Foliar phenolic glycosides from Populus fremontii,Populus angustifolia,and their hybrids

Salicortin (1) and HCH-salicortin (2) were isolated and identified from the foliage of Populus fremontii and its F₁ hybrids with Populus angustifolia. Salicortin, but not HCH-salicortin, also occurred in P. angustifolia and complex backcrosses to angustifolia. Concentrations ranged from 0 to 17.5% dry weight for salicortin and 0 to 5.9% dry weight for HCH-salicortin. HCH-salicortin may possess potent anti-herbivore activity as it contains two of the hydroxycyclohexen-on-oyl moieties known to confer such activity to salicortin. Further, this compound may be a useful chemotaxonomic character within the genus Populus, since it appears to occur in section Aigeiros but not in section Tacamahaca.     

Phylogeny Reconstruction and Hybrid Analysis of Populus (Salicaceae) Based on Nucleotide Sequences of Multiple Single-Copy Nuclear Genes and Plastid Fragments

Populus (Salicaceae) is one of the most economically and ecologically important genera of forest trees. The complex reticulate evolution and lack of highly variable orthologous single-copy DNA markers have posed difficulties in resolving the phylogeny of this genus. Based on a large data set of nuclear and plastid DNA sequences, we reconstructed robust phylogeny of Populus using parsimony, maximum likelihood and Bayesian inference methods. The resulting phylogenetic trees showed better resolution at both inter- and intra-sectional level than previous studies. The results revealed that (1) the plastid-based phylogenetic tree resulted in two main clades, suggesting an early divergence of the maternal progenitors of Populus; (2) three advanced sections (Populus, Aigeiros and Tacamahaca) are of hybrid origin; (3) species of the section Tacamahaca could be divided into two major groups based on plastid and nuclear DNA data, suggesting a polyphyletic nature of the section; and (4) many species proved to be of hybrid origin based on the incongruence between plastid and nuclear DNA trees. Reticulate evolution may have played a significant role in the evolution history of Populus by facilitating rapid adaptive radiations into different environments.     

Clonal fingerprinting in the genus <Emphasis Type="Italic">Populus</Emphasis> L. by nuclear microsatellite loci regarding differences between sections,species and hybrids

Reliable methods for clone identification are desired to characterise and distinguish breeding products within the genus Populus L. (Salicaceae). Ten nuclear microsatellite loci (PMGC14, PMGC456, PMGC2163, PTR2, PTR7, WPMS05, WPMS09, WPMS14, WPMS15 and WPMS20) were applied on a clone collection with several species and hybrids belonging to the sections Tacamahaca (balsam poplars), Aigeiros (black poplars, cottonwoods) and Populus (white poplars and aspens) and intersectional hybrids between black and balsam poplars. The members of the different sections and species do not always share their allelic ladders. Some shifts of one or two nucleotides in allele length were observed for several loci. This could be explained by nucleotide sequence differences in the flanking regions of loci in diverse taxonomic groups. Such shifts of allelic ladders result in irregular patterns in hybrid genotypes. The set of loci should have a sufficient amount of variation for a differentiation between clones, even if they are full siblings originating from crossing experiments.     

Genetic linkage mapping in aspen (Populus tremula L. and Populus tremuloides Michx.)

A large number of simple sequence repeat (SSR) marker-containing genetic maps are available for several Populus species. For aspen however, no SSR-containing map has been published so far. In this study, genetic linkage mapping was carried out with an interspecific mapping pedigree of 61 full-sib hybrids of European × quaking aspen (Populus tremula L. × Populus tremuloides Michx.), using the two-way pseudo-testcross strategy. Amplified fragment-length polymorphism (AFLP) and SSR markers were used for mapping, resulting in the first SSR-containing genetic linkage maps for aspen. The maps allow comparisons with a Populus consensus map and other published genetic maps of the genus Populus. The maps showed good collinearity to each other and to the Populus consensus map and provide a direct link to the Populus trichocarpa genomic sequence. Sex as a morphological trait was assessed in the mapping population and mapped on a non-terminal position of linkage group XIX on the male P. tremuloides map.     

High-throughput genotyping in citrus accessions using an SNP genotyping array

We developed a 384 multiplexed SNP array, named CitSGA-1, for the genotyping of Citrus cultivars, and evaluated the performance and reliability of the genotyping. SNPs were surveyed by direct sequence comparison of the sequence tagged site (STS) fragment amplified from genomic DNA of cultivars representing the genetic diversity of citrus breeding in Japan. Among 1497 SNPs candidates, 384 SNPs for a high-throughput genotyping array were selected based on physical parameters of Illumina’s bead array criteria. The assay using CitSGA-1 was applied to a hybrid population of 88 progeny and 103 citrus accessions for breeding in Japan, which resulted in 73,726 SNP calls. A total of 351 SNPs (91 %) could call different genotypes among the DNA samples, resulting in a success rate for the assay comparable to previously reported rates for other plant species. To confirm the reliability of SNP genotype calls, parentage analysis was applied, and it indicated that the number of reliable SNPs and corresponding STSs were 276 and 213, respectively. The multiplexed SNP genotyping array reported here will be useful for the efficient construction of linkage map, for the detection of markers for marker-assisted breeding, and for the identification of cultivars.     

