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The North American Betula lenta L. (sweet birch) has been used for medicinal reasons for centuries by native Americans. Although sophisticated technologies have rapidly been developed, a large information gap has been observed regarding genetic regulators of medicinally important compounds in sweet birch. Very little is known on the different genes involved in secondary metabolic biosynthesis in sweet birch. To gain a deeper insight into genetic factors, we performed a transcriptome analysis of each three biological samples from different independent trees of sweet and European silver birch (B. pendula Roth). This allowed us to precisely quantify the transcripts of about 24,000 expressed genes including 29 prominent candidate genes putatively involved in the biosynthesis of secondary metabolites like terpenoids, and aromatic benzoic acids. A total number of 597 genes were differentially expressed between B. lenta and B. pendula, while 264 and 210 genes showed upregulation in the bark and leaf of B. lenta, respectively. Moreover, we identified 39 transcriptional regulatory elements, involved in secondary metabolite biosynthesis, upregulated in B. lenta. Our study demonstrated the potential of RNA sequencing to identify candidate genes interacting in secondary metabolite biosynthesis in sweet birch. The candidate genes identified in this study could be subjected to genetic engineering to functionally characterize them in sweet birch. This knowledge can be beneficial to the increase of therapeutically important compounds.

The large-scale spatial genetic structure of European beech, Fagus sylvatica, has been until now poorly studied. We conducted double digest RAD sequencing (ddRADseq) on 54 beech individuals stemming from 36 provenances to discover spatially informative nuclear SNP loci. In addition, two pools derived from 14 early and 14 late flushing individuals each were sequenced with Illumina HiSeq. From an initial amount of 5,464 loci detected by ddRADseq, we selected 559 informative loci. Further 27 additional loci showing significant allelic differences among early and late flushing individuals could be identified after a genotyping on 95 test individuals. The final selection of 578 loci was submitted to probe design for targeted genotyping by sequencing, which yielded 543 loci. The new set of SNP loci should be, after validation on a larger sample size, useful for large-scale genetic studies in this economically-important species.

Many dioecious angiosperms are trees, which only flower after years of vegetative development and do not usually exhibit marked secondary sexual dimorphism. Nevertheless, if the genetic basis of sex determination is known, the sex of an individual can be determined using molecular markers. Here, we report that in the genus Populus sect. Populus an XY system of sex determination, which is found in P. tremula and P. tremuloides, likely re-evolved from a ZW system present in P. alba, P. adenopoda and P. qiongdaoensis. Strikingly, this new XY system is mechanistically identical to the older system found in several species of the Populus sections Tacamahaca, Aigeiros and Turanga demonstrating a remarkable example of convergent evolution. In both XY systems, male-specific inversely repeated sequences appear to silence the ARR17 gene, which functions as a sex switch, via small interfering RNAs and DNA methylation. In the ZW system, female-specific copies of ARR17 appear to regulate dioecy. With this detailed information on the genetic basis of sex determination it was possible to develop molecular markers that can be utilized to determine the sex in seedlings and non-flowering trees of different poplar species. We used the female-specific ARR17 gene to develop a sex marker for P. alba and P. adenopoda. For P. grandidentata, we employed the male-specific ARR17 inverted repeat. Finally, we summarize previously described markers for P. tremula, P. tremuloides, P. trichocarpa, P. deltoides and P. nigra. These markers can be useful for poplar ecologists, geneticists and breeders.

Although hundreds of plant lineages have independently evolved dioecy (that is, separation of the sexes), the underlying genetic basis remains largely elusive1. Here we show that diverse poplar species carry partial duplicates of the ARABIDOPSIS RESPONSE REGULATOR 17 (ARR17) orthologue in the male-specific region of the Y chromosome. These duplicates give rise to small RNAs apparently causing male-specific DNA methylation and silencing of the ARR17 gene. CRISPR-Cas9-induced mutations demonstrate that ARR17 functions as a sex switch, triggering female development when on and male development when off. Despite repeated turnover events, including a transition from the XY system to a ZW system, the sex-specific regulation of ARR17 is conserved across the poplar genus and probably beyond. Our data reveal how a single-gene-based mechanism of dioecy can enable highly dynamic sex-linked regions and contribute to maintaining recombination and integrity of sex chromosomes.

