Preselection of QTL markers enhances accuracy of genomic selection in Norway spruce.

Genomic prediction (GP) or genomic selection is a method to predict the accumulative effect of all quantitative trait loci (QTLs) in a population by estimating the realized genomic relationships between the individuals and by capturing the linkage disequilibrium between markers and QTLs. Thus, marker preselection is considered a promising method to capture Mendelian segregation effects. Using QTLs detected in a genome-wide association study (GWAS) may improve GP. Here, we performed GWAS and GP in a population with 904 clones from 32 full-sib families using a newly developed 50 k SNP Norway spruce array. Through GWAS we identified 41 SNPs associated with budburst stage (BB) and the largest effect association explained 5.1% of the phenotypic variation (PVE). For the other five traits such as growth and wood quality traits, only 2 - 13 associations were observed and the PVE of the strongest effects ranged from 1.2% to 2.0%. GP using approximately 100 preselected SNPs, based on the smallest p-values from GWAS showed the greatest predictive ability (PA) for the trait BB. For the other traits, a preselection of 2000-4000 SNPs, was found to offer the best model fit according to the Akaike information criterion being minimized. But PA-magnitudes from GP using such selections were still similar to that of GP using all markers. Analyses on both real-life and simulated data also showed that the inclusion of a large QTL SNP in the model as a fixed effect could improve PA and accuracy of GP provided that the PVE of the QTL was ≥ 2.5%.

Development of a universal height response model for transfer of Norway spruce (Picea abies L. Karst) in Fennoscandia.

Norway spruce is a major industrial tree species in Fennoscandia and future productivity of the species must be secured by matching the variation in adaptation of the species with suitable sites for optimized performance. An appropriate transfer model for forest reproductive material (FRM) is crucial for regeneration of productive forests in the changing climatic conditions that are predicted to occur in Fennoscandia. We have developed a transfer model for prediction of height of Norway spruce in Norway, Sweden, and Finland, using data acquired from 438 progeny and provenance trials with 1919 genetic entries of local and transferred origins. Transfer of genetic material at a given site was expressed in terms of the difference in daylength (photoperiod) between the site and its origin. This variable best reflected the nonlinear response to transfer that has been commonly reported in previous studies. Apart from the transfer variable, the height prediction model included the age of material when height measurements were acquired, annual temperature sum over 5 °C, precipitation during the vegetation period, and interaction terms between test site and transfer variables. The results show that long northward transfers (4-5° latitude) seem to be optimal for relatively mild sites in southern parts of the countries where growing season is longer, and shorter northward transfers (2-4° latitude) for harsher northern sites with shorter growing seasons. The transfer model also predicts that southward transfers of Norway spruce would result in height growth reductions. The developed model provides foundations for development of common or national recommendations for genetically improving Norway spruce material in Fennoscandia.

Genome-wide association mapping uncovers sex-associated copy number variation markers and female hemizygous regions on the W chromosome in Salix viminalis.

BACKGROUND: Sex chromosomes are in some species largely undifferentiated (homomorphic) with restricted sex determination regions. Homomorphic but different sex chromosomes are found in the closely related genera Populus and Salix indicating flexible sex determination systems, ideal for studies of processes involved in sex chromosome evolution. We have performed genome-wide association studies of sex and analysed sex chromosomes in a population of 265 wild collected Salix viminalis accessions and studied the sex determining locus. RESULTS: A total of 19,592 markers were used in association analyses using both Fisher's exact tests and a single-marker mixed linear model, which resulted in 48 and 41 sex-associated (SA) markers respectively. Across all 48 SA markers, females were much more often heterozygous than males, which is expected if females were the heterogametic sex. The majority of the SA markers were, based on positions in the S. purpurea genome, located on chromosome 15, previously demonstrated to be the sex chromosome. Interestingly, when mapping the genotyping-by-sequencing sequence tag harbouring the two SA markers with the highest significance to the S. viminalis genomic scaffolds, five regions of very high similarity were found: three on a scaffold that represents a part of chromosome 15, one on a scaffold that represents a part of chromosome 9 and one on a scaffold not anchored to the genome. Based on segregation differences of the alleles at the two marker positions and on differences in PCR amplification between females and males we conclude that females had multiple copies of this DNA fragment (chromosome 9 and 15), whereas males only had one (chromosome 9). We therefore postulate that the female specific sequences have been copied from chromosome 9 and inserted on chromosome 15, subsequently developing into a hemizygous W chromosome linked region. CONCLUSIONS: Our results support that sex determination in S. viminalis is controlled by one locus on chromosome 15. The segregation patterns observed at the SA markers furthermore confirm that S. viminalis females are the heterogametic sex. We also identified a translocation from chromosome 9 to the W chromosome.

