October 24, 2023, Prague – In a collaboration between Czech scientists from the Czech University of Life Sciences and the RESDiNET team, a recent groundbreaking scientific publication sheds light on the genomic markers that determine the resistance of Norway spruce to the notorious European spruce bark beetle Ips typographus. The study employs a sophisticated 50K SNP genotyping array, revealing crucial insights into the genetic basis of tree survival during severe bark beetle outbreaks.
Norway spruce, one of the most economically important coniferous species in the Northern Hemisphere, faces escalating threats from drought and bark beetle infestation exacerbated by climate change. Historically, bark beetle outbreaks were sporadic, but recent climatic shifts have led to widespread and recurrent infestations since the 1990s, causing extensive damage to forests. Despite widespread mortality, a small proportion of mature trees, termed the “Last Trees Standing” (LTS), remarkably survive bark beetle gradations. The researchers from the Faculty of Forestry and Wood Sciences of the Czech University of Life Sciences in Prague in collaboration with RESDiNET team members aimed to understand the genetic factors influencing the resistance of mature Norway spruce to bark beetle infestation. “Our goal is to find the genetic markers associated with spruce resistance and survival during bark beetle outbreaks,” says Dr. Rastislav Jakuš, the Head of the Forest Disturbance Ecology team at the Institute of Forest Ecology of the Slovak Academy of Sciences and the RESDiNET project coordinator. “A better understanding of the genetic premises behind bark beetle – spruce interactions will help increase the efficiency of decision-making processes in forest disturbance management.”
The scientists explored the genetic variation related to bark beetle resistance in surviving trees, identifying 12 significant markers (single nucleotide polymorphisms, or SNPs). SNP is a genetic variation occurring at a specific position of a single DNA base. Typically situated in non-coding regions between genes, these genomic variants serve as valuable biological markers. SNPs are used to identify genes linked to various aspects such as health, diseases, and stress responses. In cases where SNPs manifest within a gene or in a nearby regulatory region, they can exert a more direct influence on physiological condition or responses to external challenges by influencing the gene’s functionality.
Field sampling
The study was conducted in the spruce-dominated Bohemian Forest region, a mountainous area located at the cross-border of Czechia, Austria, and Germany. At present, a substantial part of the region’s ecosystems falls under the management of two reserves: the Bavarian Forest National Park in Germany and the Šumava National Park in Czechia. Initially, 400 trees in total were sampled, including 229 surviving trees (LTS), and 171 reference trees. Out of the initially sampled 400 trees, DNA genotyping of sufficient quality was obtained from 383 individuals for analysis, including 102 resistant and 281 susceptible reference trees. Resistant trees (LTS) represent mature, lone-standing trees in the main canopy layer with a diameter at breast height exceeding 35 cm, positioned amidst standing dead beetle-killed trees or decaying wood on the ground—potential hosts for bark beetles. These resistant trees exhibited no signs of bark beetle attacks. Reference trees include mature trees from adjacent unaffected stands or juvenile trees (seedlings) from natural regeneration near identified LTS, maintaining a minimum distance of 30 m to avoid sampling closely related individuals. The samples of mature trees were collected a 15 mm dimeter hole punch, while the seedlings’ needles were cut from the juvenile trees. The cutouts were preserved using silica gel and subsequently stored at a temperature of −80 °C in the laboratory.
Data analysis
The extracted DNA was genotyped on the 50K SNPchip Axiom at the Thermo Fisher genomics facility. In total, 47,445 SNPs were generated. After filtering out tens of thousands of SNPs and quantifying the matrix of kinship, the researchers designed a GWAS (genome-wide association study) model, in which tree status (LTS vs. reference tree) was a response variable. The genotype was considered a random effect, and the study site was a fixed effect. SNPs that demonstrated a significant association with survival underwent further investigation using PlantGenIE and PLAZA 5.0, web-based platforms that leverage the accessibility of the Norway spruce genome.
Genetic markers of spruce resistance to bark beetles
Out of twelve significant SNPs, the researchers identified three, namely MA_35335g0010 (PAB00038352), MA_51088g0010 (PAB00045124), and MA_77097g0010 (PAB00054584), for which Arabidopsis thaliana gene orthologs are known. Orthologs are genes that originated from a shared ancestral gene through speciation and typically maintain a similar function across various species. Interestingly, all three genes appeared to be directly or indirectly involved in stress-induced plant cellular regulation.
