Functional Characterization of PmDXR, a Critical Rate-Limiting Enzyme, for Turpentine Biosynthesis in Masson Pine (Pinus massoniana Lamb.).

As one of the largest and most diverse classes of specialized metabolites in plants, terpenoids (oprenoid compounds, a type of bio-based material) are widely used in the fields of medicine and light chemical products. They are the most important secondary metabolites in coniferous species and play an important role in the defense system of conifers. Terpene synthesis can be promoted by regulating the expressions of terpene synthase genes, and the terpene biosynthesis pathway has basically been clarified in Pinus massoniana, in which there are multiple rate-limiting enzymes and the rate-limiting steps are difficult to determine, so the terpene synthase gene regulation mechanism has become a hot spot in research. Herein, we amplified a PmDXR gene (GenBank accession no. MK969119.1) of the MEP pathway (methyl-erythritol 4-phosphate) from Pinus massoniana. The DXR enzyme activity and chlorophyll a, chlorophyll b and carotenoid contents of overexpressed Arabidopsis showed positive regulation. The PmDXR gene promoter was a tissue-specific promoter and can respond to ABA, MeJA and GA stresses to drive the expression of the GUS reporter gene in N. benthamiana. The DXR enzyme was identified as a key rate-limiting enzyme in the MEP pathway and an effective target for terpene synthesis regulation in coniferous species, which can further lay the theoretical foundation for the molecularly assisted selection of high-yielding lipid germplasm of P. massoniana, as well as provide help in the pathogenesis of pine wood nematode disease.

Genome-wide identification and expression analysis of ARF gene family in embryonic development of Korean pine (Pinus koraiensis).

BACKGROUND: The Auxin Responsive Factor (ARF) family plays a crucial role in mediating auxin signal transduction and is vital for plant growth and development. However, the function of ARF genes in Korean pine (Pinus koraiensis), a conifer species of significant economic value, remains unclear. RESULTS: This study utilized the whole genome of Korean pine to conduct bioinformatics analysis, resulting in the identification of 13 ARF genes. A phylogenetic analysis revealed that these 13 PkorARF genes can be classified into 4 subfamilies, indicating the presence of conserved structural characteristics within each subfamily. Protein interaction prediction indicated that Pkor01G00962.1 and Pkor07G00704.1 may have a significant role in regulating plant growth and development as core components of the PkorARFs family. Additionally, the analysis of RNA-seq and RT-qPCR expression patterns suggested that PkorARF genes play a crucial role in the development process of Korean pine. CONCLUSION: Pkor01G00962.1 and Pkor07G00704.1, which are core genes of the PkorARFs family, play a potentially crucial role in regulating the fertilization and developmental process of Korean pine. This study provides a valuable reference for investigating the molecular mechanism of embryonic development in Korean pine and establishes a foundation for cultivating high-quality Korean pine.

Phytophthora pluvialis maintenance, spore production and detached needle assays.

Phytophthora pluvialis is an oomycete that primarily infects Pinus radiata and Pseudotsuga menziesii causing the destructive foliar disease red needle cast (RNC). Recent observations show that P. pluvialis can also infect western hemlock inducing resinous cankers. High-throughput and reproducible infection assays are integral to find key information on tree health and oomycete pathogenicity. In this protocol, we describe the propagation and spore induction of P. pluvialis, followed by detached needle assays for verification and quantification of virulence of P. pluvialis in P. radiata needles. These needle assays can be employed for high-throughput screening of tree needles with diverse genetic backgrounds. In downstream analysis, Quantitative PCR (qPCR) was utilized to assess relative gene expression, as exemplified by candidate RxLR effector protein PpR01. Additional techniques like RNA sequencing, metabolomics, and proteomics can be combined with needle assays and can offer comprehensive insights into P. pluvialis infection mechanisms.

Transcriptome Study of Bursaphelenchus xylophilus Treated with Fomepizole Reveals a Serine/Threonine-Protein Phosphatase Gene that Is Substantially Linked with Vitality and Pathogenicity.

