The use of open-pollinated seeds from seed orchards is a common strategy for the deployment of genetically improved eucalypts, including
In Portugal, breeding programs for this species have been in place for over 40 years, relying mostly on clonal and controlled crossing schemes (
The aims of this study were to: (i) evaluate the selfing rate and impact of inbreeding depression in
To accomplish this, several field tested open-pollinated family progenies between 2 and 5 years were evaluated for growth rate and presence of abnormalities. Given the relatively young age, height, rather than diameter, was considered a better proxy of later age tree volume. Early height was shown to be strongly correlated with later age volume growth, with correlations ranging between 0.8 and 0.9 (
The tested
Up to eleven open pollinated families were collected in the orchard, at three consecutive years (2014, 2015 and 2016). These families were selected because either they resulted from the most represented mothers in the orchard, with between 14 and 40 flowering ramets each (see Tab. S1 in Supplementary material for detailed information) or they had progeny with large number of abnormal phenotypes (see below).
The progeny from these families (n=324) was tested across five locations, covering a range of sites in Central and Southern Portugal. All trials were designed as randomized complete blocks with up to 9 replicates, with each family represented in each replicate by 1 to 5 tree plots, with a standard spacing of 0 × 2 m (see Tab. S2 in Supplementary material for trials’ edaphoclimatic conditions). As a result of initial variation in progeny numbers and subsequent mortality, each family was represented in the dataset varying between 1 and 24 offsprings per site.
Survival across all trials was high being on average 95%. The mixed model analysis of growth included all trees with normal phenotype coming from the 7 most representative families of the orchard. They were measured for height, with ages between 1.6 to 4.5 years (n=283 -
DNA was extracted from leaves of 324 progeny and 27 putative parents in this study, with an optimized CTAB protocol (as described in
Parental assignment (
If a seedling had no assigned pollen donor from any of the orchard parental population, pollen parents were assumed to be originated from unrelated trees from outside the orchard, hence resulting in foreign pollen contamination. In pedigree reconstruction for each seed, the possibility of having one single mismatch (or reading error) was considered in the simulation, as recommended in
Selfing and contamination rates were estimated for the most representative families in each of the three different seed collection years. Percentage selfing per open-pollinated family (
Since trials at the time of measurement had different ages, means and variances of height varied considerably between trials. Mean annual height increments, rather than total height, was used in the linear model, to ensure measurements are more comparable and variances are homogeneous across sites. The following mixed model was fitted (
where
This model assumed mean annual height increments from height measurements taken at different ages (as height measurements divided by their corresponding age) would be an expression of the same trait. High genetic correlations between height at different early ages have been reported for
Across-site heritability for height growth was estimated without explicitly fitting a genotype by site interaction term, since the number of genotypes was small and there was unbalance in their representation across sites. It is expected that such effects, if present, would be captured in the residual term. Heritability was estimated as (
Taylor series expansion was used to obtain approximate standard errors for the variance parameters estimated from the linear mixed model, as well as for derived linear combinations or ratios of the variance estimates, as implemented on the “vpredict” function in AsReml v. 4.2 (
The association between frequency of abnormal phenotypes and inbreeding was tested based on a χ2 independence test, using the R function “chisq.test” with data from Tab. S4 (Supplementary material) as input.
Amplification was successful across the 12 SSR markers, corroborating their importance in paternity tests in
In the present study, at the time of measurement, between 2 and 4 years, the degree of selfing across families was on average 30%. This estimate is in the upper range of what has been reported in other orchards (
Contamination rates, from pollen outside the orchard, were low in absolute terms (average of 10% across years) but seem to be increasing over time. It was 0% in 2014, 8% in 2015 and 16% in 2016 (
The causes for the substantial annual variation in selfing and to a lesser extent in contamination rates are hard to interpret since no data on the phenology of trees inside and around the orchard, and insect activity has been recorded in detail. The flowering synchrony, the length and intensity of flowering, the weather conditions at critical pollination periods and the intensity of insect activity are all plausible factors affecting pollen dispersal in a seed orchard, hence influencing outcrossing and contamination rates.