One of these genes (MA_51088g0010 (PAB00045124)) is associated with methylation processes. The gene acts in macromolecule biosynthetic processes and enables S-adenosylmethionine-dependent methyltransferase activity. Methyltransferases play an critical role in the functioning of living systems. This large family of ubiquitous enzymes is crucial for the transfer of methyl groups from AdoMet to nitrogen, carbon, or oxygen compounds of a wide variety of substrates, including DNA, proteins and small-molecule secondary metabolites. DNA methylation is important for tree responses to various stresses. Trees, like other organisms, employ epigenetic modifications, including DNA methylation, as a means of adapting to environmental changes. DNA methylation is a critical epigenetic mechanism for regulating gene expression. In response to stress, certain genes may be upregulated or downregulated through changes in DNA methylation patterns. This regulation allows trees to activate stress-responsive genes and pathways. DNA methylation patterns can act as a form of epigenetic memory. Trees exposed to stress may undergo changes in DNA methylation that persist even after the stress has been alleviated. This “memory” can influence the tree’s future responses to similar stressors, providing a form of stress priming. Epigenetic changes, including DNA methylation, can contribute to the adaptation and evolution of tree populations in response to long-term environmental stress. These changes may be passed on to subsequent generations, influencing the overall resilience of tree populations. DNA methylation can be involved in the regulation of defence-related genes. For example, in response to bark beetle attacks, resistant trees may undergo changes in DNA methylation to enhance their defence mechanisms, such as the production of antifungicides, resin and secondary metabolites toxic to beetles. DNA methylation works in concert with other epigenetic modifications, such as histone modifications and small RNA pathways. The interplay between these mechanisms contributes to the overall epigenetic landscape that shapes the tree’s stress responses.
The second gene, MA_77097g0010 (PAB00054584) and its best ortholog AT3G06010, mediates the temporary growth arrest that is induced by stress perception. The third gene, MA_35335g0010 (ortholog AT5G13240), regulates transcription from RNA polymerase III (Pol III) promoter. Transcription in eukaryotic cells is performed by three RNA polymerases, with the largest among them being RNA polymerase III. This polymerase is responsible for transcribing a diverse array of short non-coding RNAs, including transfer RNAs (tRNAs) and the 5S ribosomal RNA (rRNA), along with other small RNAs. Pol III-mediated transcription is highly dynamic and regulated in response to changes in cell growth, cell proliferation and stress. Recent investigations have identified Pol III as an evolutionarily conserved determinant influencing organismal lifespan, operating downstream of the mechanistic target of rapamycin complex 1 (mTORC1). Inhibition of Pol III has been demonstrated to extend lifespan in various organisms, encompassing yeast, nematodes, and fruit flies. Intriguingly, the activation of Pol III, achieved through the impediment of its master repressor, Maf1, has also been documented to confer longevity benefits in model organisms, including mice.
Thus, the identified genes play vital roles in transcription regulation, methyltransferase activity, and growth response to environmental stresses, providing insights into the complex mechanisms of spruce resistance to bark beetles.
Geographic differentiation of Norway spruce populations
Additionally, the population–genetic structure of all studied trees was investigated using Discriminant Analysis of Principal Components analysis. The results indicate that trees originating from the targeted sites differed more among the sites than they did between the groups by tree status (resistant vs reference trees). The whole genetic differentiation was low yet significant among all compared trees grouped by status and site. This discovery aligns with various studies documenting minimal genetic differentiation among subpopulations of Norway spruce, a pattern observed in investigations using both microsatellite and SNP markers. Typically, this phenomenon is ascribed to inherent species characteristics, particularly the robust influence of gene flow. Factors such as human-assisted regeneration and the deliberate spread of the species beyond its natural habitat can also significantly contribute to maintaining a notable genetic similarity across various subpopulations.
In conclusion, the research unveils the potential genetic markers for Norway spruce resistance to bark beetles, offering applications in selective breeding, forest management, and forest health monitoring to mitigate the impact of bark beetle infestations. The findings point to intricate regulatory mechanisms connected to defence against bark beetle attacks, emphasizing the need for further exploration and understanding of the complex spruce-bark beetle interactions.
The full research paper can be found here.