Genetic Improvement of Sawn-Board Stiffness and Strength in Scots Pine (Pinus sylvestris L.).

Given an overall aim of improving Scots pine structural wood quality by selective tree breeding, we investigated the potential of non-destructive acoustic sensing tools to accurately predict wood stiffness (modulus of elasticity, MOE) and strength (modulus of rupture, MOR) of sawn boards. Non-destructive measurements of wood density (DEN), acoustic velocity (VEL) and MOE were carried out at different stages of wood processing chain (standing trees, felled logs and sawn boards), whilst destructively measured stiffness and strength served as benchmark traits. All acoustic based MOE and VEL estimates proved to be good proxies (rA > 0.65) for sawn-board stiffness while MOETREE, VELHIT and resistograph wood density (DENRES) measured on standing trees and MOELOG and VELFAK measured on felled logs well reflected board strength. Individual-tree narrow-sense heritability ( h i 2 ) for VEL, MOE and MOR were weak (0.05-0.26) but were substantially stronger for wood density (0.34-0.40). Moreover, additive genetic coefficients of variation for MOE and MOR were in the range from 5.4% to 9.1%, offering potential targets for exploitation by selective breeding. Consequently, selective breeding based on MOETREE, DENRES or stem straightness (STR) could improve several structural wood traits simultaneously.

Analysis of phenotypic- and Estimated Breeding Values (EBV) to dissect the genetic architecture of complex traits in a Scots pine three-generation pedigree design.

In forest tree breeding, family-based Quantitative Trait Loci (QTL) studies are valuable as methods to dissect the complexity of a trait and as a source of candidate genes. In the field of conifer research, our study contributes to the evaluation of phenotypic and predicted breeding values for the identification of QTL linked to complex traits in a three-generation pedigree population in Scots pine (Pinus sylvestris L.). A total of 11 470 open pollinated F2-progeny trees established at three different locations, were measured for growth and adaptive traits. Breeding values were predicted for their 360 mothers, originating from a single cross of two grand-parents. A multilevel LASSO association analysis was conducted to detect QTL using genotypes of the mothers with the corresponding phenotypes and Estimated Breeding Values (EBV). Different levels of genotype-by-environment (G × E) effects among sites at different years, were detected for survival and height. Moderate-to-low narrow sense heritabilities and EBV accuracies were found for all traits and all sites. We identified 18 AFLPs and 12 SNPs to be associated with QTL for one or more traits. 62 QTL were significant with percentages of variance explained ranging from 1.7 to 18.9%. In those cases where the same marker was associated to a phenotypic or an ebvQTL, the ebvQTL always explained higher proportion of the variance, maybe due to the more accurate nature of Estimated Breeding Values (EBV). Two SNP-QTL showed pleiotropic effects for traits related with hardiness, seed, cone and flower production. Furthermore, we detected several QTL with significant effects across multiple ages, which could be considered as strong candidate loci for early selection. The lack of reproducibility of some QTL detected across sites may be due to environmental heterogeneity reflected by the genotype- and QTL-by-environment effects.

Allelic incompatibility can explain female biased sex ratios in dioecious plants.

BACKGROUND: Biased sex ratios are common among dioecious plant species despite the theoretical prediction of selective advantage of even sex ratios. Albeit the high prevalence of deviations from even sex ratios, the genetic causes to sex biases are rarely known outside of a few model species. Here we present a mechanism underlying the female biased sex ratio in the dioecious willow species Salix viminalis. RESULTS: We compared the segregation pattern of genome-wide single nucleotide polymorphism markers in two contrasting bi-parental pedigree populations, the S3 with even sex ratio and the S5 with a female biased sex ratio. With the segregation analysis and comparison between the two populations, we were able to demonstrate that sex determination and sex ratio distortion are controlled by different genetic mechanisms. We furthermore located the sex ratio distorter locus to a Z/W-gametologous region on chromosome 15, which was in close linkage with the sex determination locus. Interestingly, all males in the population with biased sex ratio have in this sex ratio distorter locus the same genotype, meaning that males with the Z1/Z3-genotype were missing from the population, thereby creating the 2:1 female biased sex ratio. CONCLUSIONS: We attribute the absence of Z1/Z3 males to an allelic incompatibility between maternally and paternally inherited alleles in this sex ratio distorter locus. Due to the tight linkage with the sex determination locus only male individuals are purged from the population at an early age, presumably before or during seed development. We showed that such allelic incompatibility could be stably maintained over evolutionary times through a system of overdominant or pseudooverdominant alleles. Thus, it is possible that the same mechanism generates the female biased sex ratio in natural willow populations.

