A Small Set of Nuclear Markers for Reliable Differentiation of the Two Closely Related Oak Species Quercus Robur and Q. Petraea.

Quercus robur and Q. petraea are, in addition to Fagus sylvatica, the main economically used deciduous tree species in Europe. Identification of these two species is crucial because they differ in their ecological demands. Because of a changing climate, foresters must know more than ever which species will perform better under given environmental conditions. The search for differentiating molecular markers between these two species has already lasted for decades. Until now, differentiation has only been possible in approaches with a combination of several molecular markers and a subsequent statistical analysis to calculate the probability of being one or the other species. Here, we used MiSeq Illumina data from pools of Q. robur and Q. petraea specimens and identified nuclear SNPs and small InDels versus the Q. robur reference genome. Selected sequence variants with 100% allele frequency difference between the two pools were further validated in an extended set of Q. robur and Q. petraea specimens, and then the number of markers was deliberately reduced to the smallest possible set for species differentiation. A combination of six markers from four nuclear regions is enough to identify Q. robur, Q. petraea or hybrids between these two species quite well and represents a marker set that is cost-efficient and useable in every laboratory.

The mitochondrial genome sequence of Abies alba Mill. reveals a high structural and combinatorial variation.

BACKGROUND: Plant mitogenomes vary widely in size and genomic architecture. Although hundreds of plant mitogenomes of angiosperm species have already been sequence-characterized, only a few mitogenomes are available from gymnosperms. Silver fir (Abies alba) is an economically important gymnosperm species that is widely distributed in Europe and occupies a large range of environmental conditions. Reference sequences of the nuclear and chloroplast genome of A. alba are available, however, the mitogenome has not yet been assembled and studied. RESULTS: Here, we used paired-end Illumina short reads generated from a single haploid megagametophyte in combination with PacBio long reads from high molecular weight DNA of needles to assemble the first mitogenome sequence of A. alba. Assembly and scaffolding resulted in 11 mitogenome scaffolds, with the largest scaffold being 0.25 Mbp long. Two of the scaffolds displayed a potential circular structure supported by PCR. The total size of the A. alba mitogenome was estimated at 1.43 Mbp, similar to the size (1.33 Mbp) of a draft assembly of the Abies firma mitogenome. In total, 53 distinct genes of known function were annotated in the A. alba mitogenome, comprising 41 protein-coding genes, nine tRNA, and three rRNA genes. The proportion of highly repetitive elements (REs) was 0.168. The mitogenome seems to have a complex and dynamic structure featured by high combinatorial variation, which was specifically confirmed by PCR for the contig with the highest mapping coverage. Comparative analysis of all sequenced mitogenomes of gymnosperms revealed a moderate, but significant positive correlation between mitogenome size and proportion of REs. CONCLUSIONS: The A. alba mitogenome provides a basis for new comparative studies and will allow to answer important structural, phylogenetic and other evolutionary questions. Future long-read sequencing with higher coverage of the A. alba mitogenome will be the key to further resolve its physical structure. The observed positive correlation between mitogenome size and proportion of REs will be further validated once available mitogenomes of gymnosperms would become more numerous. To test whether a higher proportion of REs in a mitogenome leads to an increased recombination and higher structural complexity and variability is a prospective avenue for future research.

Tree Genetic Engineering, Genome Editing and Genomics.

In this Special Issue [...].

ARR17 controls dioecy in Populus by repressing B-class MADS-box gene expression.

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 (Populus tremula) lines differing exclusively for the sex switch gene ARR17, we show that a narrowly defined genetic network controls differential development of female and male flowers. Although ARR17 encodes a type-A response regulator supposedly involved in cytokinin (CK) hormone signalling, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated arr17 knockout only affected the expression of a strikingly small number of genes, indicating a specific role in the regulation of floral development rather than a generic function in hormone signalling. Notably, the UNUSUAL FLORAL ORGANS (UFO) gene, encoding an F-box protein acting as a transcriptional cofactor with LEAFY (LFY) to activate B-class MADS-box gene expression, and the B-class gene PISTILLATA (PI), necessary for male floral organ development, were strongly de-repressed in the arr17 CRISPR mutants. Our data highlight a CK-independent role of the poplar response regulator ARR17 and further emphasize the minimal differences between female and male individuals. This article is part of the theme issue 'Sex determination and sex chromosome evolution in land plants'.

