased in seedlings from all
origins (cf. Grossnickle 1989, Colombo and Teng 1992). The
spring period was the only time during the year when osmotic
potentials were lower in Sitka spruce populations than in
interior spruce populations. Seedlings with a higher Si-rDNA
index broke bud earlier than seedlings with a lower Si-rDNA
index (Grossnickle and Sutton, unpublished observations) and
this was reflected in their earlier seasonal increase in osmotic
potential. Osmotic potentials decreased in seedlings from all

Figure 1. Seasonal changes in osmotic potential at saturation
(Ψ sat ) (mean ± SE) of seedlings
from four Sitka × interior spruce
seed sources together with their
corresponding Si-rDNA index.
An asterisk indicates a measurement on that Julian Day is significantly different within all tested
seed sources as determined by a
one-way ANOVA (P = 0.05). The
relationships between Ψ sat (mean
± SE) and the Si-rDNA index
(mean ± SE) for all Sitka × interior spruce seed sources are given
for selected days. Shoot phenological stages are defined by:
(BB) bud break, (SE) shoot elongation and (BI) bud initiation.

RELATIONSHIP OF DNA MARKERS TO PHYSIOLOGICAL PARAMETERS

551

Figure 2. Seasonal changes in osmotic potential at turgor loss point
(Ψ tlp ) (mean ± SE) in seedlings of
four Sitka × interior spruce seed
sources together with their corresponding Si-rDNA index. An asterisk indicates a measurement on
that Julian Day is significantly different within all tested seed
sources as determined by a oneway ANOVA (P = 0.05). The relationships between Ψtlp (mean ±
SE) and Si-rDNA index (mean ±
SE) for all Sitka × interior spruce
seed sources are given for selected
days.

sources throughout the summer (JD 146--201) and early fall
(JD 279) and Sitka spruce populations had higher Ψsat (up to
0.20--0.35 MPa higher) and higher Ψtlp (up to 0.35--0.6 MPa
higher) than interior spruce populations. After bud set and
during fall acclimation, Ψsat and Ψ tlp decreased in all Sitka ×
interior spruce populations (Figures 1 and 2, respectively).
Similar declines in osmotic potential during fall acclimation
have been reported for Sitka spruce (Hellkvist et al. 1974) and
white spruce (Grossnickle 1989, Colombo and Teng 1992).
In all populations at all times of year, there was an inverse
relationship between the Si-rDNA index and the values of
Ψsat and Ψ tlp ; i.e., populations with a high Si-rDNA index had
low values of Ψ sat and Ψtlp (Figures 1 and 2, respectively). The
difference in Ψ sat between the Sitka spruce and interior spruce
populations remained between 0.20 and 0.30 MPa throughout
the year, indicating that there were only minor differences in
active osmotic adjustment between Sitka spruce and interior
spruce populations across the season. On the other hand, the
difference in Ψ tlp between Sitka spruce and interior spruce
populations changed seasonally from 0.50 MPa in the late fall
when seedlings were in a dormant state (JD 279), to 0.30 MPa
during the spring growth phase (JD 146), and up to 0.60 MPa
just after summer bud set (JD 201). Interior spruce populations
had greater seasonal changes in Ψtlp than Sitka spruce populations, indicating a greater capacity for passive osmotic adjustment through the concentration of existing solutes. Ecotypic
differences in osmotic adjustment within a tree species have
been related to soil water availability at the location of origin
of the plant source (Bongarten and Teskey 1986, Abrams 1988,
Collier and Boyer 1989, Russell 1993).
All seedlings had higher total turgor (Ψ PTotal ) in late winter
(JD 47) than at other times of year, and seedlings from populations that were more interior in origin had higher values than
more coastal populations (Figure 3). During bud break and
shoot elongation (JD 90--140), ΨPTotal decreased in seedlings