Genetics of phenotypic plasticity and biomass traits in hybrid willows across contrasting environments and years.

Background and Aims: Phenotypic plasticity can affect the geographical distribution of taxa and greatly impact the productivity of crops across contrasting and variable environments. The main objectives of this study were to identify genotype-phenotype associations in key biomass and phenology traits and the strength of phenotypic plasticity of these traits in a short-rotation coppice willow population across multiple years and contrasting environments to facilitate marker-assisted selection for these traits. Methods: A hybrid Salix viminalis × ( S. viminalis × Salix schwerinii ) population with 463 individuals was clonally propagated and planted in three common garden experiments comprising one climatic contrast between Sweden and Italy and one water availability contrast in Italy. Several key phenotypic traits were measured and phenotypic plasticity was estimated as the trait value difference between experiments. Quantitative trait locus (QTL) mapping analyses were conducted using a dense linkage map and phenotypic effects of S. schwerinii haplotypes derived from detected QTL were assessed. Key Results: Across the climatic contrast, clone predictor correlations for biomass traits were low and few common biomass QTL were detected. This indicates that the genetic regulation of biomass traits was sensitive to environmental variation. Biomass QTL were, however, frequently shared across years and across the water availability contrast. Phenology QTL were generally shared between all experiments. Substantial phenotypic plasticity was found among the hybrid offspring, that to a large extent had a genetic origin. Individuals carrying influential S. schwerinii haplotypes generally performed well in Sweden but less well in Italy in terms of biomass production. Conclusions: The results indicate that specific genetic elements of S. schwerinii are more suited to Swedish conditions than to those of Italy. Therefore, selection should preferably be conducted separately for such environments in order to maximize biomass production in admixed S. viminalis × S. schwerinii populations.

Implications of accounting for marker-based population structure in the quantitative genetic evaluation of genetic parameters related to growth and wood properties in Norway spruce.

BACKGROUND: Forest geneticists typically use provenances to account for population differences in their improvement schemes; however, the historical records of the imported materials might not be very precise or well-aligned with the genetic clusters derived from advanced molecular techniques. The main objective of this study was to assess the impact of marker-based population structure on genetic parameter estimates related to growth and wood properties and their trade-offs in Norway spruce, by either incorporating it as a fixed effect (model-A) or excluding it entirely from the analysis (model-B). RESULTS: Our results indicate that models incorporating population structure significantly reduce estimates of additive genetic variance, resulting in substantial reduction of narrow-sense heritability. However, these models considerably improve prediction accuracies. This was particularly significant for growth and solid-wood properties, which showed to have the highest population genetic differentiation (QST) among the studied traits. Additionally, although the pattern of correlations remained similar across the models, their magnitude was slightly lower for models that included population structure as a fixed effect. This suggests that selection, consistently performed within populations, might be less affected by unfavourable genetic correlations compared to mass selection conducted without pedigree restrictions. CONCLUSION: We conclude that the results of models properly accounting for population structure are more accurate and less biased compared to those neglecting this effect. This might have practical implications for breeders and forest managers where, decisions based on imprecise selections can pose a high risk to economic efficiency.

Genome Wide Associations of Growth, Phenology, and Plasticity Traits in Willow [Salix viminalis (L.)].

