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.

Inbreeding Effects on the Performance and Genomic Prediction for Polysomic Tetraploid Potato Offspring Grown at High Nordic Latitudes.

Inbreeding depression (ID) is caused by increased homozygosity in the offspring after selfing. Although the self-compatible, highly heterozygous, tetrasomic polyploid potato (Solanum tuberosum L.) suffers from ID, some argue that the potential genetic gains from using inbred lines in a sexual propagation system of potato are too large to be ignored. The aim of this research was to assess the effects of inbreeding on potato offspring performance under a high latitude and the accuracy of the genomic prediction of breeding values (GEBVs) for further use in selection. Four inbred (S1) and two hybrid (F1) offspring and their parents (S0) were used in the experiment, with a field layout of an augmented design with the four S0 replicated in nine incomplete blocks comprising 100, four-plant plots at Umeå (63°49'30″ N 20°15'50″ E), Sweden. S0 was significantly (p < 0.01) better than both S1 and F1 offspring for tuber weight (total and according to five grading sizes), tuber shape and size uniformity, tuber eye depth and reducing sugars in the tuber flesh, while F1 was significantly (p < 0.01) better than S1 for all tuber weight and uniformity traits. Some F1 hybrid offspring (15-19%) had better total tuber yield than the best-performing parent. The GEBV accuracy ranged from -0.3928 to 0.4436. Overall, tuber shape uniformity had the highest GEBV accuracy, while tuber weight traits exhibited the lowest accuracy. The F1 full sib's GEBV accuracy was higher, on average, than that of S1. Genomic prediction may facilitate eliminating undesired inbred or hybrid offspring for further use in the genetic betterment of potato.

Life stage-specific inbreeding depression in long-lived Pinaceae species depends on population connectivity.

Inbreeding depression (ID) is a fundamental selective pressure that shapes mating systems and population genetic structures in plants. Although it has been shown that ID varies over the life stages of shorter-lived plants, less is known about how the fitness effects of inbreeding vary across life stages in long-lived species. We conducted a literature survey in the Pinaceae, a tree family known to harbour some of the highest mutational loads ever reported. Using a meta-regression model, we investigated distributions of inbreeding depression over life stages, adjusting for effects of inbreeding levels and the genetic differentiation of populations within species. The final dataset contained 147 estimates of ID across life stages from 41 studies. 44 Fst estimates were collected from 40 peer-reviewed studies for the 18 species to aid genetic differentiation modelling. Partitioning species into fragmented and well-connected groups using Fst resulted in the best way (i.e. trade-off between high goodness-of-fit of the model to the data and reduced model complexity) to incorporate genetic connectivity in the meta-regression analysis. Inclusion of a life stage term and its interaction with the inbreeding coefficient (F) dramatically increased model precision. We observed that the correlation between ID and F was significant at the earliest life stage. Although partitioning of species populations into fragmented and well-connected groups explained little of the between-study heterogeneity, the inclusion of an interaction between life stage and population differentiation revealed that populations with fragmented distributions suffered lower inbreeding depression at early embryonic stages than species with well-connected populations. There was no evidence for increased ID in late life stages in well-connected populations, although ID tended to increase across life stages in the fragmented group. These findings suggest that life stage data should be included in inbreeding depression studies and that inbreeding needs to be managed over life stages in commercial populations of long-lived plants.

Annulohypoxylon sp. strain MUS1, an endophytic fungus isolated from Taxus wallichiana Zucc., produces taxol and other bioactive metabolites.