from all origins (Figure 3). As a result of the decrease in
Ψ PTotal during shoot elongation, seedlings were susceptible to
water stress during this period (Grossnickle 1988, Grossnickle
and Major 1994). During shoot elongation (JD146), the interior and Sitka spruce populations had similar ΨPTotal values.
Total turgor increased in all seedlings during the summer and
fall, and the increase was greater in the interior spruce populations than in the Sitka spruce populations. By JD 201 and 279,
the differences in ΨPTotal between the Sitka spruce and interior
spruce populations were 5.0 and 9.0 MPa, respectively. A
similar increase in ΨPTotal occurs after bud set in white spruce
(Grossnickle 1988, Colombo and Teng 1992). The F1 cross
between interior and Sitka spruce had ΨPTotal values that were
intermediate between those of interior spruce and Sitka spruce
throughout the year.
In summary, interior spruce populations had greater osmotic
adjustment (i.e., lower Ψsat and Ψtlp , Figures 1 and 2, respectively) and greater turgor maintenance capacity (i.e., higher
Ψ PTotal , Figure 3) throughout most of the year than Sitka spruce
populations. Seasonal precipitation patterns for sites across the
Nass Skeena Transition area indicated that precipitation was
high in maritime locations and declined through the mountainous transition area and was lowest at continental locations
(Environment Canada 1980). Differences in drought tolerance
among Sitka × interior spruce populations conformed to these
regional precipitation patterns.
Freezing tolerance
As daylength and air temperatures decreased in the fall, all
Sitka × interior spruce populations exhibited a decrease in the
temperature at which 50% needle electrolyte leakage occurred
(LT50) (Figure 4) and in the critical temperature (Figure 5). A
rapid decline in freezing tolerance of spruce species in the fall
has been observed in many studies (e.g., Cannell and Sheppard
1982, Cannell et al. 1985, Burr et al. 1989, Simpson 1990,

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GROSSNICKLE, SUTTON, FOLK AND GAWLEY

Figure 3. Seasonals change in total turgor potential (Ψ PTotal )
(mean ± SE) in seedlings of four
Sitka × interior spruce seed
sources together with their corresponding Si-rDNA index. An asterisk indicates a measurement on
that Julian Day is significantly different within all tested seed
sources as determined by a oneway ANOVA (P = 0.05). The relationships between ΨPTotal (mean ±
SE) and Si-rDNA index (mean ±
SE) for all Sitka × interior spruce
seed sources are given for selected
days.

Bigras and D’Aoust 1992, Grossnickle et al. 1994).
Throughout the fall, populations with a high Si-rDNA index
usually had lower LT50 (Figure 4) and critical (Figure 5) temperatures than populations with a low Si-rDNA index. In the
early fall (JD 250), there was up to a 10 °C difference in LT50
temperatures between Sitka and interior spruce populations.
As the fall progressed (JD 305 and 347), LT50 values for
interior spruce populations were up to 30 °C lower than those
of Sitka spruce populations. There were large differences in
critical temperature between Sitka and interior spruce populations in early (7 °C on JD 250) and mid (15 °C on JD 305) fall;
however, by late fall (JD 347) the critical temperatures of Sitka

and interior spruce populations were similar. These results
support earlier findings that Sitka spruce develops freezing
tolerance more slowly in the fall than Engelmann spruce or
white spruce (Sheppard and Cannell 1985, Kolotelo 1991) and
that its survival under severe winter conditions is lower (Ying
and Morgenstern 1982). At most measurement times, hybrids
of the Sitka × interior spruce complex had an intermediate
degree of freezing tolerance compared with the Sitka and
interior spruce populations (cf. Ying and Morgenstern 1982,
Sheppard and Cannell 1985, Kolotelo 1991). The difference in
freezing tolerance patterns among spruce species and their
hybrids has been explained on the basis of the theory of

Figure 4. Freezing temperature resulting in 50% needle electrolyte
leakage (LT50) (mean ± SE) of
seedlings of four Sitka × interior
spruce hybrids together with their
corresponding Si-rDNA index. An
asterisk indicates a measurement
on that Julian Day is significantly
different within all tested seed
sources as determined by a oneway ANOVA (P = 0.05). The relationship between LT50 (mean ±
SE) and all Sitka × interior spruce
seed sources in relation to their SirDNA index (mean ± SE) are described for selected days
throughout the fall. Note that full
sib F1 crosses (filled symbols)
were not included in the relationship with other spruce hybrid
populations.

RELATIONSHIP OF DNA MARKERS TO PHYSIOLOGICAL PARAMETERS

553

Figure 5. Critical temperature
(mean ± SE) resulting in initial needle electrolyte leakage of seedlings
of four Sitka × interior spruce hybrids during the fall together with
their corresponding Si-rDNA index. An asterisk indicates a measurement on that Julian Day is
significantly different within all
tested seed sources as determined
by a one-way ANOVA (P = 0.05).
The relationship between critical
temperature (mean ± SE) and all
Sitka × interior spruce seed sources
in relation to their Si-rDNA index
(mean ± SE) are described for selected days throughout the fall. The
full-sib F1 crosses (filled symbols)
were not included in the relationship with other spruce hybrid populations.