All unrelated base parents in the orchard were assumed to have an inbreeding of F=0. However, some parents in the orchard are second- and third-generation selections, with some degree of kinship among them although not inbred themselves. The average co-ancestry among the 15 orchard parents contributing to the studied progeny, derived from pedigree records, was greater than zero (θP=0.05) and corresponds to a Status Number of
The reconstructed pedigree for all sampled trees allowed the estimation of the inbreeding coefficient (F) for each tree. Of the 283 trees with normal phenotypes used in the mixed model analysis, 54% had unrelated parents (F=0), 11% had F=0.0625, 4% had F=0.125, 1% had F=0.25 and 30% had F=0.5. Mean annual increment across the five trials was 2.9 m, with site effects and replicate effects within site being significant (p<0.001). The population mean inbreeding depression slope (
The inbreeding depression slopes for each of the seven maternal parents studied (
Despite this large variation in the inbreeding depression slope between parents, an effect fitted as a random term in the model, differences between them and the mean population slope
Across-site additive genetic effects for height were significant, with a variance of σA2 = 0.017 (LRT=7.12; p=0.008 -
Among the 324 trees assessed, 30 were classified as having abnormal phenotypes, showing dwarfism and a mallee appearance, as a result of partial or total loss of apical dominance and excessive proliferation of axillary buds. However, it was clear from the pedigree records that there was no obvious association between such abnormalities and selfed individuals, as many abnormal phenotypes were found in unrelated outcrossed progeny (where F=0). The chi-square test confirmed the independency observed between abnormal phenotypes and estimated levels of inbreeding (null hypothesis: χ2[4] = 2.4277; p = 0.6576). To clarify any putative association between abnormal phenotypes and parents, we looked at crosses with 10 or more offsprings. This analysis showed however that abnormal phenotypes were more frequent in specific, but otherwise unrelated, crosses (
The studied open-pollinated
All authors contributed to the conceptualization, methodology definition, data analysis and interpretation, as well as the review and edition of the manuscript. All authors have read and agreed to the published version of the manuscript. Laboratory work was performed by JF supervised by HT, fieldwork supervised by JA; writing of the original draft was first outlined by JF and HT, review and editing was performed by all the authors.
The authors would like to thank to The Navigator Company for providing access to the study sites, to Luis Ferreira and José Cardoso for trial assessments and Fátima Cunha for expert lab assistance.
This research was funded by
The authors declare no conflicts of interest.
Frequency of male contribution (pollen) to the progeny in the field-tested offspring. The counting includes progeny from seed collected over a period of three flowering seasons (2014-2016) and tested in five different locations, with ages ranging between 1.6 and 4.5 years.
Mean annual height increments (given as percentage of the trial means) for all trees in the five-progeny trial (n=283), relative to their corresponding inbreeding coefficient.
Details of the trial location, age of measurements and experimental design in the five field trials included in the mixed model analysis. Number of Randomized complete blocks and families tested, and the total across family progeny measured are also given (n=283).
Trial Location | Seedyear | Age | # Replicates | # Families | Total progeny size |
---|---|---|---|---|---|
Fundão | 2014 | 4.5 | 4 | 4 | 53 |
Abrantes | 2015 | 3.2 | 9 | 4 | 75 |
Oleiros | 2015 | 3.2 | 9 | 5 | 50 |
Gorda | 2016 | 2.3 | 9 | 6 | 54 |
Nisa | 2016 | 1.6 | 9 | 6 | 51 |
Details of the nuclear SSR markers used in the analysis, and the number of alleles, the percentage of null alleles and the expected heterozygosity (He) found across the seven open-pollinated families tested.
Microsatellite | NoAlleles | Null(%) | He |
---|---|---|---|
Embra11 | 17 | 0.10 | 0.87 |
Embra119 | 14 | 0.14 | 0.86 |
Emcrc8 | 17 | 0.04 | 0.88 |
Emcrc7 | 8 | 0.08 | 0.77 |
Embra23 | 15 | 0.08 | 0.90 |
Embra41 | 15 | 0.07 | 0.86 |
Embra227 | 10 | 0.04 | 0.78 |
En15 | 11 | 0.03 | 0.84 |
Es76 | 18 | 0.07 | 0.90 |
Eg65 | 18 | 0.05 | 0.89 |
Embra37 | 23 | 0.06 | 0.91 |
En12 | 17 | 0.08 | 0.87 |
Average | 15.25 | 0.07 | 0.86 |
Total number of progenies sampled from each family (ID Mother) and seed collection year, as well as the number and respective percentage of selfed and contamination with foreign pollen. The trial’s location where the progenies were tested are also given (in parentheses). The progeny listed only includes the most representative families within each year (n = 311).