Bursaphelenchus xylophilus, the pine wood nematode (PWN), is the causal agent of pine wilt disease (PWD), which causes enormous economic loss annually. According to our previous research, fomepizole, as a selective inhibitor of PWN alcohol dehydrogenase (ADH), has the potential to be a preferable lead compound for developing novel nematicides. However, the underlying molecular mechanism is still unclear. The result of molecular docking showed that the stronger interactions between fomepizole and PWN ADH at the active site of ADH were attributed to hydrogen bonds. Low-dose fomepizole had a substantial negative impact on the egg hatchability, development, oviposition, and lifespan of PWN. Transcriptome analysis indicated that 2,124 upregulated genes and 490 downregulated genes in fomepizole-treated PWN were obtained. Kyoto Encyclopedia of Genes and Genomes enrichment analysis of differentially expressed genes indicated that fomepizole could be involved in controlling PWN vitality mainly by regulating key signaling pathways, such as the ribosome, hippo signaling pathway, and lysosome. Remarkably, the results of RNA interference indicated that the downregulated serine/threonine-protein phosphatase gene (stpp) could reduce the egg hatchability, development, oviposition, and lifespan of PWN, which was closely similar to the consequences of nematodes with low-dose fomepizole treatment. In addition, the silencing of stpp resulted in weakness of PWN pathogenicity, which indicated that stpp could be a potential drug target to control PWN.

Physiological responses to drought stress of three pine species and comparative transcriptome analysis of Pinus yunnanensis var. pygmaea.

Drought stress can significantly affect plant growth, development, and yield. Fewer comparative studies have been conducted between different species of pines, particularly involving Pinus yunnanensis var. pygmaea (P. pygmaea). In this study, the physiological indices, photosynthetic pigment and related antioxidant enzyme changes in needles from P. pygmaea, P. elliottii and P. massoniana under drought at 0, 7, 14, 21, 28 and 35 d, as well as 7 days after rehydration, were measured. The PacBio single-molecule real-time (SMRT) and Illumina RNA sequencing were used to uncover the gene expression differences in P. pygmaea under drought and rehydration conditions. The results showed that the total antioxidant capacity (TAOC) of P. pygmaea was significantly higher than P. massoniana and P. elliottii. TAOC showed a continuous increase trend across all species. Soluble sugar (SS), starch content and non-structural carbohydrate (NSC) of all three pines displayed a "W" pattern, declining initially, increasing, and then decreasing again. P. pygmaea exhibits stronger drought tolerance and greater recovery ability under prolonged drought conditions. Through the PacBio SMRT-seq, a total of 50,979 high-quality transcripts were generated, and 6,521 SSR and 5,561 long non-coding RNAs (LncRNAs) were identified. A total of 2310, 1849, 5271, 5947, 7710, and 6854 differentially expressed genes (DEGs) were identified compared to the control (Pp0D) in six pair-wise comparisons of treatment versus control. bHLH, NAC, ERF, MYB_related, C3H transcription factors (TFs) play an important role in drought tolerance of P. pygmaea. KEGG enrichment analysis and Gene set enrichment analysis (GSEA) analysis showed that P. pygmaea may respond to drought by enhancing metabolic processes such as ABA signaling pathway, alpha-linolenic acid. Weighted gene co-expression network analysis (WGCNA) revealed GST, CAT, LEC14B, SEC23 were associated with antioxidant enzyme activity and TAOC. This study provides a basis for further research on drought tolerance differences among coniferous species.

Comparative analysis of differential gene expression reveals novel insights into the heteroblastic foliage functional traits of Pinus massoniana seedlings.

Pinus massoniana needles, rich in medicinal polysaccharides and flavonoids, undergo heteroblastic foliage, transitioning from primary needles (PN) to secondary needles (SN) during growth, resulting in altered functional traits. Despite its significance, the molecular regulatory mechanisms governing these traits remain unclear. This study employs Iso-Seq and RNA-Seq analyses to explore differentially expressed genes (DEGs) associated with functional traits throughout the main growth season of heteroblastic foliage. Co-expression network analysis identified 34 hub genes and 17 key transcription factors (TFs) influencing light-harvesting antenna, photosystem I and II, crucial in photosynthesis regulation. Additionally, 14 genes involved in polysaccharide metabolism pathways, synthesizing sucrose, glucose, UDP sugars, and xylan, along with four genes in flavonoid biosynthesis pathways, regulating p-coumaroyl-CoA, quercetin, galangin, and myricetin production, exhibited differential expression between PN and SN. Further analysis unveils a highly interconnected network among these genes, forming a pivotal cascade of TFs and DEGs. Therefore, heteroblastic changes significantly impact needle functional traits, potentially affecting the pharmacological properties of PN and SN. Thus, these genomic insights into understanding the molecular-level differences of heteroblastic foliage, thereby establishing a foundation for advancements in the pharmaceutical industry related to needle-derived products.