The number of dioecious species for which the genetic basis of sex determination has been resolved is rapidly increasing. Nevertheless, the molecular mechanisms downstream of the sex determinants remain largely elusive. Here, by RNA-sequencing early-flowering isogenic aspen (

INISEFOR Swietenia species are the most valuable American tropical timbers and have been heavily overexploited for decades. The three species are listed as either vulnerable or endangered by IUCN and are included on Appendix II of CITES, yet illegal exploitation continues. Here, we used restriction associated DNA sequencing to develop a new set of 120 SNP markers for Swietenia sp., suitable for MassARRAY®iPLEX™ genotyping. These markers can be used for population genetic studies and timber tracking purposes. Las especies de Swietenia son las maderas tropicales estadounidenses más valiosas y han sido muy sobreexplotadas durante décadas. los Tres especies están incluidas como vulnerables o en peligro de extinción por la UICN y están incluidas en el Apéndice II de la CITES, pero son ilegales. la explotación continúa. Aquí, utilizamos la secuenciación de ADN asociada a la restricción para desarrollar un nuevo conjunto de 120 marcadores SNP para Swietenia sp., Adecuada para el genotipado MassARRAY®iPLEX ™. Estos marcadores se pueden utilizar para estudios genéticos de poblaciones. y seguimiento de la madera. As espécies de Swietenia são as madeiras tropicais americanas mais valiosas e têm sido fortemente superexploradas por décadas. As três espécies estão listadas como vulneráveis ​​ou em perigo pela IUCN e estão incluídas no Apêndice II da CITES, mas a exploração ilegal continua. Aqui, usamos o sequenciamento de DNA associado à restrição para desenvolver um novo conjunto de 120 marcadores SNP para Swietenia sp., Adequado para genotipagem MassARRAY®iPLEX ™. Esses marcadores podem ser usados ​​para estudos de genética populacional e fins de rastreamento de madeira. Thünen Institute of Forest Genetics, Germany Instituto Nacional de Pesquisas da Amazônia, Brazil Universidad Nacional, Costa Rica Centre for Ecology & Hydrology, United Kingdom Instituto Florestal de São Paulo, Brazil University of Bordeaux, France Mahogany for the Future Inc., USA Bush Estate, United Kingdom Instituto de Investigación y Servicios Forestales

Populus species are dioecious, but deviations from dioecy are reported in some cases. The objectives of this study were to investigate the phenotypic expression and the inheritance of subdioecy in a Populus ×canescens pedigree. The F1 progeny was monitored for sex during 14 years. Thirty per cent of individuals expressed deviations from dioecy and long-term plasticity of sex. Some plants started flowering as male, then became cosexual, and finally turned female. Two cosexual individuals were self-pollinated and generated a selfed progeny markedly impaired by inbreeding depression, but able to reproduce by outcrossing. Sex segregation of the F1 progeny statistically fitted the expected ratio 1:2:1 (female:male:cosexual). By analysis of DNA markers, the cosexual individuals were genetically clustered with the females. The segregation ratio and the genetic profile indicated that cosexual plants were female with altered sex phenotype. Linkage analysis identified a putative sex-determining region with suppressed recombination on chromosome 19 of the male Populus tremula parent. The male sex trait was linked to the pericentromeric region of the P. tremula chromosome 19, whereas the cosexual trait was linked to chromosome 19 of the female Populus alba parent. A genetic model is proposed to explain inheritance and phenotypic expression of sex.

RNA editing occurs in the endosymbiont organelles of higher plants as C-to-U conversions of defined nucleotides. The availability of large quantities of RNA sequencing data makes it possible to identify RNA editing sites and to quantify their editing extent. We have investigated RNA editing in 34 protein-coding mitochondrial transcripts of four Populus species, a genus noteworthy for its remarkably small number of RNA editing sites compared to other angiosperms. 27 of these transcripts were subject to RNA editing in at least one species. In total, 355 RNA editing sites were identified with high confidence, their editing extents ranging from 10 to 100%. The most heavily edited transcripts were ccmB with the highest density of RNA editing sites (53.7 sites / kb) and ccmFn with the highest number of sites (39 sites). Most of the editing events are at position 1 or 2 of the codons, usually altering the encoded amino acid, and are highly conserved among the species, also with regard to their editing extent. However, one SNP was found in the newly sequenced and annotated mitochondrial genome of P. alba resulting in the loss of an RNA editing site compared to P. tremula and P. davidiana. This SNP causes a C-to-T transition and an amino acid exchange from Ser to Phe, highlighting the widely discussed role of RNA editing in compensating mutations.