Year (Trial) | ID Mother | Progeny | Self | %Selfing | Contaminated | %Contamination |
---|---|---|---|---|---|---|
2014 (Fundão) | M74 | 9 | 0 | 0.0 | 0 | 0.0 |
M07 | 13 | 0 | 0.0 | 0 | 0.0 | |
M13 | 15 | 0 | 0.0 | 0 | 0.0 | |
M18 | 18 | 0 | 0.0 | 0 | 0.0 | |
2015 (Abrantes and Oleiros) | M74 | 27 | 3 | 11.1 | 0 | 0.0 |
M07 | 31 | 3 | 9.7 | 5 | 16.1 | |
M13 | 34 | 3 | 8.8 | 5 | 14.7 | |
M49 | 27 | 26 | 96.3 | 1 | 3.7 | |
M28 | 17 | 15 | 88.2 | 1 | 5.9 | |
2016 (Gorda and Nisa) | M74 | 19 | 7 | 36.8 | 3 | 15.7 |
M07 | 15 | 1 | 6.7 | 5 | 33.3 | |
M13 | 20 | 2 | 10.0 | 4 | 20 | |
M89 | 24 | 16 | 66.7 | 0 | 0.0 | |
M49 | 21 | 12 | 57.1 | 2 | 9.5 | |
M28 | 21 | 3 | 14.3 | 4 | 19 |
Variance components for the random effects in the mixed linear model for annual height growth and corresponding likelihood ratio tests (LRT) and probability of significance. Effects include the trees’ additive genetic effects (
Effects | Variance | LRT | Prob |
---|---|---|---|
|
0.017 | 7.12 | 0.008 |
|
0.476 | 6.16 | 0.013 |
|
0.064 | - | - |
Estimates of the solutions for the overall and site mean, the parent’s additive genetics effects (a), the mean population inbreeding coefficient slope (bF), and the individual parent inbreeding coefficient slopes (bFi) for the seven parents in the study.
Effects | Mother ID | Estimate | Standard error |
---|---|---|---|
Mean | - | 2.929 | 0.145 |
Site | 394 | 2.929 | 0.145 |
407 | 2.367 | 0.156 | |
416 | 3.355 | 0.210 | |
421 | 2.853 | 0.185 | |
423 | 1.596 | 0.193 | |
Parents breeding value ( |
M74 | 0.231 | 0.089 |
M18 | 0.032 | 0.106 | |
M49 | 0.097 | 0.099 | |
M28 | 0.013 | 0.099 | |
M07 | -0.117 | 0.087 | |
M13 | -0.024 | 0.086 | |
M89 | -0.012 | 0.080 | |
bF | -0.867 | 0.327 | |
|
M74 | -1.404 | 0.392 |
M18 | -1.310 | 0.628 | |
M49 | -0.795 | 0.375 | |
M28 | -0.580 | 0.392 | |
M07 | -0.743 | 0.460 | |
M13 | -0.432 | 0.423 | |
M89 | -0.724 | 0.386 |
Progeny with normal or abnormal phenotypes resulting from crosses with 10 or more offspring present in the five progeny trials (aged between 1.6 and 4.5 years). Parent A and Parent B columns are not indicative of female or male parents and merely represent the cross between the two clones.
Parent A | Parent B | Normal phenotype | Abnormal phenotype |
---|---|---|---|
M49 | M28 | 15 | 7 |
M49 | M49 | 37 | 1 |
100841010 | M18 | 6 | 4 |
M07 | M89 | 12 | 0 |
M07 | 100841010 | 11 | 1 |
M07 | 100881019 | 13 | 0 |
M13 | M89 | 33 | 1 |
M89 | M89 | 13 | 3 |
M28 | M28 | 18 | 0 |
M74 | M89 | 16 | 0 |
M74 | 100841010 | 10 | 0 |
M74 | M74 | 10 | 0 |
Tab. S1 - Number of ramets of the parent trees included in the orchard and progeny analysed in each field trial.
Tab. S2 - Field trials brief characterization, including year of establishment, lithology, soil classification according to FAO, range of annual precipitation and temperatures.
Tab. S3 - Microsatellite identification, forward and reverse primer sequences, fragment size and multiplexing.
Tab. S4 - Contingency table with the number of normal and abnormal phenotypes per class of inbreeding coefficient.
Tab. S5 - Values of within family group co-ancestry and corresponding intraclass correlation for the seven open pollinated families sampled in the