Measurements of solid-wood quality traits and fiber properties traits in the field progeny materials
Two open-pollinated field progeny trials, S21F9021146 (F1146) and S21F9021147 (F1147), were established in 1990 in southern Sweden (Chen et al. 2014). Increment cores were sampled at age 21 years from 12 trees for each of 524 families. The material was analyzed with a SilviScan instrument (Evans 1994, 2006) at Innventia, Stockholm, for radial variations from pith to bark of wood quality traits. The traits measured were wood density (WD), microfibril angle (MFA), modulus of elasticity (MOE), radial fiber width (RFW), tangential fiber width (TFW), fiber wall thickness (FWT), and fiber coarseness (FC; Chen et al. 2014). Fiber length (FL) was measured with a laboratory fiber analyzer, the Kajaani FiberLab at SLU, Umeå (Table 1; Chen et al. 2016).
Table 1 Summary of growth, disease resistance- and wood quality traits
Phenotyping for H. parviporum resistance in the nursery progeny materials
Plant material
Open-pollinated families from 500 mother trees (genotypes) in the Swedish Norway spruce breeding program with an origin from south to central Sweden were sown in 2015. Of these, 243 families were in common with the progenies in the two field trials, S21F9021146 (F1146) and S21F9021147 (F1147). After the first growth season, the seedlings of 446 families with 6–12 progenies were randomized into a complete block design with three replications, where each family was planted in 4-tree row-plots in plastic trays consisting of 24 individual 0.124 L plastic pots. The seedlings were grown for another season in Skogforsk’s experimental forest tree nursery at Ekebo (55°56′53.1″N 13°6′52.2″E) following ordinary watering and fertilization routines. No fungicides were used during cultivation.
Inoculation experiment
To prepare the inoculum, the heterokaryotic H. parviporum Rb175 (Stenlid 1987) grown on Hagem medium (Stenlid 1985) was allowed to colonize 6 mm diameter wood dowels for 6 weeks prior to the inoculation experiment.
At the day of inoculation, the vitality of the seedlings was measured according to the following score: (1) fully vital; (2) some loss of vitality (Fig. S1a) and (3) pronounced loss of vitality (Fig. S1b). To allow the fungus to enter the plant, bark was removed with a 6 mm diameter cork borer at 10 cm from the base of the seedling. A wooden dowel colonized by H. parviporum was fixated at the wound with Parafilm®. The plants were kept under ambient light and temperature in the forest tree nursery and 21 days post-inoculation the seedlings were harvested.
At harvest the diameter at the point of inoculation (D) was measured and the induced defence responses in the phloem were estimated by measuring the lesion length (LL) spread upwards and downwards from the edge of the inoculation point on the inside of the bark. Fungal growth (FG) was measured according to the established protocols (Arnerup et al. 2010; Stenlid and Swedjemark 1988). The inoculated stem was then cut up into 5 mm discs and placed on moist filter papers in Petri dishes. To avoid contamination, the stem was cut from the top towards the point of inoculation and then from the bottom and towards the point of inoculation. After approximately 1 weeks’ incubation under humid conditions, the presence of H. parviporum on the discs was determined by observation under the stereo-microscope (Arnerup et al. 2010; Stenlid and Swedjemark 1988). Samples with no conidia detected on the inoculation plug and a total lesion length of 2 mm or shorter were removed from the analysis as the inoculation was deemed as non-successful (Lind et al. 2014).
Statistical analyses
Due to the deviation from normal distribution of the lesion length data, a natural logarithm was used to transform the data to an approximation of a normal distribution (Steffenrem et al. 2016). Variance and covariance components for genetic analyses were estimated using ASReml4.1 (Gilmour et al. 2015) and the following linear mixed model for nursery data analysis was fitted:
$$y_{ijklm} = \mu + B_{j} + V_{i} + P_{k} + D_{jklm} + F_{l\left( k \right)} + e_{jklm}$$
where \(y_{ijklm}\) is the observation on the mth tree from the lth family within the kth provenance in the jth block and belongs to the ith vitality class (Vi), μ is the general mean, \(B_{j}\), \(V_{i}\), and \(P_{k}\) are the fixed effects of the jth block, the ith vitality and the kth provenance, respectively. The variable \(F_{l\left( k \right)}\) is the random effect of the lth family within the kth provenance, and ejklm is the random residual effect. \(D_{ijklm}\) is a covariate for diameter at inoculation point. Significance of the fixed effects was tested by Wald F-test. Non-significant fixed effects were dropped from the model after preliminary analysis. Estimates of heritability were obtained for each trait using variance components from the univariate analysis. Standard errors were estimated using the Taylor series expansion method (Gilmour et al. 2015).