polygenic inheritance of hardiness (Ying and Morgenstern
1982), i.e., the combining of hardiness characteristics through
independent acquisition of genetic information derived from
more than one source.
Based on their Si-rDNA index of 0.63, progeny from the F1
cross between Sitka and interior spruce did not fit the patterns
for LT50 and critical temperatures during the fall acclimation
period (Figures 4 and 5, respectively), but exhibited freezing
tolerance characteristics more like those of Sitka spruce. However, their drought tolerance characteristics did conform to
their Si-rDNA index patterns. The origin of parent material for
the F1 hybrids may account, at least in part, for their freezing
tolerance behavior. Seeds from four of the six crosses tested
were derived from Sitka spruce located on Vancouver Island;
which is 4 to 5° further south than the Nass Skeena seed
sources. Thus, these results indicate that the rDNA index also
reflects differences between populations with respect to overall hybrid nature, and this difference is only one factor that
contributes to differences in drought or freezing tolerance
among populations.
With declining mean daily air temperatures in the fall, both
LT50 and critical temperature decreased in all Sitka × interior
spruce populations (Figure 6). The rate of freezing tolerance
acclimation was related to the Si-rDNA index. Among spruce
populations, differences in LT50 values increased as the mean
daily air temperature declined. At a mean daily air temperature
of 6 °C, seedlings with Si-rDNA indices of 0.07 and 0.94 had
LT50 values of −33 and −69 °C, respectively. Differences in
critical temperature among spruce populations was smaller
than differences in LT50 values. All seedlings with an Si-rDNA
index > 0.36 exhibited similar decreases in their critical temperatures, with a critical temperature of around −25 °C com-

Figure 6. Relationship between (a) freezing temperature resulting in
50% needle electrolyte leakage (LT50) (mean ± SE) and (b) critical
temperature (mean ± SE) resulting in initial needle electrolyte leakage
and mean daily air temperature of seedlings from four Sitka × interior
spruce seed sources during the fall. The Si-rDNA index of each seed
source is also presented.

554

GROSSNICKLE, SUTTON, FOLK AND GAWLEY

pared to −17 °C for seedlings with an Si-rDNA index of 0.07,
at a mean daily air temperature of 6 °C. Field sites in the Nass
Skeena Transition zone have average daily temperatures of
between 4 and 5 °C by October, and extreme minimum temperatures range between −8 °C for maritime and −18 °C for
interior locations (Environment Canada 1980). Extreme minimum temperatures in November can drop to −18 °C for maritime and −37 °C for interior locations. From December to
March, extreme minimum temperatures below −40 °C occur
in the coastal--interior transition and interior locations. Spruce
seedlings with an Si-rDNA index of at least 0.35 have a better
chance of withstanding freezing temperatures that cause initial
needle damage. Furthermore, a spruce population with a high
Si-rDNA index can have low seasonal LT50 values and thus
greater freezing tolerance during fall acclimation.
Variation in freezing tolerance among populations of a species that spans coastal and interior ecosystems is not unique to
the transition from Sitka to interior spruce populations. Interior
populations of both Douglas-fir (Pseudotsuga menziesii
(Mirb.) Franco) (Rehfeldt 1977) and western white pine (Pinus
monticola Dougl. ex D Don) (Thomas and Lester 1992) have
greater acclimation to freezing temperatures than coastal
populations.

Conclusions
Our findings confirm previous work by Sutton et al. (1991a,
1991b, 1994) that DNA analysis is an effective way to identify
genotypic differences in Sitka × interior spruce populations
covering the Nass Skeena Transition zone within British Columbia, Canada. Thus, a high Si-rDNA index was associated
with high drought and freezing tolerance. These ecophysiological patterns were consistent throughout the yearly cycle
indicating that screening Sitka × interior spruce populations
based on their Si-rDNA index is a valid selection procedure.
The use of genetic markers provides the opportunity to screen
for a desired genetic makeup without having to subject all
populations to ecophysiological testing. The incorporation of
genetic marker procedures, based on freezing and drought
tolerance patterns, should improve seed transfer guidelines for
Sitka and interior spruce populations (Thomas and Lester
1992).
Acknowledgments
This research was supported by the Science Council of British Columbia (Contract No. 175 (T-5)) and British Columbia Ministry of Forests
(Contract No. 18605-20/SXSS). The authors thank Drs. Shihe Fan,
Kurt Johnsen and John Major for reviewing earlier versions of the
manuscript.
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