Even though “supermale” (YY) poplar individuals are rarely found in natural populations or breeding collections, they can arise and can be as viable as regular male and female plants. Nearly all species of the genus Populus are dioecious, i.e., form male and female flowers on separate individuals. However, bisexual poplar can occasionally be found under natural conditions or during the breeding process. According to the XX/XY model of sex determination, “supermale” (YY) individuals should only occur in the selfed progeny of a genetically male (XY) tree and not in the one of a genetically female (XX) parent. We performed detailed molecular genetic analyses of a bisexual Populus tremula XY-individual (ZP8) and its selfed progeny. The ZP8 tree was confirmed as P. tremula by chloroplast and nuclear markers, and the progeny was genotyped by microsatellite markers to verify self-pollination. In the selfed progeny but also in about 420 different poplar individuals from several breeding programmes and the wild, the X and Y chromosomes were tracked using previously reported and newly developed markers. From two self-pollination experiments of the bisexual ZP8 tree, 39 S1-individuals were obtained. Application of X- and Y-specific molecular markers revealed 15 XY, 17 YY, and 7 XX individuals. Additional investigations of about 300 different poplar clones employed in several breeding programmes as well as about 120 individuals from wild populations revealed only three trees with two Y chromosomes. Indications for inbreeding depression in the selfed progeny were obtained during germination and glasshouse cultivation, but are independent from the sex chromosome combination. Even though YY-poplar individuals are rarely found in natural populations or breeding collections, they can arise and can be as viable as regular XY- and XX-plants.

We developed a new set of 25 nuclear (nc), 12 chloroplast (cp) and 7 mitochondrial (mt) SNPs and used it to genotype 371 Mongolian oak (Quercus mongolica Fisch. ex Ledeb.) trees from seven locations in a 200 km by 400 km area in the Russian Far East. One of the locations in an area of 15 km by 25 km east of the city Ussuriusk was analyzed more intensively with 188 collected trees. The genetic differentiation at the nuclear SNPs was small to moderate and for the plastid SNPs it was high when considering all trees from the seven locations. The gene pool distances between locations were for 19 out of 21 pairs statistically highly significant. There was no correlation of genetic and spatial distances. Only three different multilocus-haplotypes could be identified and 42 two-loci-combinations of plastid SNPs could be used to identify them. Conclusions for the practical application such as timber tracking and gene conservation are discussed.

Climate change is increasing insect pressure and forcing plants to adapt. Although chemotypic differentiation and phenotypic plasticity in spatially separated tree populations are known for decades, understanding their importance in herbivory resistance across forests remains challenging. We studied four oak forest stands in Germany using nontarget metabolomics, elemental analysis, and chemometrics and mapped the leaf metabolome of herbivore-resistant (T-) and herbivore-susceptible (S-) European oaks (Quercus robur) to Tortrix viridana, an oak pest that causes severe forest defoliation. Among the detected metabolites, we identified reliable metabolic biomarkers to distinguish S- and T-oak trees. Chemotypic differentiation resulted in metabolic shifts of primary and secondary leaf metabolism. Across forests, T-oaks allocate resources towards constitutive chemical defense enriched of polyphenolic compounds, e.g. the flavonoids kaempferol, kaempferol and quercetin glucosides, while S-oaks towards growth-promoting substances such as carbohydrates and amino-acid derivatives. This extensive work across natural forests shows that oaks' resistance and susceptibility to herbivory are linked to growth-defense trade-offs of leaf metabolism. The discovery of biomarkers and the developed predictive model pave the way to understand Quercus robur's susceptibility to herbivore attack and to support forest management, contributing to the preservation of oak forests in Europe.