The short rotation biomass crop willow (Salix genera) has been of interest for bioenergy but recently also for biofuel production. For a faster development of new varieties molecular markers could be used as selection tool in an early stage of the breeding cycle. To identify markers associated with growth traits, genome-wide association mapping was conducted using a population of 291 Salix viminalis accessions collected across Europe and Russia and a large set of genotyping-by-sequencing markers. The accessions were vegetatively propagated and planted in replicated field experiments, one in Southern Sweden and one in Central Sweden. Phenology data, including bud burst and leaf senescence, as well as different growth traits were collected and measured repeatedly between 2010 and 2017 at both field environments. A value of the plasticity for each accession was calculated for all traits that were measured the same year in both environments as the normalized accession value in one environment subtracted by the corresponding value in the other environment. Broad-sense accession heritabilities and narrow-sense chip heritabilities ranged from 0.68 to 0.95 and 0.45 to 0.99, respectively for phenology traits and from 0.56 to 0.85 and 0.24 to 0.97 for growth traits indicating a considerable genetic component for most traits. Population structure and kinship between accessions were taken into account in the association analyses. In total, 39 marker-trait associations were found where four were specifically connected to plasticity and interestingly one particular marker was associated to several different plasticity growth traits. Otherwise association consistency was poor, possibly due to accession by environment interactions which were demonstrated by the low structure adjusted accession correlations across environments (ranging from 0.40 to 0.58). However, one marker association with biomass fresh weight was repeatedly observed in the same environment over two harvest years. For some traits where several associations were found, the markers jointly explained over 20% of the accession variation. The result from this study using a population of unrelated accessions has given useful information about marker-trait associations especially highlighting marker-plasticity associations and genotype-by-environment interactions as important factors to take account of in future strategies of Salix breeding.

Genetic variation of biomass recalcitrance in a natural Salix viminalis (L.) population.

BACKGROUND: Salix spp. are high-productivity crops potentially used for lignocellulosic biofuels such as bioethanol. In general, pretreatment is needed to facilitate the enzymatic depolymerization process. Biomass resistance to degradation, i.e., biomass recalcitrance, is a trait which can be assessed by measuring the sugar released after combined pretreatment and enzymatic hydrolysis. We have examined genetic parameters of enzymatic sugar release and other traits related to biorefinery use in a population of 286 natural Salix viminalis clones. Furthermore, we have evaluated phenotypic and genetic correlations between these traits and performed a genomewide association mapping analysis using a set of 19,411 markers. RESULTS: Sugar release (glucose and xylose) after pretreatment and enzymatic saccharification proved highly variable with large genetic and phenotypic variations, and chip heritability estimates (h 2) of 0.23-0.29. Lignin syringyl/guaiacyl (S/G) ratio and wood density were the most heritable traits (h 2 = 0.42 and 0.59, respectively). Sugar release traits were positively correlated, phenotypically and genetically, with biomass yield and lignin S/G ratio. Association mapping revealed seven marker-trait associations below a suggestive significance threshold, including one marker associated with glucose release. CONCLUSIONS: We identified lignin S/G ratio and shoot diameter as heritable traits that could be relatively easily evaluated by breeders, making them suitable proxy traits for developing low-recalcitrance varieties. One marker below the suggestive threshold for marker associations was identified for sugar release, meriting further investigation while also highlighting the difficulties in employing genomewide association mapping for complex traits.

Performance of Seven Tree Breeding Strategies Under Conditions of Inbreeding Depression.

In the domestication and breeding of tree species that suffer from inbreeding depression (ID), the long-term performance of different breeding strategies is poorly known. Therefore, seven tree breeding strategies including single population, subline, selfing, and nucleus breeding were simulated using a multi-locus model with additive, partial, and complete dominance allele effects, and with intermediate, U-shaped, and major allele distributions. The strategies were compared for genetic gain, inbreeding accumulation, capacity to show ID, the frequencies and fixations of unfavorable alleles, and genetic variances in breeding and production populations. Measured by genetic gain of production population, the nucleus breeding and the single breeding population with mass selection strategies were equal or superior to subline and single breeding population with within-family selection strategies in all simulated scenarios, in spite of their higher inbreeding coefficients. Inbreeding and cross-breeding effectively decreased ID and could in some scenarios produce genetic gains during the first few generations. However, in all scenarios, considerable fixation of unfavorable alleles rendered the purging performance of selfing and cross-breeding strategies ineffective, and resulted in substantial inferiority in comparison to the other strategies in the long-term.

Association mapping in Salix viminalis L. (Salicaceae) - identification of candidate genes associated with growth and phenology.