Evolutionary radiation of the Eurasian Pinus species under pervasive gene flow.

Evolutionary radiation, a pivotal aspect of macroevolution, offers valuable insights into evolutionary processes. The genus Pinus is the largest genus in conifers with c . 90% of the extant species emerged in the Miocene, which signifies a case of rapid diversification. Despite this remarkable history, our understanding of the mechanisms driving radiation within this expansive genus has remained limited. Using exome capture sequencing and a fossil-calibrated phylogeny, we investigated the divergence history, niche diversification, and introgression among 13 closely related Eurasian species spanning climate zones from the tropics to the boreal Arctic. We detected complex introgression among lineages in subsection Pinus at all stages of the phylogeny. Despite this widespread gene exchange, each species maintained its genetic identity and showed clear niche differentiation. Demographic analysis unveiled distinct population histories among these species, which further influenced the nucleotide diversity and efficacy of purifying and positive selection in each species. Our findings suggest that radiation in the Eurasian pines was likely fueled by interspecific recombination and further reinforced by their adaptation to distinct environments. Our study highlights the constraints and opportunities for evolutionary change, and the expectations of future adaptation in response to environmental changes in different lineages.

A genome sequence for the threatened whitebark pine.

Whitebark pine (WBP, Pinus albicaulis) is a white pine of subalpine regions in the Western contiguous United States and Canada. WBP has become critically threatened throughout a significant part of its natural range due to mortality from the introduced fungal pathogen white pine blister rust (WPBR, Cronartium ribicola) and additional threats from mountain pine beetle (Dendroctonus ponderosae), wildfire, and maladaptation due to changing climate. Vast acreages of WBP have suffered nearly complete mortality. Genomic technologies can contribute to a faster, more cost-effective approach to the traditional practices of identifying disease-resistant, climate-adapted seed sources for restoration. With deep-coverage Illumina short reads of haploid megagametophyte tissue and Oxford Nanopore long reads of diploid needle tissue, followed by a hybrid, multistep assembly approach, we produced a final assembly containing 27.6 Gb of sequence in 92,740 contigs (N50 537,007 bp) and 34,716 scaffolds (N50 2.0 Gb). Approximately 87.2% (24.0 Gb) of total sequence was placed on the 12 WBP chromosomes. Annotation yielded 25,362 protein-coding genes, and over 77% of the genome was characterized as repeats. WBP has demonstrated the greatest variation in resistance to WPBR among the North American white pines. Candidate genes for quantitative resistance include disease resistance genes known as nucleotide-binding leucine-rich repeat receptors (NLRs). A combination of protein domain alignments and direct genome scanning was employed to fully describe the 3 subclasses of NLRs. Our high-quality reference sequence and annotation provide a marked improvement in NLR identification compared to previous assessments that leveraged de novo-assembled transcriptomes.

Genomics of the expanding pine pathogen Lecanosticta acicola reveals patterns of ongoing genetic admixture.

Lecanosticta acicola is the causal agent for brown spot needle blight that affects pine trees across the northern hemisphere. Based on marker genes and microsatellite data, two distinct lineages have been identified that were introduced into Europe on two separate occasions. Despite their overall distinct geographic distribution, they have been found to coexist in regions of northern Spain and France. Here, we present the first genome-wide study of Lecanosticta acicola, including assembly of the reference genome and a population genomics analysis of 70 natural isolates from northern Spain. We show that most of the isolates belong to the southern lineage but show signs of introgression with northern lineage isolates, indicating mating between the two lineages. We also identify phenotypic differences between the two lineages based on the activity profiles of 20 enzymes, with introgressed strains being more phenotypically similar to members of the southern lineage. In conclusion, we show undergoing genetic admixture between the two main lineages of L. acicola in a region of recent expansion. IMPORTANCE: Lecanosticta acicola is a fungal pathogen causing severe defoliation, growth reduction, and even death in more than 70 conifer species. Despite the increasing incidence of this species, little is known about its population dynamics. Two divergent lineages have been described that have now been found together in regions of France and Spain, but it is unknown how these mixed populations evolve. Here we present the first reference genome for this important plant pathogenic fungi and use it to study the population genomics of 70 isolates from an affected forest in the north of Spain. We find signs of introgression between the two main lineages, indicating that active mating is occurring in this region which could propitiate the appearance of novel traits in this species. We also study the phenotypic differences across this population based on enzymatic activities on 20 compounds.