The diversity of inflorescences among flowering plants is captivating. Such charm is not only due to the variety of sizes, shapes, colors, and flowers displayed, but also to the range of reproductive systems. For instance, hermaphrodites occur abundantly throughout the plant kingdom with both stamens and carpels within the same flower. Nevertheless, 10% of flowering plants have separate unisexual flowers, either in different locations of the same individual (monoecy) or on different individuals (dioecy). Despite their rarity, dioecious plants provide an excellent opportunity to investigate the mechanisms involved in sex expression and the evolution of sex-determining regions (SDRs) and sex chromosomes. The SDRs and the evolution of dioecy have been studied in many species ranging from Ginkgo to important fruit crops. Some of these studies, for example in asparagus or kiwifruit, identified two sex-determining genes within the non-recombining SDR and may thus be consistent with the classical model for the evolution of dioecy from hermaphroditism via gynodioecy, that predicts two successive mutations, the first one affecting male and the second one female function, becoming linked in a region of suppressed recombination. On the other hand, aided by genome sequencing and gene editing, single factor sex determination has emerged in other species, such as persimmon or poplar. Despite the diversity of sex-determining mechanisms, a tentative comparative analysis of the known sex-determining genes and candidates in different species suggests that similar genes and pathways may be employed repeatedly for the evolution of dioecy. The cytokinin signaling pathway appears important for sex determination in several species regardless of the underlying genetic system. Additionally, tapetum-related genes often seem to act as male-promoting factors when sex is determined via two genes. We present a unified model that synthesizes the genetic networks of sex determination in monoecious and dioecious plants and will support the generation of hypothesis regarding candidate sex determinants in future studies.

The Swiss pine (Pinus cembra) is a montane tree in Central Europe and, therefore, known for its hardiness against severe winter colds. The seeds are harvested and eaten as pine nuts. We assembled and characterized the complete chloroplast genome of P. cembra to serve as a valuable resource in future genetic studies. The complete plastome sequence is 116,609 bp in length and contains 113 genes including 79 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. A phylogenetic analysis of 34 Pinus plastome sequences shows that Pinus sibirica is the nearest relative to P. cembra and that there is a distinct clustering together with the other members of the section Quinquefoliae.

European beech, Fagus sylvatica L., is one of the most important and widespread deciduous tree species in Central Europe and is widely managed for its hard wood. The complete DNA sequence of the mitochondrial genome of Fagus sylvatica L. was assembled and annotated based on Illumina MiSeq reads and validated using long reads from nanopore MinION sequencing. The genome assembled into a single DNA sequence of 504,715 bp in length containing 58 genes with predicted function, including 35 protein-coding, 20 tRNA and three rRNA genes. Additionally, 23 putative protein-coding genes were predicted supported by RNA-Seq data. Aiming at the development of taxon-specific mitochondrial genetic markers, the tool SNPtax was developed and applied to select genic SNPs potentially specific for different taxa within the Fagales. Further validation of a small SNP set resulted in the development of four CAPS markers specific for Fagus, Fagaceae, or Fagales, respectively, when considering over 100 individuals from a total of 69 species of deciduous trees and conifers from up to 15 families included in the marker validation. The CAPS marker set is suitable to identify the genus Fagus in DNA samples from tree tissues or wood products, including wood composite products.

Next-generation sequencing (NGS) approaches are attractive alternatives to the PCR-based characterisation of genetically modified plants for safety assessment and labelling since NGS is highly sensitive to the detection of T-DNA inserts as well as vector backbone sequences in transgenic plants. In this study, two independent transgenic male Populus tremula lines, T193-2 and T195-1, both carrying the FLOWERING LOCUS T gene from Arabidopsis thaliana under control of a heat-inducible promoter (pHSP::AtFT) and the non-transgenic control clone W52, were further characterised by NGS and third-generation sequencing. The results support previous findings that the T-DNA was hemizygously inserted in one genomic locus of each line. However, the T-DNA insertions consist of conglomerations of one or two T-DNA copies together with a small T-DNA fragment without AtFT parts. Based on NGS data, no additional T-DNA splinters or vector backbone sequences could be identified in the genome of the two transgenic lines. Seedlings derived from crosses between the pHSP::AtFT transgenic male parents and female wild type plants are therefore expected to be T-DNA splinter or vector backbone free. Thus, PCR analyses amplifying a partial T-DNA fragment with AtFT-specific primers are sufficient to determine whether the seedlings are transgenic or not. An analysis of 72 second generation-seedlings clearly showed that about 50% of them still reveal the presence of the T-DNA, confirming data already published. To prove if unanticipated genomic changes were induced by T-DNA integration, extended future studies using long-range sequencing technologies are required once a suitable chromosome-level P. tremula reference genome sequence is available.

Key message This review gives a comprehensive overview on the genomics of sex determination in dioecious woody plants and plants with a tree-like habitus, in particular considering species where sex-linked regions and/or markers have been identified.

