A molecular analysis of the population structure, mating system and demography of Eucalyptus

This thesis aimed to study the evolutionary forces affecting populations of Eucalyptus, with special emphasis on the compounds implicated in plant defence, notably terpenes. In Chapter 1, I explored the genetic structure in Eucalyptus globulus to understand how processes such as gene flow and genetic drift have shaped the genetic distribution of the species. Using microsatellite-derived genotypes of individuals covering the range of the species, I separated E. globulus into five geographically distinct regions. Furthermore, local-scale spatial genetic analysis using two of the regions detected spatial genetic structure of over 40 km, indicating long-distance gene flow and a larger population than previously thought. The five regions provided the units of study in Chapters 2 and 3. This chapter also provided data on population structure for the association studies of candidate genes from secondary metabolic pathways (Appendix 5). In Chapter 2, I turned my attention to genes of known function - dxs and dxr genes from the non-mevalonate terpene biosynthesis pathway because these had been previously implicated as bottlenecks to terpene production in other species. The aim was to determine whether the genes revealed the variation we observed in the concentrations of foliar terpenes. Using gene sequences from 104 E. globulus individuals, I showed that the dxr and two copies of dxs genes were under purifying selection but there was no evidence that these genes cause the variation we see in foliar terpene production. I also found that different enzymatic domains encoded by the genes have taken different evolutionary pathways. In Chapter 3, I reconstructed the demographic history of E. globulus using information from introns and third coding sites of exons of dxr and two copies of dxs, with the aim of estimating the timing of major demographic events for the entire species and for the five regions from Chapter 1. To do this, I applied a novel analysis using Bayesian Skyline plots. The demographic reconstruction of the regions showed two trends of exponential expansion, which started around the early-mid Pleistocene transition. These trends suggested that the island populations expanded earlier while those on the mainland expanded faster. These appeared as two continuous expansions when the entire species was analysed. The results of this study excluded early human activity as an important cause of expansion of Eucalyptus globulus. In Chapter 4, I examined the genetic variation in families within populations over shorter and contemporary evolutionary time-frames. The aim was to examine the correspondence between mating systems and heritability of foliar terpenes and to test if all populations of a species are equally suited for inferring marker-based heritability. Using microsatellite genotypes and the foliar terpene profile from three disparate populations of E. tricarpa, I found that the estimates of the heritability of foliar terpenes differed among populations and were not correlated with outcrossing rates or pollen heterogeneity among females. The variable mating systems and structure of the pollen pool resulted in some populations providing more reliable heritability estimates, which is important for most studies of community genetics. I concluded my thesis by discussing the value of next generation sequencing technology in expanding the population genetic aspects covered in this project

Comparative SNP diversity among four Eucalyptus species for genes from secondary metabolite biosynthetic pathways

<p>Abstract</p> <p>Background</p> <p>There is little information about the DNA sequence variation within and between closely related plant species. The combination of re-sequencing technologies, large-scale DNA pools and availability of reference gene sequences allowed the extensive characterisation of single nucleotide polymorphisms (SNPs) in genes of four biosynthetic pathways leading to the formation of ecologically relevant secondary metabolites in <it>Eucalyptus</it>. With this approach the occurrence and patterns of SNP variation for a set of genes can be compared across different species from the same genus.</p> <p>Results</p> <p>In a single GS-FLX run, we sequenced over 103 Mbp and assembled them to approximately 50 kbp of reference sequences. An average sequencing depth of 315 reads per nucleotide site was achieved for all four eucalypt species, <it>Eucalyptus globulus</it>, <it>E. nitens</it>, <it>E. camaldulensis </it>and <it>E. loxophleba</it>. We sequenced 23 genes from 1,764 individuals and discovered 8,631 SNPs across the species, with about 1.5 times as many SNPs per kbp in the introns compared to exons. The exons of the two closely related species (<it>E. globulus </it>and <it>E. nitens</it>) had similar numbers of SNPs at synonymous and non-synonymous sites. These species also had similar levels of SNP diversity, whereas <it>E. camaldulensis </it>and <it>E. loxophleba </it>had much higher SNP diversity. Neither the pathway nor the position in the pathway influenced gene diversity. The four species share between 20 and 43% of the SNPs in these genes.</p> <p>Conclusion</p> <p>By using conservative statistical detection methods, we were confident about the validity of each SNP. With numerous individuals sampled over the geographical range of each species, we discovered one SNP in every 33 bp for <it>E. nitens </it>and one in every 31 bp in <it>E. globulus</it>. In contrast, the more distantly related species contained more SNPs: one in every 16 bp for <it>E. camaldulensis </it>and one in 17 bp for <it>E. loxophleba</it>, which is, to the best of our knowledge, the highest frequency of SNPs described in woody plant species.</p