Pine wilt disease: what do we know from proteomics?

Pine wilt disease (PWD) is a devastating forest disease caused by the pinewood nematode (PWN), Bursaphelenchus xylophilus, a migratory endoparasite that infects several coniferous species. During the last 20 years, advances have been made for understanding the molecular bases of PWN-host trees interactions. Major advances emerged from transcriptomic and genomic studies, which revealed some unique features related to PWN pathogenicity and constituted fundamental data that allowed the development of postgenomic studies. Here we review the proteomic approaches that were applied to study PWD and integrated the current knowledge on the molecular basis of the PWN pathogenicity. Proteomics has been useful for understanding cellular activities and protein functions involved in PWN-host trees interactions, shedding light into the mechanisms associated with PWN pathogenicity and being promising tools to better clarify host trees PWN resistance/susceptibility.

Analyses of high spatial resolution datasets identify genes associated with multi-layered secondary cell wall thickening in Pinus bungeana.

BACKGROUND AND AIMS: Secondary cell wall (SCW) thickening is a major cellular developmental stage determining wood structure and properties. Although the molecular regulation of cell wall deposition during tracheary element differentiation has been well established in primary growth systems, less is known about the gene regulatory processes involved in the multi-layered SCW thickening of mature trees. METHODS: Using third-generation [long-read single-molecule real-time (SMRT)] and second-generation [short-read sequencing by synthesis (SBS)] sequencing methods, we established a Pinus bungeana transcriptome resource with comprehensive functional and structural annotation for the first time. Using these approaches, we generated high spatial resolution datasets for the vascular cambium, xylem expansion regions, early SCW thickening, late SCW thickening and mature xylem tissues of 71-year-old Pinus bungeana trees. KEY RESULTS: A total of 79 390 non-redundant transcripts, 31 808 long non-coding RNAs and 5147 transcription factors were annotated and quantified in different xylem tissues at all growth and differentiation stages. Furthermore, using this high spatial resolution dataset, we established a comprehensive transcriptomic profile and found that members of the NAC, WRKY, SUS, CESA and LAC gene families are major players in early SCW formation in tracheids, whereas members of the MYB and LBD transcription factor families are highly expressed during late SCW thickening. CONCLUSIONS: Our results provide new molecular insights into the regulation of multi-layered SCW thickening in conifers. The high spatial resolution datasets provided can serve as important gene resources for improving softwoods.

Genetic parameter changes and age-age correlations in Pinus koraiensis growth over 40-year progeny testing.

BACKGROUND: Early selection in tree breeding could be achieved by addressing the longevity of tree improvement activities. Genetic parameter changes and age-age correlations are essential for determining the optimal timing of early selection. Practical tracking of genetic parameters of Pinus koraiensis, a major timber species with economic and ecological value, has become feasible as its progeny testing has entered the mid-term age in Korea. However, research on the age-age correlation of P. koraiensis as progeny trials approach rotation age is limited. This study aimed to investigate genetic parameter trends and age-age correlations in P. koraiensis progeny. P. koraiensis progeny were assessed at two sites using a linear mixed-effects model with two-dimensional spatial autoregressive structure. Height, diameter, and volume growth were measured in 11 assessments over 40 years. RESULTS: Genetic parameters, such as height and diameter, showed different patterns of change. The heritability ranged for the three growth traits in 0.083-0.710, 0.288-0.781, and 0.299-0.755 across the sites and age. Height heritability and its coefficient of variance decreased, whereas the diameter and volume estimates remained relatively constant. Correlations with Age 40 for phenotypic, genetic, and rank of breeding values ranged between 0.16 and 0.92, 0.594 and 0.988, and 0.412 and 0.965, respectively. These correlations generally increased as the age approached Age 40, with particularly high levels observed at Age 26 and Age 30. CONCLUSION: The observed genetic trends in P. koraiensis progeny testing offer valuable insights for early and precise selection. Notably, selecting superior genotypes at Ages 26-30 is supported by discernible genetic gains and robust correlations. Future research should integrate unbalanced data for selecting mother trees or families and conduct a comprehensive economic analysis of early selection to validate its practical benefits.