Silver fir (Abies alba Mill.) is a keystone conifer of European montane forest ecosystems that has experienced large fluctuations in population size during during the Quaternary and, more recently, due to land-use change. To forecast the species’ future distribution and survival, it is important to investigate the genetic basis of adaptation to environmental change, notably to extreme events. For this purpose, we here provide a first draft genome assembly and annotation of the silver fir genome, established through a community-based initiative. DNA obtained from haploid megagametophyte and diploid needle tissue was used to construct and sequence Illumina paired-end and mate-pair libraries, respectively, to high depth. The assembled A. alba genome sequence accounted for over 37 million scaffolds corresponding to 18.16 Gb, with a scaffold N50 of 14,051 bp. Despite the fragmented nature of the assembly, a total of 50,757 full-length genes were functionally annotated in the nuclear genome. The chloroplast genome was also assembled into a single scaffold (120,908 bp) that shows a high collinearity with both the A. koreana and A. sibirica complete chloroplast genomes. This first genome assembly of silver fir is an important genomic resource that is now publicly available in support of a new generation of research. By genome-enabling this important conifer, this resource will open the gate for new research and more precise genetic monitoring of European silver fir forests.

100 SNP and one Indel markers were developed for Khaya using a combination of restriction associated DNA sequencing and low coverage MiSeq genome sequencing. The marker set was successfully used to genotype a set of 1919 individuals, representing 5 Khaya species from 18 African countries, using MassARRAY®iPLEX™ genotyping.

182 SNP markers were developed for Ayous (Triplochiton scleroxylon K. Schum) by incorporating information from two next generation sequencing approaches (RADseq Floragenex and AFLPseq IonTorrent PGM) into a single genotyping panel for MassARRAY® iPLEX™. This set of markers was successfully used to genotype 753 individuals from 43 populations in five Tropical West and Central African Countries. These loci have an expected heterozygosity range of 0.007–0.501 and F ST from 0 to 0.306.

Poplar trees displayed an increased plant height due to the transgenic knockdown of PCBER1, a gene of lignan biosynthesis. The wood composition was slightly altered in both overexpression and knockdown lines. The gene PHENYLCOUMARAN BENZYLIC ETHER REDUCTASE1 (PCBER1) is well known as an important gene in the synthesis of lignans, a group of diverse phenylpropanoid derivatives. They are widely distributed in the plant kingdom and may have a role in both plant defense and growth regulation. To analyze its role in biomass formation and wood composition in poplar, both overexpression and knockdown approaches have been performed. Transgenic lines were analyzed on genetic and phenotypic levels, and partly in regard to their biomass composition. While the PCBER1 overexpression approach remaine d unremarkable concerning the plant height, biomass composition of obtained transgenic lines was modified. They had a significantly increased amount of ethanol extractives. The PCBER1 knockdown resulted in significantly deviating plants; after 17 months of greenhouse cultivation, transgenic plants were up to 38% higher compared to non-transgenic wild type. Most examined transgenic lines did not reveal a significantly enhanced stem diameter after three vegetation periods in the greenhouse. Significant changes were not obtained with regard to the three major wood components, lignin, cellulose and hemicelluloses. As a slight but not significant reduction in ethanol extractives was detected, the hypothesis arises that the lignan content could be influenced. Lignans become important in the phar maceutical industry and clinical studies concerning cancer and other diseases, thus further investigations on lignan formation in poplar and its connection to biomass formation seem promising.

International audience; We describe the development of new nuclear SNP markers for the genetic timber tracking of the geographical origin of Sapelli, Entandrophragma cylindricum (Meliaceae). Restriction associated DNA sequencing (RADseq) of two reference individuals yielded 1131 putative SNPs. Among those, 131 were selected to design four MassARRAY multiplexes and screened at 178 individuals. Seventy-two loci were selected for further use in genetic tracking.

Table 4 in the original publication reports incomplete genotype names in the column "Cross" and wrong codes in the column "Generation".

The Swiss pine (Pinus cembra) is a montane tree in Central Europe and, therefore, known for its hardiness against severe winter colds. The seeds are harvested and eaten as pine nuts. We assembled and characterized the complete chloroplast genome of P. cembra to serve as a valuable resource in future genetic studies. The complete plastome sequence is 116,609 bp in length and contains 113 genes including 79 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. A phylogenetic analysis of 34 Pinus plastome sequences shows that Pinus sibirica is the nearest relative to P. cembra and that there is a distinct clustering together with the other members of the section Quinquefoliae.

The European beech (Fagus sylvatica) is one of the most important and widespread tree species in Central Europe and is widely managed for its valuable hard wood. We assembled and characterized the ...