title: Linking genetics and chemistry to minimise bark stripping in Pinus radiata
Creators: name: Nantongo, JS
Publisher: name: University of Tasmania
Year: 2021

description: Pinus radiata (radiata pine) is native to California but the main plantation softwood in both Australia and New Zealand where it has been subject to breeding to improve productivity and wood quality. However, in many plantations in Australia trees are subject to bark stripping by mammalian (mainly marsupial) herbivores, which can markedly reduce the genetic gain achieved in breeding programmes. This thesis explores the mechanisms and potential for exploiting natural genetic variation in resistance/susceptibility to minimise bark stripping in P. radiata. The study uses field and nursery experiments, integrating results from quantitative genetics, genomics, gene expression, analytical chemistry and rapid phenotyping to understand the genetic basis of variation in bark stripping and associated physical and chemical traits. To understand the genetic control and the stability of the genetic signal, bark stripping was scored in three Pinus radiata family trials (Chapter 2). Quantitative genetic analysis, using pedigree-based mixed linear models (ABLUP) revealed significant additive genetic variation in bark stripping. Non additive genetic effects were insignificant. While narrow-sense heritability estimates were low, the significant genetic signal was relatively stable across sites. The highest damaged families had approximately two-fold more bark removed than the least browsed families. Selecting the top 20% least susceptible families for planting could potentially reduce bark stripping by up to 22%. In the two older trials, reduced bark stripping was genetically associated with the presence of thick and rough bark while the presence of obstructive branches and needles on the lower stem (stem access) reduced bark stripping in the younger field trial. These important physical traits were also under significant genetic control. A positive additive correlation between prior height and bark stripping in the younger trial suggests that selecting faster growing trees may make P. radiata more vulnerable in the early stages of tree growth. However, when accounting for these physical and growth traits as covariates, significant additive genetic variation in bark stripping was still evident suggesting that genetic-based chemical properties of the bark were also important. To provide a framework to assess if chemical traits mediated variation in bark-stripping, the plant wide constitutive and induced chemistry was first assessed. In a shade house experiment, induction over a 4-week period was achieved by treating trees with methyl jasmonate (a chemical stressor) and artificial bark stripping (Chapter 3), following which 81 chemical compounds were quantified in the needles, stem and roots. These plant parts had different constitutive chemical profiles, with quantitatively and qualitatively more secondary plant compounds in the bark. After treatment, an overall upregulation of terpenes and phenolics and a down-regulation of sugars and fatty acids was observed. However, the quantitative and qualitative chemical responses differed between plant parts, treatments and time period over the 4 weeks of the experiment. Stronger responses were observed for primary compared to secondary metabolites suggesting their potential roles in plant stress responses, including bark stripping. To identify the specific constitutive and induced chemical traits that differentiated families with extreme levels of bark stripping, 21 of the most damaged and 21 of the least damaged families were selected from a fenced area within the younger trial used in Chapter 2. This field experiment examined the constitutive and induced chemistry with 83 compounds quantified (Chapter 4). Of the constitutive chemical traits in the bark, specific sugars, phenolics and terpenes were significantly different between the resistant and susceptible families. The bark sugars - fructose and glucose - and the phenolics - phenyl ethanol and benzene acetic acid - increased in the more susceptible families. The bark sesquiterpenoids - bicyclogermacrene and an unknown sesquiterpenoid alcohol - increased in the less susceptible families. The resistant and susceptible families could not be separated based on induced bark chemistry nor constitutive and induced needle chemistry. An important aim of this thesis was to generate genetic parameters for the chemical traits and identify those that are genetically correlated with bark stripping. To gain sufficient sample size for the genetic study, Chapter 5 explored the potential of near infra-red spectroscopy (NIRS) in qualitative classification and in quantifying the amounts of compounds identified in the samples from the methyl jasmonate treated, artificially bark stripped and non-treated trees used in Chapter 3. NIRS was successful in qualitatively separating samples from different plant parts as well as separating the treated from non-treated samples. NIRS models with high accuracy were developed for individual sugars, terpenes, phenolics and fatty acids. Highest accuracy models were developed for the sugars - glucose and fructose, suggesting practical application of such models, while models for most secondary compounds were able to give proximate amounts. The NIRS modelling was extended to quantify the chemistry for all samples in Chapter 6 for quantitative genetic analysis. Quantitative genetic analysis of the NIRS predicted values of 65 compounds in the bark using pedigree-based mixed models (ABLUP) showed significant additive genetic variation for individual chemical traits with low to moderate narrow-sense heritability estimates (Chapter 6). Results further showed strong positive genetic correlation of the sugar – glucose with bark stripping and a strong negative correlation with the unknown sesquiterpenoid alcohol. The results strengthen the findings based on the wet chemistry of the extreme families in Chapter 4. More positive genetic correlations were detected between bark stripping and fatty acids and an unknown diterpenoid, possibly due to the increased sample size gained from using NIRS prediction. No additive genetic variation in inducibility was detected and non-additive genetic variation in the constitutive chemistry was also not significant. However, most of the heritability estimates were low, implying that response to selection will be slow for these traits. Therefore, the potential improvement in the heritability estimates was tested using genomic models. To develop the genomic models, SNP genotyping was performed on needles from trees collected in Chapter 6; giving a total of 15,624 SNPs (Chapter 7). Using linear mixed models, the narrow-sense heritability estimates based on genomic models, genomic best linear unbiased prediction (GBLUP) and single-step GBLUP (ssGBLUP) were substantially better for both resistance and chemical traits compared to estimates obtained from the pedigree-based models (ABLUP). For the chemical traits, the average of the univariate GBLUP heritability estimates was1.6- fold higher than the average of the univariate ABLUP heritabilities, suggesting that the SNPs were able to capture additional genetic information. Similarly, the heritability of all the compounds based on the trivariate ssGBLUP was 1.7 – fold higher than the trivariate ABLUP estimates. The predictive ability (PA) of the ssGBLUP was comparable to the trivariate ABLUP model. Similarly, the PA of univariate GBLUP was mostly comparable to the univariate generalised ridge regression (GRR) with a few exceptions. The better performance of the GBLUP over the GRR for most traits suggests that the traits are quantitative in nature, influenced by many genes. The final chapter examined the expression of genes following methyl jasmonate and bark stripping with the aim of linking the chemical phenotypes in Chapter 3 to the underlying molecular activity, both in the needles and bark samples (Chapter 8). RNA was extracted and sequenced to yield 100bp paired-end sequences and each sample was sequenced to a depth of 20m reads per sample. After aligning the project transcriptome with the available P. radiata transcriptome, gene expression analysis showed up- and downregulation of genes associated with primary and secondary metabolism, with differences in transcript expression in the needles and the bark, between treatments and at different times. Consistent with the chemistry results, the genes that were related to secondary metabolism were also mainly up-regulated. Genes related to primary metabolism were more responsive than those related to secondary metabolism by up-regulation or down-regulation. Methyl jasmonate and bark-stripping showed many non-overlapping responses. Whereas maximum expression of the transcripts was observed 7 days after treatment, on the same population stronger chemical changes were detected 14 and 21 days after treatment, suggesting a time-lag between gene and phenotypic expression. Overall, the results indicated the potential for selection of less susceptible germplasm for operational plantings as a strategy to reduce the effects of bark stripping in plantation forestry. Selection against bark stripping in P. radiata can also be performed indirectly based on physical or chemical traits. Further tests may be required to establish the stability of the less susceptible families when planted as a monoculture.