Response mechanism of carbon metabolism of Pinus massoniana to gradient high temperature and drought stress.

BACKGROUND: The carbon metabolism pathway is of paramount importance for the growth and development of plants, exerting a pivotal regulatory role in stress responses. The exacerbation of drought impacts on the plant carbon cycle due to global warming necessitates comprehensive investigation into the response mechanisms of Masson Pine (Pinus massoniana Lamb.), an exemplary pioneer drought-tolerant tree, thereby establishing a foundation for predicting future forest ecosystem responses to climate change. RESULTS: The seedlings of Masson Pine were utilized as experimental materials in this study, and the transcriptome, metabolome, and photosynthesis were assessed under varying temperatures and drought intensities. The findings demonstrated that the impact of high temperature and drought on the photosynthetic rate and transpiration rate of Masson Pine seedlings was more pronounced compared to individual stressors. The analysis of transcriptome data revealed that the carbon metabolic pathways of Masson Pine seedlings were significantly influenced by high temperature and drought co-stress, with a particular impact on genes involved in starch and sucrose metabolism. The metabolome analysis revealed that only trehalose and Galactose 1-phosphate were specifically associated with the starch and sucrose metabolic pathways. Furthermore, the trehalose metabolic heat map was constructed by integrating metabolome and transcriptome data, revealing a significant increase in trehalose levels across all three comparison groups. Additionally, the PmTPS1, PmTPS5, and PmTPPD genes were identified as key regulatory genes governing trehalose accumulation. CONCLUSIONS: The combined effects of high temperature and drought on photosynthetic rate, transpiration rate, transcriptome, and metabolome were more pronounced than those induced by either high temperature or drought alone. Starch and sucrose metabolism emerged as the pivotal carbon metabolic pathways in response to high temperature and drought stress in Masson pine. Trehalose along with PmTPS1, PmTPS5, and PmTPPD genes played crucial roles as metabolites and key regulators within the starch and sucrose metabolism.

Identification and Expression Patterns of WOX Transcription Factors under Abiotic Stresses in Pinus massoniana.

WUSCHEL-related homeobox (WOX) transcription factors (TFs) play a crucial role in regulating plant development and responding to various abiotic stresses. However, the members and functions of WOX proteins in Pinus massoniana remain unclear. In this study, a total of 11 WOX genes were identified, and bioinformatics methods were used for preliminary identification and analysis. The phylogenetic tree revealed that most PmWOXs were distributed in ancient and WUS clades, with only one member found in the intermediate clade. We selected four highly conserved WOX genes within plants for further expression analysis. These genes exhibited expressions across almost all tissues, while PmWOX2, PmWOX3, and PmWOX4 showed high expression levels in the callus, suggesting their potential involvement in specific functions during callus development. Expression patterns under different abiotic stresses indicated that PmWOXs could participate in resisting multiple stresses in P. massoniana. The identification and preliminary analysis of PmWOXs lay the foundation for further research on analyzing the resistance molecular mechanism of P. massoniana to abiotic stresses.

Like mother like son: Transgenerational memory and cross-tolerance from drought to heat stress are identified in chloroplast proteome and seed provisioning in Pinus radiata.

How different stressors impact plant health and memory when they are imposed in different generations in wild ecosystems is still scarce. Here, we address how different environments shape heritable memory for the next generation in seeds and seedlings of Pinus radiata, a long-lived species with economic interest. The performance of the seedlings belonging to two wild clonal subpopulations (optimal fertirrigation vs. slightly stressful conditions) was tested under heat stress through physiological profiling and comparative time-series chloroplast proteomics. In addition, we explored the seeds conducting a physiological characterization and targeted transcriptomic profiling in both subpopulations. Our results showed differential responses between them, evidencing a cross-stress transgenerational memory. Seedlings belonging to the stressed subpopulation retained key proteins related to Photosystem II, chloroplast-to-nucleus signalling and osmoprotection which helped to overcome the applied heat stress. The seeds also showed a differential gene expression profile for targeted genes and microRNAs, as well as an increased content of starch and secondary metabolites, molecules which showed potential interest as biomarkers for early selection of primed plants. Thus, these finds not only delve into transgenerational cross-stress memory in trees, but also provide a new biotechnological tool for forest design.