Evaluation of methods and marker systems in genomic selection of oil palm (Elaeis guineensis Jacq.)

Background Genomic selection (GS) uses genome-wide markers as an attempt to accelerate genetic gain in breeding programs of both animals and plants. This approach is particularly useful for perennial crops such as oil palm, which have long breeding cycles, and for which the optimal method for GS is still under debate. In this study, we evaluated the effect of different marker systems and modeling methods for implementing GS in an introgressed dura family derived from a Deli dura x Nigerian dura (Deli x Nigerian) with 112 individuals. This family is an important breeding source for developing new mother palms for superior oil yield and bunch characters. The traits of interest selected for this study were fruit-to-bunch (F/B), shell-to-fruit (S/F), kernel-to-fruit (K/F), mesocarp-to-fruit (M/F), oil per palm (O/P) and oil-to-dry mesocarp (O/DM). The marker systems evaluated were simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs). RR-BLUP, Bayesian A, B, Cπ, LASSO, Ridge Regression and two machine learning methods (SVM and Random Forest) were used to evaluate GS accuracy of the traits. Results The kinship coefficient between individuals in this family ranged from 0.35 to 0.62. S/F and O/DM had the highest genomic heritability, whereas F/B and O/P had the lowest. The accuracies using 135 SSRs were low, with accuracies of the traits around 0.20. The average accuracy of machine learning methods was 0.24, as compared to 0.20 achieved by other methods. The trait with the highest mean accuracy was F/B (0.28), while the lowest were both M/F and O/P (0.18). By using whole genomic SNPs, the accuracies for all traits, especially for O/DM (0.43), S/F (0.39) and M/F (0.30) were improved. The average accuracy of machine learning methods was 0.32, compared to 0.31 achieved by other methods. Conclusion Due to high genomic resolution, the use of whole-genome SNPs improved the efficiency of GS dramatically for oil palm and is recommended for dura breeding programs. Machine learning slightly outperformed other methods, but required parameters optimization for GS implementation

Regional population expansion in Eucalyptus globulus

Foundation tree species define the structure of forest habitat and influence their ecosystem dynamics. However, there is limited understanding of both the patterns and timing of population fluctuations in foundation trees and how they vary among geograph

Regional population expansion in Eucalyptus globulus

Foundation tree species define the structure of forest habitat and influence their ecosystem dynamics. However, there is limited understanding of both the patterns and timing of population fluctuations in foundation trees and how they vary among geographical regions. We have reconstructed the demographic history of five genetically distinct populations of the Tasmanian blue gum (Eucalyptus globulus ssp. globulus) at the species and regional levels, using three nuclear loci sequenced from 104 individuals. Analysis using a Bayesian skyline plot indicated that the species experienced two periods of expansion, commencing in the Pliocene. Regional analyses showed that island populations expanded earlier, but that the rate of expansion was relatively slow when compared to that of the mainland group. This highlights the need for local demographic history to be taken into account when inferring local adaptation for candidate genes. Population growth throughout the Quaternary signals the ability of the species to persist and thrive under the predominantly harsh conditions of this period