As there is a good genetic connection between nursery trial and the two field progeny trials (243 common parents), the following linear mixed model for joint nursery and progeny data was fitted:
$$y_{hijklmn} = \mu + S_{i} + B_{j\left( i \right)} + V_{h} + P_{l\left( k \right)} + D_{hijklmn} + F_{{m\left( {l\left( K \right)} \right)}} + e_{hijklmn}$$
where \(y_{ijklmn}\) is the observation on the nth tree from the mth family within the lth provenance in the jth block within the ith trial belongs to hth vitality (Vh), μ is the general mean, \(S_{i}\), \(B_{j\left( i \right)}\), Vh, and \(P_{l\left( k \right)}\), are the fixed effects of the ith trial, the jth block within the ith trial, the hth vitality and the lth provenance within the kth materials (including two types of data, from nursery and progeny), respectively. \(D_{hijklm}\) is the covariate for diameter at inoculation point. The variable \(F_{{m \left( {l\left( k \right)} \right)}}\) is the random effect of the mth family within the lth provenance within the kth materials and ehijklmn is the random residual effect. The random \(F_{{m\left( {l\left( k \right)} \right)}}\) is assumed to be normally distributed with the expectation value at zero and structured as: \(\text{var} \left( {F_{{m\left( {l(k} \right))}} } \right) = \left[ {\begin{array}{*{20}c} {\sigma_{aP}^{2} } & {\sigma_{aPN} } \\ {\sigma_{aPN} } & {\sigma_{aN}^{2} } \\ \end{array} } \right] \otimes I\), where σ
2
aP
, σ
2
aN
, σaPN are the additive variance for progeny data, additive variance for nursery data, and covariance for progeny and nursery data, respectively. The random \(e_{hijklm}\) is assumed to be normally distributed with the expectation value at zero and structured as: \(\text{var} \left( e \right) = I \otimes \left[ {\begin{array}{*{20}l} {\sigma_{eP1}^{2} } \hfill & 0 \hfill & 0 \hfill \\ 0 \hfill & {\sigma_{eP2}^{2} } \hfill & 0 \hfill \\ 0 \hfill & 0 \hfill & {\sigma_{eN}^{2} } \hfill \\ \end{array} } \right]\), where σ
2
eP1
, σ
2
eP2
and σ
2
eN
are the residual variances for progeny trials F1146, F1147, and nursery data, respectively. Preliminary analyses indicated that there was no significant provenance by site effect for all traits and also no any provenance effect for lesion length and sapwood fungal growth. Therefore, these effects were removed in the final model. A two-tailed likelihood ratio test (LRT) against the null hypothesis of genetic correlation of zero was used to check the significance of genetic correlations between disease traits in the nursery and wood quality traits in the progeny trials.
The individual-tree narrow-sense heritability for each trait was estimated by
$$\widehat{h}_{\text{i}}^{2} = \frac{{\widehat{\sigma }_{\text{a}}^{2} }}{{\widehat{\sigma }_{p}^{2} }} = \frac{{4 \times \widehat{\sigma }_{\text{f}}^{2} }}{{\widehat{\sigma }_{\text{f}}^{2} + \widehat{\sigma }_{\text{e}}^{2} }}$$
assuming half-sib relationship and where \(h_{\text{i}}^{2}\), \(\widehat{\sigma }_{a}^{2}\), \(\widehat{\sigma }_{f}^{2}\), \(\widehat{\sigma }_{e}^{2}\), and \(\widehat{\sigma }_{p}^{2}\) were narrow-sense heritability and additive genetic, family within provenance, residual, and phenotypic variance components, respectively. Phenotypic and genetic correlations between traits were calculated as:
$$r = \frac{{\widehat{\text{Cov}}\left( {x,y} \right)}}{{\sqrt {\widehat{\sigma }_{\left( x \right)}^{2} \times \widehat{\sigma }_{\left( y \right)}^{2} } }}$$
where \(\widehat{\sigma }_{\left( x \right)}^{2}\) and \(\widehat{\sigma }_{\left( y \right)}^{2}\) are the estimated phenotypic or genetic variances for traits x and y or the same trait variances at two different ages, respectively, and \(\widehat{\text{Cov}}_{{\left( {x,y} \right)}}\) is the estimated phenotypic or genetic covariance between traits x and y.
Coefficients of additive variation (CVa) and phenotypic variation (CVp) were calculated by dividing square root of additive and phenotypic variances by mean value of that trait, respectively.
Genetic gain (G) was calculated using a selection intensity of 1% \((i = 2.67)\):
$$G = i \times {\text{CV}}_{\text{p}} \times h_{i}^{2}$$
where CVp is coefficient of variation of phenotypic effect (calculated as the phenotypic standard deviation divided by the mean of a specific trait) and \(h_{\text{i}}^{2}\) is individual narrow-sense heritability.
To study the impact of early selection of disease resistance at late-age performance of tree growth and wood quality traits, the correlated response (CR) expressed as the percentage of the mean for those traits (y) in field progeny trials to early selection based on nursery resistance traits (x) is calculated as:
$${\text{CR}}_{y} = \frac{{ih_{x} h_{y} r_{a} \sigma_{{p_{y} }} }}{{M_{y} }} \times 100$$
where hx and hy are the square roots of individual narrow-sense heritability for early selection trait x and correlated late-age trait y, ra is the genetic correlation between traits x and y, and \(\sigma_{{p_{y} }}\) is the phenotypic standard deviation for trait y, My is the mean value of trait y (Falconer and Mackay 1996).