Transcriptome analysis of North American sweet birch (Betula lenta) revealed a higher expression of genes involved in the biosynthesis of secondary metabolites than European silver birch (B. pendula).

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.

European oak chemical diversity - from ecotypes to herbivore resistance.

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.

Mitochondrial Genome of Fagus sylvatica L. as a Source for Taxonomic Marker Development in the Fagales.

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.

A single gene underlies the dynamic evolution of poplar sex determination.

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.

Sequencing of two transgenic early-flowering poplar lines confirmed vector-free single-locus T-DNA integration.

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.

The Diversity and Dynamics of Sex Determination in Dioecious Plants.

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.

Correction to: Long-term study of a subdioecious Populus ×canescens family reveals sex lability of females and reproduction behaviour of cosexual plants.

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

Long-term study of a subdioecious Populus ×canescens family reveals sex lability of females and reproduction behaviour of cosexual plants.

KEY MESSAGE: Cosexual Populus ×canescens plants are inconstant females with life course plasticity of sex phenotype and can reproduce by selfing. 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-seq of eight different poplar clones reveals conserved up-regulation of gene expression in response to insect herbivory.

BACKGROUND: Herbivorous insects can have a profound impact on plant growth performance. In some years, canopy damage in poplar plantations exceeds 50% of the total leaf surface, thereby possibly compromising carbon fixation and biomass yield. To assess the transcriptional response of elite poplar clones to insect feeding and to test whether this response varies between different genotypes, we performed an RNA-sequencing experiment. We deeply sequenced the transcriptomes of eight elite clones belonging to three poplar species (Populus trichocarpa, P. nigra and P. maximowiczii), under Phratora vitellinae feeding and control conditions. This allowed us to precisely quantify transcript levels of about 24,000 expressed genes. RESULTS: Our data reveal a striking overall up-regulation of gene expression under insect attack in all eight poplar clones studied. The up-regulated genes were markedly enriched for the biological process 'regulation of transcription' indicating a highly concerted restructuring of the transcriptome. A search for potential cis-regulatory elements (CREs) that may be involved in this process identified the G-box (CACGTG) as the most significant motif in the promoters of the induced genes. In line with the role of the G-box in jasmonate (JA)-mediated activation of gene expression by MYC2, several genes involved in JA biosynthesis and signaling were up-regulated in our dataset. A co-expression network analysis additionally highlighted WRKY transcription factors. Within the most prominent expression module, WRKYs were strongly overrepresented and occupied several network hubs. Finally, the insect-induced genes comprised several protein families known to be involved in plant defenses, e.g. cytochrome P450s, chitinases and protease inhibitors. CONCLUSIONS: Our data represent a comprehensive characterization of the transcriptional response of selected elite poplar clones to insect herbivory. Our results suggest that the concerted up-regulation of gene expression is controlled by JA signaling and WRKY transcription factors, and activates several defense mechanisms. Our data highlight potential targets of selection and may thus contribute to breeding insect-resistant poplar clones.

A Reference Genome Sequence for the European Silver Fir (Abies alba Mill.): A Community-Generated Genomic Resource.

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.

High Level of Conservation of Mitochondrial RNA Editing Sites Among Four Populus Species.

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.

Knockdown of PCBER1, a gene of neolignan biosynthesis, resulted in increased poplar growth.

MAIN CONCLUSION: 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 remained 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 pharmaceutical 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.

The complete chloroplast genome sequence of Pinus cembra L. (Pinaceae).

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.

Complete Chloroplast Genome Sequences of Four Meliaceae Species and Comparative Analyses.