The current study focuses on the isolation and in vitro characterization of bioactive metabolites produced by endophytic fungi isolated from the Himalayan yew (Taxus wallichiana Zucc.). The endophytic fungi were isolated on artificial media from inner tissues of bark and needles. Antimicrobial and antioxidant activity, along with total phenolic- and flavonoid-content assays were used in the evaluation of bioactivity of the fermented crude extracts. The ability of the endophytes to produce the anticancer compound Taxol was also analyzed using thin-layer chromatography (TLC) and reverse-phase high-performance liquid chromatography (RP-HPLC). A total of 16 fungal morphotypes were obtained from asymptomatic inner tissues of the bark and needles of T. wallichiana. Among the 16 isolates, the ethyl acetate (EA) fraction of isolate MUS1, showed antibacterial and antifungal activity against all test-pathogens used (Streptococcus faecalis ATCC 19433, Staphylococcus aureus ATCC 12600, Bacillus subtilis ATCC 6633, Escherichia coli ATCC 25922, Salmonella enterica ATCC 13076, Pseudomonas aeruginosa ATCC 27853, and Candida albicans). MUS1 showed significant inhibition against Pseudomonas aeruginosa ATCC 27853 (minimum inhibitory concentration (MIC): 250 µg/ml) and the pathogenic yeast, Candida albicans (MIC: 125 µg/ml). Antioxidant activity, total phenolic, and total flavonoid content as well as in vitro Taxol production were evaluated for EA fraction of isolate MUS1. Antioxidant activity was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. At a concentration of 100 µg/ml, the % DPPH radical scavenging activity was 83.15 ± 0.40, 81.62 ± 0.11, and 62.36 ± 0.29, for ascorbic acid, butylated hydroxytoluene (BHT), and the EA fraction of MUS1, respectively. The DPPH-Half maximal inhibitory concentration (DPPH-IC50) value for the EA fraction was 81.52 ± 0.23 µg/ml, compared to BHT (62.87 ± 0.08 µg/ml) and ascorbic acid (56.15 ± 0.19 µg/ml). The total phenolic and flavonoid content in the EA fraction were 16.90 ± 0.075 µg gallic acid equivalent (GAE) and 11.59 ± 0.148 µg rutin equivalent (RE), per mg of dry crude extract, respectively. TLC and RP-HPLC analysis showed that the isolate MUS1 also produces Taxol (282.05 µg/l of fermentation broth). Isolate MUS1 was identified as Annulohypoxylon sp. by internal transcribed spacer (ITS) sequencing. Having the ability to produce antimicrobial and antioxidant metabolites, as well as the anticancer compound Taxol, makes Annulohypoxylon sp. strain MUS1, a promising candidate for further study of naturally occurring bioactive metabolites. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02693-z.

Genotypic variation in Norway spruce correlates to fungal communities in vegetative buds.

The taxonomically diverse phyllosphere fungi inhabit leaves of plants. Thus, apart from the fungi's dispersal capacities and environmental factors, the assembly of the phyllosphere community associated with a given host plant depends on factors encoded by the host's genome. The host genetic factors and their influence on the assembly of phyllosphere communities under natural conditions are poorly understood, especially in trees. Recent work indicates that Norway spruce (Picea abies) vegetative buds harbour active fungal communities, but these are hitherto largely uncharacterized. This study combines internal transcribed spacer sequencing of the fungal communities associated with dormant vegetative buds with a genome-wide association study (GWAS) in 478 unrelated Norway spruce trees. The aim was to detect host loci associated with variation in the fungal communities across the population, and to identify loci correlating with the presence of specific, latent, pathogens. The fungal communities were dominated by known Norway spruce phyllosphere endophytes and pathogens. We identified six quantitative trait loci (QTLs) associated with the relative abundance of the dominating taxa (i.e., top 1% most abundant taxa). Three additional QTLs associated with colonization by the spruce needle cast pathogen Lirula macrospora or the cherry spruce rust (Thekopsora areolata) in asymptomatic tissues were detected. The identification of the nine QTLs shows that the genetic variation in Norway spruce influences the fungal community in dormant buds and that mechanisms underlying the assembly of the communities and the colonization of latent pathogens in trees may be uncovered by combining molecular identification of fungi with GWAS.

Global gene expression analysis in etiolated and de-etiolated seedlings in conifers.

Plant life cycle begins with germination of seed below the ground. This is followed by seedling's development in the dark: skotomorphogenesis; and then a light-mediated growth: photomorphogenesis. After germination, hypocotyl grows rapidly to reach the sun, which involves elongation of shoot at the expense of root and cotyledons. Upon reaching ground level, seedling gets exposed to sunlight following a switch from the etiolated (skotomorphogenesis) to the de-etiolated (photomorphogenesis) stage, involving a series of molecular and physiological changes. Gymnosperms have evolved very differently and adopted diverse strategies as compared to angiosperms; with regards to response to light quality, conifers display a very mild high-irradiance response as compared to angiosperms. Absence of apical hook and synthesis of chlorophyll during skotomorphogenesis are two typical features in gymnosperms which differentiate them from angiosperms (dicots). Information regarding etiolation and de-etiolation processes are well understood in angiosperms, but these mechanisms are less explored in conifer species. It is, therefore, interesting to know how similar these processes are in conifers as compared to angiosperms. We performed a global expression analysis (RNA sequencing) on etiolated and de-etiolated seedlings of two economically important conifer species in Sweden to review the differentially expressed genes associated with the two processes. Based on the results, we propose that high levels of HY5 in conifers under DARK condition coupled with expression of few other genes associated with de-etiolation in angiosperms e.g. SPA, DET1 (lower expression under DARK) and CRY1 (higher expression under DARK), leads to partial expression of photomorphogenic genes in the DARK phenotype in conifers as displayed by absence of apical hook, opening of cotyledons and synthesis of chlorophyll.