The impact of insect egg deposition on Pinus sylvestris transcriptomic and phytohormonal responses to larval herbivory.

Plants can improve their resistance to feeding damage by insects if they have perceived insect egg deposition prior to larval feeding. Molecular analyses of these egg-mediated defence mechanisms have until now focused on angiosperm species. It is unknown how the transcriptome of a gymnosperm species responds to insect eggs and subsequent larval feeding. Scots pine (Pinus sylvestris L.) is known to improve its defences against larvae of the herbivorous sawfly Diprion pini L. if it has previously received sawfly eggs. Here, we analysed the transcriptomic and phytohormonal responses of Scots pine needles to D. pini eggs (E-pine), larval feeding (F-pine) and to both eggs and larval feeding (EF-pine). Pine showed strong transcriptomic responses to sawfly eggs and-as expected-to larval feeding. Many egg-responsive genes were also differentially expressed in response to feeding damage, and these genes play an important role in biological processes related to cell wall modification, cell death and jasmonic acid signalling. EF-pine showed fewer transcriptomic changes than F-pine, whereas EF-treated angiosperm species studied so far showed more transcriptional changes to the initial phase of larval feeding than only feeding-damaged F-angiosperms. However, as with responses of EF-angiosperms, EF-pine showed higher salicylic acid concentrations than F-pine. Based on the considerable overlap of the transcriptomes of E- and F-pine, we suggest that the weaker transcriptomic response of EF-pine than F-pine to larval feeding damage is compensated by the strong, egg-induced response, which might result in maintained pine defences against larval feeding.

Stochastic processes and changes in evolutionary rate are associated with diversification in a lineage of tropical hard pines (Pinus).

The study of the patterns of polymorphism and molecular evolution among closely related species is key to understanding the evolutionary forces involved in the diversification of lineages. This point is a big challenge in species with slow evolutionary rates, long life cycles, and ancient, shared polymorphisms such as conifers. Under the premise of divergence in a stepwise migration process, we expect clinal geographical patterns of purifying selection efficiency, and genetic structure related to latitude or longitude. If migration is accompanied by changes in the environment, we could further expect a role of positive selection in driving species divergence. Here, we infer patterns of polymorphism, efficiency of purifying selection, and molecular evolution using a dataset of 161 nuclear genes (∼71 Kb) in a lineage of hard pines from North America, the Caribbean, Mexico, and Central America presumed to have migrated from North America toward lower latitudes with tropical conditions. Under the premise of differences in selective pressures, we also look for possible signals of positive selection. To test our hypothesis, first we estimated different indices to infer patterns of polymorphism and efficiency of purifying selection (Ka, Ks, Ka/Ks, dN, dS, dN/dS, and dxy) and compared these metrics across five clades. Also, we investigated possible clinal patterns in these indices and morphological traits (needle length and cone length). Then we inferred genetic structure and environmental differences among species to test for possible signals of positive selection using phylogenetic methods in specific clades. We found differences among clades using Ka, Ks, and Ka/Ks with a relaxation of purifying selection, especially in the Elliotti and Patula clades. We also found environmental differences related to geographic distance, and among clades suggesting differences in selective pressures. The indices Ks, dxy, and needle length had relationships with geography but not ovulate cone length. Finally, we found that most analyzed genes are under purifying selection, but there was an exception of faster evolutionary rate in some pine species, suggesting the possible action of positive selection in divergence. Our study indicated that stochastic processes have played a key role in the diversification of the group, with a possible input of positive selection in pines from Mexico and Central America.

Mitochondrial genome provides species-specific targets for the rapid detection of early invasive populations of Hylurgus ligniperda in China.