Estimating population boundaries using regional and local-scale spatial genetic structure: An example in Eucalyptus globulus

Eucalyptus globulus Labill is a foundation tree species over its disjunct distribution in southeastern Australia. The quality of its pulp makes it the most important hardwood species in the world. The importance of E. globulus prompted the establishment of common gardens from seed collected across its geographic range. This enabled us to study the genetic structure of the species, its population boundaries, and gene flow using 444 trees from different open-pollinated families that were genotyped at 16 microsatellite loci. A Bayesian clustering method was used to resolve five genetically distinct groups across the geographical range. These groups were identified as regions, which varied in diameter from 38 to 294 km and contain 4 to 16 putative populations. For two of these regional groups, we used spatial autocorrelation analysis based on assignment of trees to their natural stands to examine gene flow within each region. Consistent significant local-scale spatial structure occurred in both regions. Pairs of individuals within a region showed significant genetic similarity that extended beyond 40 km, suggesting distant movement of pollen. This suggests that breeding populations in E. globulus are much bigger than traditionally accepted in eucalypts. Our results are important for the management of genetic diversity and breeding populations in E. globulus. Similar studies of a variety of eucalypts pollinated by insects and birds will determine whether the local-scale genetic structure of E. globulus is unusual

Regional population expansion in Eucalyptus globulus

Foundation tree species define the structure of forest habitat and influence their ecosystem dynamics. However, there is limited understanding of both the patterns and timing of population fluctuations in foundation trees and how they vary among geographical regions. We have reconstructed the demographic history of five genetically distinct populations of the Tasmanian blue gum (Eucalyptus globulus ssp. globulus) at the species and regional levels, using three nuclear loci sequenced from 104 individuals. Analysis using a Bayesian skyline plot indicated that the species experienced two periods of expansion, commencing in the Pliocene. Regional analyses showed that island populations expanded earlier, but that the rate of expansion was relatively slow when compared to that of the mainland group. This highlights the need for local demographic history to be taken into account when inferring local adaptation for candidate genes. Population growth throughout the Quaternary signals the ability of the species to persist and thrive under the predominantly harsh conditions of this period

The molecular basis of quantitative variation in foliar secondary metabolites in Eucalyptus globulus

Eucalyptus is characterized by high foliar concentrations of plant secondary metabolites with marked qualitative and quantitative variation within a single species. Secondary metabolites in eucalypts are important mediators of a diverse community of herbivores. We used a candidate gene approach to investigate genetic associations between 195 single nucleotide polymorphisms (SNPs) from 24 candidate genes and 33 traits related to secondary metabolites in the Tasmanian Blue Gum (Eucalyptus globulus). We discovered 37 significant associations (false discovery rate (FDR) Q<0.05) across 11 candidate genes and 19 traits. The effects of SNPs on phenotypic variation were within the expected range (0.018<r2<0.061) for forest trees. Whereas most marker effects were nonadditive, two alleles from two consecutive genes in the methylerythritol phosphate pathway (MEP) showed additive effects. This study successfully links allelic variants to ecologically important phenotypes which can have a large impact on the entire community. It is one of very few studies to identify the genetic variants of a foundation tree that influences ecosystem function

Characterization of leaf transcriptome in a tropical tree species, Shorea curtisii, over a flowering season