Host effect on genetic variation of Marssonina brunnea pathogenic to poplars

A broad collection was made for 42 isolates of Marssonina brunnea affecting poplar trees from three different sections (Leuce, Aigeiros and Tacamahaca) within the same Populus genus in China. Genetic diversity among these isolates was analyzed for morphological traits, cultural features, pathogenicity, hyphal anastomosis and randomly amplified polymorphic DNA markers (RAPDs). No significant difference was found in conidial morphological features, such as size, shape and septum location. Yet, considerable differences occur in other characteristics, which leads to the classification of the 42 isolates into two distinct groups, M. brunnea f.sp. monogermtubi and M. brunnea f.sp. multigermtubi. Isolates of M. brunnea f.sp. monogermtubi, derived from section Leuce, germinate only one germ tube, grow fast, produce dark-reddish conidiosorus clusters on the PDA medium, and are highly pathogenic to Populus tomentosa of section Leuce. By contrast, isolates of M. brunnea f.sp. multigermtubi, derived from sections Aigeiros and Tacamahaca, germinate 1–5 germ tubes, grow slowly, produce yellow-greenish conidiosorus clusters on PDA medium, and are pathogenic to Populus ×euramericana cv I-45 and Populus canadensis of section Aigeiros. DNA amplification using 11 RAPD primers generate 78 polymorphic bands among isolates. Cluster analyses based on RAPD markers broadly support such a classification by phenotypes, but provide a new insight into the possible origins of M. brunnea. It is proposed that the pathogen co-evolves with the poplars of section Leuce and has been subsequently distributed to the poplars of sections Aigeiros and Tacamahaca. An isolate from Populus adenopoda of section Leuce is placed in the third group, which is most likely a transmission type from M. brunnea f.sp. monogermtubi to M. brunnea f.sp. multigermtubi. Received: 20 May 1999 / Accepted: 30 July 1999     

Chloroplast phylogenomic analyses maternal relationships among sections in the genus Populus

The genus Populus L. has been divided into five sections based on morphological characters, but the phylogenetic relationships among sections remain uncertain. Topological discrepancies have been reported between trees obtained using nuclear and plastid sequences. We selected nine chloroplast genomes from all five sections, including two new sequenced species in this study for analyses of maternal phylogenetic relationships in the genus Populus at the sectional level. Phylogenetic analyses were performed using various subsets of data, coding sequences, noncoding sequences, and different districts of the genome, yielding contradictory outcomes for various subsets. According to our phylogenetic analyses, (1) a robust maternal phylogenetic relationship among sections based on complete chloroplast genomes was obtained; (2) Sect. Tacamahaca can be divided into two clades based on maternally inherited loci, i.e. cladeⅠ, distributed in North America and northeast China, and cladeⅡ, distributed in southwest China; (3) SSC-noncoding regions revealed an inconsistent topology compared with all other subsets; (4) this discrepancy may be resulted from incomplete lineage sorting between species of Populus. We tested multiple partitioning schemes to resolve deep-level phylogenetic relationships in Populus, and complete noncoding subset is most recommended.     

Genetic analysis of admixture and patterns of introgression in foundation cottonwood trees (Salicaceae) in southwestern Colorado,USA

Cottonwoods are well known as foundation riparian trees that support diverse communities and drive ecosystem processes. Although hybridization naturally occurs when the distributions of two or more cottonwood species overlap, few cottonwood hybrid zones have been genetically characterized. We use genetic and genomic analyses to characterize patterns of admixture and introgression for a newly described hybrid zone at the intersection of three species (Populus L. Salicaceae—Populus deltoides, Populus fremontii, and Populus angustifolia) in southwestern Colorado, USA. Analysis of nuclear and chloroplast microsatellite marker data detected substantial genetic variation among individuals, revealing that (1) hybridization is occurring between two, not three, species (P. deltoides and P. angustifolia); (2) gene flow is bidirectional; (3) hybrids are not abundant (admixture detected in only 34 of 270 trees), with most being early-generation F₁ hybrids; (4) cytonuclear disequilibria exists and F₁ hybrids tend to retain P. deltoides—like chloroplasts; and (5) roughly 30 % of the nuclear markers deviated from a neutral pattern of introgression, suggesting that selection may play a role in shaping the genetic structure of the hybrid zone in this region. Overall, our results show that despite strong selection maintaining species divergence, transfer of allelic variants across species boundaries can occur. Our study assesses the fine-scale genetic structure of hybridization between P. angustifolia and P. deltoides and lays the foundation for examining how geographic differences in hybrid zone dynamics arise and may influence subsequent ecological and evolutionary processes.     

Potentials and limitations of the cross-species transfer of nuclear microsatellite marker in six species belonging to three sections of the genus Populus L.