Genome-wide association study identified novel candidate loci affecting wood formation in Norway spruce.

Norway spruce is a boreal forest tree species of significant ecological and economic importance. Hence there is a strong imperative to dissect the genetics underlying important wood quality traits in the species. We performed a functional genome-wide association study (GWAS) of 17 wood traits in Norway spruce using 178 101 single nucleotide polymorphisms (SNPs) generated from exome genotyping of 517 mother trees. The wood traits were defined using functional modelling of wood properties across annual growth rings. We applied a Least Absolute Shrinkage and Selection Operator (LASSO-based) association mapping method using a functional multilocus mapping approach that utilizes latent traits, with a stability selection probability method as the hypothesis testing approach to determine a significant quantitative trait locus. The analysis provided 52 significant SNPs from 39 candidate genes, including genes previously implicated in wood formation and tree growth in spruce and other species. Our study represents a multilocus GWAS for complex wood traits in Norway spruce. The results advance our understanding of the genetics influencing wood traits and identifies candidate genes for future functional studies.

An Ultra-Dense Haploid Genetic Map for Evaluating the Highly Fragmented Genome Assembly of Norway Spruce (Picea abies).

Norway spruce (Picea abies (L.) Karst.) is a conifer species of substanital economic and ecological importance. In common with most conifers, the P. abies genome is very large (∼20 Gbp) and contains a high fraction of repetitive DNA. The current P. abies genome assembly (v1.0) covers approximately 60% of the total genome size but is highly fragmented, consisting of >10 million scaffolds. The genome annotation contains 66,632 gene models that are at least partially validated (www.congenie.org), however, the fragmented nature of the assembly means that there is currently little information available on how these genes are physically distributed over the 12 P. abies chromosomes. By creating an ultra-dense genetic linkage map, we anchored and ordered scaffolds into linkage groups, which complements the fine-scale information available in assembly contigs. Our ultra-dense haploid consensus genetic map consists of 21,056 markers derived from 14,336 scaffolds that contain 17,079 gene models (25.6% of the validated gene models) that we have anchored to the 12 linkage groups. We used data from three independent component maps, as well as comparisons with previously published Picea maps to evaluate the accuracy and marker ordering of the linkage groups. We demonstrate that approximately 3.8% of the anchored scaffolds and 1.6% of the gene models covered by the consensus map have likely assembly errors as they contain genetic markers that map to different regions within or between linkage groups. We further evaluate the utility of the genetic map for the conifer research community by using an independent data set of unrelated individuals to assess genome-wide variation in genetic diversity using the genomic regions anchored to linkage groups. The results show that our map is sufficiently dense to enable detailed evolutionary analyses across the P. abies genome.

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.

Demographic history has influenced nucleotide diversity in European Pinus sylvestris populations.

To infer the role of natural selection in shaping standing genetic diversity, it is necessary to assess the genomewide impact of demographic history on nucleotide diversity. In this study we analyzed sequence diversity of 16 nuclear loci in eight Pinus sylvestris populations. Populations were divided into four geographical groups on the basis of their current location and the geographical history of the region: northern Europe, central Europe, Spain, and Turkey. There were no among-group differences in the level of silent nucleotide diversity, which was approximately 0.005/bp in all groups. There was some evidence that linkage disequilibrium extended further in northern Europe than in central Europe: the estimates of the population recombination rate parameter, rho, were 0.0064 and 0.0294, respectively. The summary statistics of nucleotide diversity in central and northern European populations were compatible with an ancient bottleneck rather than the standard neutral model.