BACKGROUND: Hylurgus ligniperda, a major international forestry quarantine pest, was recently found to have invaded and posed a serious threat to the Pinus forests of the Jiaodong Peninsula in China. Continuous monitoring and vigilance of the early population is imperative, and rapid molecular detection technology is urgently needed. We focused on developing a single-gene-based species-specific PCR (SS-PCR) method. RESULTS: We sequenced and assembled the mitochondrial genome of H. ligniperda to identify suitable target genes. We identified three closely related species for detecting the specificity of SS-PCR through phylogenetic analysis based on 13 protein-coding genes (PCGs). Subsequently, we analyzed the evolution of 13 PCGs and selected four mitochondrial genes to represent slow-evolving gene (COI) and faster-evolving genes (e.g. ND2, ND4, and ND5), respectively. We developed four species-specific primers targeting COI, ND2, ND4, and ND5 to rapidly identify H. ligniperda. The results showed that the four species-specific primers exhibited excellent specificity and sensitivity in the PCR assays, with consistent performance across a broader range of species. This method demonstrates the ability to identify beetles promptly, even during their larval stage. The entire detection process can be completed within 2-3 h. CONCLUSIONS: This method is suitable for large-scale species detection in laboratory settings. Moreover, the selection of target genes in the SS-PCR method is not affected by the evolutionary rate. SS-PCR can be widely implemented at port and forestry workstations, significantly enhancing early management strategies and quarantine measures against H. ligniperda. This approach will help prevent the spread of the pest and effectively preserve the resources of Chinese pine forests.

Demographic dynamics and molecular evolution of the rare and endangered subsect. Gerardianae of Pinus: insights from chloroplast genomes and mitochondrial DNA markers.

MAIN CONCLUSION: The divergence of subsect. Gerardianae was likely triggered by the uplift of the Qinghai-Tibetan Plateau and adjacent mountains. Pinus bungeana might have probably experienced expansion since Last Interglacial period. Historical geological and climatic oscillations have profoundly affected patterns of nucleotide variability, evolutionary history, and species divergence in numerous plants of the Northern Hemisphere. However, how long-lived conifers responded to geological and climatic fluctuations in East Asia remain poorly understood. Here, based on paternally inherited chloroplast genomes and maternally inherited mitochondrial DNA markers, we investigated the population demographic history and molecular evolution of subsect. Gerardianae (only including three species, Pinus bungeana, P. gerardiana, and P. squamata) of Pinus. A low level of nucleotide diversity was found in P. bungeana (π was 0.00016 in chloroplast DNA sequences, and 0.00304 in mitochondrial DNAs). The haplotype-based phylogenetic topology and unimodal distributions of demographic analysis suggested that P. bungeana probably originated in the southern Qinling Mountains and experienced rapid population expansion since Last Interglacial period. Phylogenetic analysis revealed that P. gerardiana and P. squamata had closer genetic relationship. The species divergence of subsect. Gerardianae occurred about 27.18 million years ago (Mya) during the middle to late Oligocene, which was significantly associated with the uplift of the Qinghai-Tibetan Plateau and adjacent mountains from the Eocene to the mid-Pliocene. The molecular evolutionary analysis showed that two chloroplast genes (psaI and ycf1) were under positive selection, the genetic lineages of P. bungeana exhibited higher transition and nonsynonymous mutations, which were involved with the strongly environmental adaptation. These findings shed light on the population evolutionary history of white pine species and provide striking insights for comprehension of their species divergence and molecular evolution.

Whole-genome resequencing facilitates the development of a 50K single nucleotide polymorphism genotyping array for Scots pine (Pinus sylvestris L.) and its transferability to other pine species.

Scots pine (Pinus sylvestris L.) is one of the most widespread and economically important conifer species in the world. Applications like genomic selection and association studies, which could help accelerate breeding cycles, are challenging in Scots pine because of its large and repetitive genome. For this reason, genotyping tools for conifer species, and in particular for Scots pine, are commonly based on transcribed regions of the genome. In this article, we present the Axiom Psyl50K array, the first single nucleotide polymorphism (SNP) genotyping array for Scots pine based on whole-genome resequencing, that represents both genic and intergenic regions. This array was designed following a two-step procedure: first, 192 trees were sequenced, and a 430K SNP screening array was constructed. Then, 480 samples, including haploid megagametophytes, full-sib family trios, breeding population, and range-wide individuals from across Eurasia were genotyped with the screening array. The best 50K SNPs were selected based on quality, replicability, distribution across the draft genome assembly, balance between genic and intergenic regions, and genotype-environment and genotype-phenotype associations. Of the final 49 877 probes tiled in the array, 20 372 (40.84%) occur inside gene models, while the rest lie in intergenic regions. We also show that the Psyl50K array can yield enough high-confidence SNPs for genetic studies in pine species from North America and Eurasia. This new genotyping tool will be a valuable resource for high-throughput fundamental and applied research of Scots pine and other pine species.