The Meliaceae family mainly consists of trees and shrubs with a pantropical distribution. In this study, the complete chloroplast genomes of four Meliaceae species were sequenced and compared with each other and with the previously published Azadirachta indica plastome. The five plastomes are circular and exhibit a quadripartite structure with high conservation of gene content and order. They include 130 genes encoding 85 proteins, 37 tRNAs and 8 rRNAs. Inverted repeat expansion resulted in a duplication of rps19 in the five Meliaceae species, which is consistent with that in many other Sapindales, but different from many other rosids. Compared to Azadirachta indica, the four newly sequenced Meliaceae individuals share several large deletions, which mainly contribute to the decreased genome sizes. A whole-plastome phylogeny supports previous findings that the four species form a monophyletic sister clade to Azadirachta indica within the Meliaceae. SNPs and indels identified in all complete Meliaceae plastomes might be suitable targets for the future development of genetic markers at different taxonomic levels. The extended analysis of SNPs in the matK gene led to the identification of four potential Meliaceae-specific SNPs as a basis for future validation and marker development.

Development of Molecular Markers for Determining Continental Origin of Wood from White Oaks (Quercus L. sect. Quercus).

To detect and avoid illegal logging of valuable tree species, identification methods for the origin of timber are necessary. We used next-generation sequencing to identify chloroplast genome regions that differentiate the origin of white oaks from the three continents; Asia, Europe, and North America. By using the chloroplast genome of Asian Q. mongolica as a reference, we identified 861 variant sites (672 single nucleotide polymorphisms (SNPs); 189 insertion/deletion (indel) polymorphism) from representative species of three continents (Q. mongolica from Asia; Q. petraea and Q. robur from Europe; Q. alba from North America), and we identified additional chloroplast polymorphisms in pools of 20 individuals each from Q. mongolica (789 variant sites) and Q. robur (346 variant sites). Genome sequences were screened for indels to develop markers that identify continental origin of oak species, and that can be easily evaluated using a variety of detection methods. We identified five indels and one SNP that reliably identify continent-of-origin, based on evaluations of up to 1078 individuals representing 13 white oak species and three continents. Due to the size of length polymorphisms revealed, this marker set can be visualized using capillary electrophoresis or high resolution gel (acrylamide or agarose) electrophoresis. With these markers, we provide the wood trading market with an instrument to comply with the U.S. and European laws that require timber companies to avoid the trade of illegally harvested timber.

Genome Sequences of Populus tremula Chloroplast and Mitochondrion: Implications for Holistic Poplar Breeding.

Complete Populus genome sequences are available for the nucleus (P. trichocarpa; section Tacamahaca) and for chloroplasts (seven species), but not for mitochondria. Here, we provide the complete genome sequences of the chloroplast and the mitochondrion for the clones P. tremula W52 and P. tremula x P. alba 717-1B4 (section Populus). The organization of the chloroplast genomes of both Populus clones is described. A phylogenetic tree constructed from all available complete chloroplast DNA sequences of Populus was not congruent with the assignment of the related species to different Populus sections. In total, 3,024 variable nucleotide positions were identified among all compared Populus chloroplast DNA sequences. The 5-prime part of the LSC from trnH to atpA showed the highest frequency of variations. The variable positions included 163 positions with SNPs allowing for differentiating the two clones with P. tremula chloroplast genomes (W52, 717-1B4) from the other seven Populus individuals. These potential P. tremula-specific SNPs were displayed as a whole-plastome barcode on the P. tremula W52 chloroplast DNA sequence. Three of these SNPs and one InDel in the trnH-psbA linker were successfully validated by Sanger sequencing in an extended set of Populus individuals. The complete mitochondrial genome sequence of P. tremula is the first in the family of Salicaceae. The mitochondrial genomes of the two clones are 783,442 bp (W52) and 783,513 bp (717-1B4) in size, structurally very similar and organized as single circles. DNA sequence regions with high similarity to the W52 chloroplast sequence account for about 2% of the W52 mitochondrial genome. The mean SNP frequency was found to be nearly six fold higher in the chloroplast than in the mitochondrial genome when comparing 717-1B4 with W52. The availability of the genomic information of all three DNA-containing cell organelles will allow a holistic approach in poplar molecular breeding in the future.