&lt;p&gt;&lt;span&gt;General flowering (GF) is a synchronous flowering event in the Southeast Asian tropical rainforests that occurs at irregular intervals of multiple years. The unpredictable intervals of GF raise conservation concerns for these under-researched forests with rich economically and ecologically important species. In this study, the leaf transcriptome of a GF species, &lt;em&gt;Shorea&lt;/em&gt; &lt;em&gt;curtisii&lt;/em&gt; obtained from three time points – before and after floral initiation, and post flowering stage – was sequenced. We assembled 243,759,478 sequencing reads into 39,943 non-redundant unigenes including 677 putative homologs of &lt;em&gt;Arabidopsis&lt;/em&gt; &lt;em&gt;thaliana&lt;/em&gt; flowering-related genes. Differential expression analysis conducted on pairwise comparisons of the time points identified 930 differentially expressed unigenes, which includes 17 flowering-related homologs. The differential expression of unigenes with significant enrichments of functions related to drought corroborated the involvement of drought as an environmental cue for GF. The outcomes of this study offer an insight into the conservation of floral regulatory genes and pathways in Shorea and could be used as a model to better understand the floral initiation cues and regulation of GF trees.&lt;/span&gt;&lt;/p&gt;&lt;p&gt;Fig_S1_3_Table_S1_2.pdf: A pdf file which contains Supplementary Figure S1–S3 and Table S1–S2. &lt;/p&gt; &lt;p&gt;Fig S1 is a flow chart of research strategy employed to study the leaf transcriptome of &lt;em&gt;Shorea curtisii&lt;/em&gt; during a flowering season. Details of the methodology are described in the legend in details.&lt;/p&gt; &lt;p&gt;Fig S2 is a histogram which shows the distribution of the contig lengths of &lt;em&gt;S. curtisii&lt;/em&gt; transcriptome.&lt;/p&gt; &lt;p&gt;Fig S3 is a bar chart which depicts the classification of &lt;em&gt;S. curtisii &lt;/em&gt;unigenes into three main Gene Ontology categories.&lt;/p&gt; &lt;p&gt;Table S1 is a table which contains details of meteorological status, specifically daily minimum, maximum, and mean temperature, as well as rainfall records during sampling days. The records were obtained from the nearest meteorological and hydrological stations to the sampling site. The first line shows titles of data which include information on when the sampling was done i.e. time point, date, developmental stage, and sampling time for individual trees, as well as meteorological status i.e. daily temperature and rainfall records.&lt;/p&gt; &lt;p&gt;Table S2 is a table with the summary of BUSCO analysis which reflects the completeness of &lt;em&gt;S. curtisii &lt;/em&gt;transcriptome assembly. The first column shows the description for each category of BUSCO genes and the following columns show the number of genes recovered and percentage for each category.&lt;/p&gt; &lt;p&gt;Table_S3_4_5.xlsx is an Excel workbook which contains the following sheets:&lt;/p&gt; &lt;p&gt;Table S3 shows the raw read counts and annotations for each unigene in &lt;em&gt;S. curtisii&lt;/em&gt; leaf transcriptome. The first column shows titles of data.&lt;/p&gt; &lt;p&gt;Table S4 shows a summary of flowering-related homologs in &lt;em&gt;S. curtisii&lt;/em&gt; leaf transcriptome. The table contains information on flowering-related homologs in the transcriptome including number of homologs that were differentially expressed in the study period.&lt;/p&gt; &lt;p&gt;Table S5 shows normalized read counts, annotations, and pairwise comparisons of differentially expressed unigenes in &lt;em&gt;S. curtisii&lt;/em&gt; leaf transcriptome. The first column shows titles of data.