Willow species (Salix) are important as short-rotation biomass crops for bioenergy, which creates a demand for faster genetic improvement and breeding through deployment of molecular marker-assisted selection (MAS). To find markers associated with important adaptive traits, such as growth and phenology, for use in MAS, we genetically dissected the trait variation of a Salix viminalis (L.) population of 323 accessions. The accessions were sampled throughout northern Europe and were established at two field sites in Pustnäs, Sweden, and at Woburn, UK, offering the opportunity to assess the impact of genotype-by-environment interactions (G × E) on trait-marker associations. Field measurements were recorded for growth and phenology traits. The accessions were genotyped using 1536 SNP markers developed from phenology candidate genes and from genes previously observed to be differentially expressed in contrasting environments. Association mapping between 1233 of these SNPs and the measured traits was performed taking into account population structure and threshold selection bias. At a false discovery rate (FDR) of 0.2, 29 SNPs were associated with bud burst, leaf senescence, number of shoots or shoot diameter. The percentage of accession variation (Radj2) explained by these associations ranged from 0.3% to 4.4%, suggesting that the studied traits are controlled by many loci of limited individual impact. Despite this, a SNP in the EARLY FLOWERING 3 gene was repeatedly associated (FDR < 0.2) with bud burst. The rare homozygous genotype exhibited 0.4-1.0 lower bud burst scores than the other genotype classes on a five-grade scale. Consequently, this marker could be promising for use in MAS and the gene deserves further study. Otherwise, associations were less consistent across sites, likely due to their small Radj2 estimates and to considerable G × E interactions indicated by multivariate association analyses and modest trait accession correlations across sites (0.32-0.61).

Functional multi-locus QTL mapping of temporal trends in Scots pine wood traits.

Quantitative trait loci (QTL) mapping of wood properties in conifer species has focused on single time point measurements or on trait means based on heterogeneous wood samples (e.g., increment cores), thus ignoring systematic within-tree trends. In this study, functional QTL mapping was performed for a set of important wood properties in increment cores from a 17-yr-old Scots pine (Pinus sylvestris L.) full-sib family with the aim of detecting wood trait QTL for general intercepts (means) and for linear slopes by increasing cambial age. Two multi-locus functional QTL analysis approaches were proposed and their performances were compared on trait datasets comprising 2 to 9 time points, 91 to 455 individual tree measurements and genotype datasets of amplified length polymorphisms (AFLP), and single nucleotide polymorphism (SNP) markers. The first method was a multilevel LASSO analysis whereby trend parameter estimation and QTL mapping were conducted consecutively; the second method was our Bayesian linear mixed model whereby trends and underlying genetic effects were estimated simultaneously. We also compared several different hypothesis testing methods under either the LASSO or the Bayesian framework to perform QTL inference. In total, five and four significant QTL were observed for the intercepts and slopes, respectively, across wood traits such as earlywood percentage, wood density, radial fiberwidth, and spiral grain angle. Four of these QTL were represented by candidate gene SNPs, thus providing promising targets for future research in QTL mapping and molecular function. Bayesian and LASSO methods both detected similar sets of QTL given datasets that comprised large numbers of individuals.

Comparison of the binding and reactivity of plant and mammalian peroxidases to indole derivatives by computational docking.

The oxidation of melatonin by the mammalian myeloperoxidase (MPO) provides protection against the damaging effects of reactive oxygen species. Indole derivatives, such as melatonin and serotonin, are also substrates of the plant horseradish peroxidase (HRP), but this enzyme exhibits remarkable differences from MPO in the specificity and reaction rates for these compounds. A structural understanding of the determinants of the reactivity of these enzymes to indole derivatives would greatly aid their exploitation for biosynthetic and drug design applications. Consequently, after validation of the docking procedure, we performed computational docking of melatonin and serotonin to structural models of the ferric and compound I and II (co I and co II, respectively) states of HRP and MPO. The substrates dock at the heme edge on the distal side, but with different orientations in the two proteins. The distal cavity is larger in MPO than in HRP; however, in MPO, the substrates make closer contacts with the heme involving ring stacking, whereas in HRP, no ring stacking is observed. The observed differences in substrate binding may contribute to the higher reaction rates and lower substrate specificity of MPO relative to those of HRP. The docking results, along with the previously measured heme-protein reduction potentials, suggest that the differentially lowered reaction rates of co II of HRP and MPO with respect to those of co I could stem from as yet undetermined conformational or electrostatic differences between the co I and co II states of MPO, which are absent in HRP.