&lt;/p&gt; &lt;p&gt;Table_S6.xlsx is an Excel workbook which contains a summary of KEGG annotation in &lt;em&gt;S. curtisii&lt;/em&gt; leaf transcriptome (Table S6A) and lists of enriched KEGG pathways in differentially expressed unigenes of &lt;em&gt;S. curtisii&lt;/em&gt; for every pairwise time points comparison (Table S6B–S6D).&lt;/p&gt; &lt;p&gt;Table_S7 is an Excel workbook which contains a summary of GO annotation in &lt;em&gt;S. curtisii&lt;/em&gt; leaf transcriptome (Table S7A) and lists of enriched GO terms in differentially expressed unigenes of &lt;em&gt;S. curtisii&lt;/em&gt; for every pairwise time points comparison (Table S7B–S7D).&lt;/p&gt;&lt;p&gt;Funding provided by: MoE-HIR*&lt;br&gt;Crossref Funder Registry ID: &lt;br&gt;Award Number: UM.C/625/1/HIR/MOE/SCI/18&lt;/p&gt;&lt;p&gt;Funding provided by: BKP*&lt;br&gt;Crossref Funder Registry ID: &lt;br&gt;Award Number: BKS074-2017&lt;/p&gt;&lt;p&gt;Funding provided by: Ministry of the Environment&lt;br&gt;Crossref Funder Registry ID: https://ror.org/03rybxa06&lt;br&gt;Award Number: RFd-1101&lt;/p&gt;&lt;p&gt;Funding provided by: Japan Society for the Promotion of Science&lt;br&gt;Crossref Funder Registry ID: https://ror.org/00hhkn466&lt;br&gt;Award Number: 26251042&lt;/p&gt;&lt;p&gt;&lt;span&gt;Leaf samples at top layer of canopy from two &lt;em&gt;S. curtisii &lt;/em&gt;individuals, C1 and C2 were collected from Semangkok Forest Reserve (2°58'N, 102°18'E). We selected three time points (TPs) corresponding to before and after floral initiation, and abortion/fruiting stage. Samples were collected at each TP around midday, soaked in RNAlater reagent (Ambion, USA), and stored at –80°C.&lt;/span&gt;&lt;/p&gt; &lt;p&gt;&lt;span&gt;&lt;span&gt;Total RNA was extracted from leaf samples using the method described by Kobayashi et al. (2013). RNA-seq was performed using Illumina HiSeq 4000 Sequencer (Illumina, USA). After removal of low quality bases by Trimmomatic v0.36 (Bolger et al., 2014), the sequence reads from each sample were combined and &lt;em&gt;de&lt;/em&gt; &lt;em&gt;novo&lt;/em&gt; assembled using Trinity v2.8.5 (Grabherr et al., 2011). Only non-redundant transcripts with complete open reading frame were retained by using TransDecoder v5.5.0 (&lt;a href="http://transdecoder.github.io"&gt;http://transdecoder.github.io&lt;/a&gt;) and CD-HIT v4.8.1 (Fu et al., 2012). The quality of the assembly was assessed with BUSCO v5.2.1 (Simao et al., 2015). &lt;/span&gt;&lt;/span&gt;&lt;/p&gt; &lt;p&gt;&lt;span&gt;&lt;span&gt;The unigenes were queried against the proteome of &lt;em&gt;A. thaliana&lt;/em&gt; (Cheng et al., 2017) using BLASTx v2.10 (Camacho et al., 2009) with E-value cut-off: 1E–10. The annotated unigenes were searched against &lt;em&gt;A. thaliana&lt;/em&gt; flowering genes database (Bouché et al., 2016) to identify their homologs in &lt;em&gt;S. curtisii&lt;/em&gt;. Translated unigenes were queried against other public protein databases using BLASTp v2.10 (Camacho et al., 2009) and InterProScan v5.36 (Jones et al., 2014). We also searched the unigenes against Gene Ontology (GO; Ashburner et al., 2000) and KEGG pathways (Kanehisa &amp; Goto, 2000) to characterize the unigenes. GO classification is divided into cellular component (CC), molecular function (MF), and biological process (BP) categories.&lt;/span&gt;&lt;/span&gt;&lt;/p&gt; &lt;p&gt;&lt;span&gt;The unigenes were quantified by Salmon v1.5.1 (Patro et al., 2016) and subjected to differential expression analysis using DESeq2 v1.32.0 (Love et al., 2014). The samples were compared in a pairwise manner and differentially expressed unigenes (DEUs) with absolute log2 fold change ≥ 1 and FDR &lt; 0.05 were identified. Significantly enriched GO terms (P &lt; 0.05) and KEGG pathways (FDR &lt; 0.05) in the DEUs were also identified using topGO package v2.40.0 (Alexa &amp; Rahnenfuhrer, 2020) and KOBAS v3.0 (Bu et al., 2021), respectively. &lt;/span&gt;&lt;/p&gt