The genus Populus is classified into six different sections, and depending on the declaration of hybrids, the number of species varies between 22 and 85. Species within one section, and sometimes between sections, are crossable to each other, resulting in many naturally but also artificially produced hybrids. Morphological attributes for a clone characterisation are often difficult to evaluate when different poplar species or even hybrids are crossed; thus, molecular markers are needed to characterise the different species. Taking advantage of the large microsatellite resource developed for Populus trichocarpa, however, amplification of these microsatellite markers in other Populus species either often fails, or in the case of amplification, unrelated genomic regions are amplified. To meet this obvious problem of the species transferability of microsatellite markers, in total, 305 microsatellite loci, mainly from P. trichocarpa but also few from Populus tremuloides and Populus nigra, were tested for their transferability to diverse genotypes of six species belonging to three sections of the genus Populus. Ultimately, 209 microsatellite loci could be amplified with varying sizes in the different species. The PCR products of selected loci were separated in a polyacrylamide gel and sequenced to assure that the expected loci were derived from the database genome of P. trichocarpa. The present results constitute a large study for microsatellite transferability for Populus species. The documented microsatellite loci can be applied to species-, hybrid- and clone-specific diagnostic approaches or as universal markers for comprehensive ecological studies.     

A 34K SNP genotyping array for Populus trichocarpa: Design,application to the study of natural populations and transferability to other Populus species

Genetic mapping of quantitative traits requires genotypic data for large numbers of markers in many individuals. For such studies, the use of large single nucleotide polymorphism (SNP) genotyping arrays still offers the most cost‐effective solution. Herein we report on the design and performance of a SNP genotyping array for Populus trichocarpa (black cottonwood). This genotyping array was designed with SNPs pre‐ascertained in 34 wild accessions covering most of the species latitudinal range. We adopted a candidate gene approach to the array design that resulted in the selection of 34 131 SNPs, the majority of which are located in, or within 2 kb of, 3543 candidate genes. A subset of the SNPs on the array (539) was selected based on patterns of variation among the SNP discovery accessions. We show that more than 95% of the loci produce high quality genotypes and that the genotyping error rate for these is likely below 2%. We demonstrate that even among small numbers of samples (n = 10) from local populations over 84% of loci are polymorphic. We also tested the applicability of the array to other species in the genus and found that the number of polymorphic loci decreases rapidly with genetic distance, with the largest numbers detected in other species in section Tacamahaca. Finally, we provide evidence for the utility of the array to address evolutionary questions such as intraspecific studies of genetic differentiation, species assignment and the detection of natural hybrids.     

Genetic identification of 91 poplar cultivars based on SSR markers

Accurate genetic identification and relationship analysis of poplar cultivars is necessary to establish commercial poplar plantations and select suitable breeding strategies. In this study, 91 poplar cultivars belonging to four sections (Aigeiros, Tacamahaca, Populus and Turanga) and inter/intra-sectional hybrids were genotyped using 18 polymorphic simple sequence repeat (SSR) markers. In total, 222 alleles were amplified with an average of 12.3 alleles per marker. The mean polymorphic information content and power of discrimination were 0.706 and 0.813, respectively. Five SSR markers (ORPM_103, ORPM_247, GCPM_1048, GCPM_1255 and LG_X_19) constituted a core fingerprint and were sufficient to identify all the tested cultivars. With some notable exceptions, cultivars of the same species generally clustered together in cluster (UPGMA) and ordination (PCO) analyses. Flow cytometry indicated that 11 poplar cultivars were triploid. Among these, seven had three alleles at some loci, suggesting that SSR markers could indicate the ploidy level to some extent. This study provides useful genetic information for the identification and protection of poplar cultivars in China and offers a guideline for the selection of poplar crossing parents based on ploidy level and genetic relationships.     

杨属物种多样性在中国的地理分布格局

利用地理信息系统技术与空间统计相结合的方法,研究了杨属物种多样性在中国区域尺度上的空间分布格局,并且采用线性回归分析方法研究了杨属物种多样性与经纬度的关系。结果表明:(1)杨属物种在中国呈带状分布,东西分布幅度大于南北分布幅度;(2)纬度梯度上杨属物种集中分布区位于30—40°N,经度梯度上位于110—120°E,说明杨属物种多样性格局在经、纬度梯度上并非单调递增或递减,而是在某一最适区域具有最高的物种多样性;(3)中国杨属物种多样性的分布中心集中于东北—西南方向的黑河-腾冲线两侧,从黑腾线向西北和东南方向延伸过程中,物种多样性逐渐降低;沿黑腾线从东北向西南方向物种多样性呈增加趋势;(4)从分组、分种检索上看,白杨组和青杨组物种在中国分布范围最广,山杨、小叶杨、响叶杨和青杨4种物种分布范围最广,可见同一类群中不同分类等级的植物多样性地理分布格局存在差异。     

Efficient Agrobacterium-mediated transformation of commercial hybrid poplar Populus nigra L. × P. maximowiczii A. Henry

Many economically important species of Populus, especially those in sections Aigeiros and Tacamahaca, remain recalcitrant to genetic transformation. In this study, a simple and reliable protocol was developed for the efficient Agrobacterium-mediated transformation of a difficult-to-transform, but commercially viable, hybrid poplar Populus nigra L. × P. maximowiczii A. Henry (NM6). A plant transformation vector designed to express the β-glucuronidase (GUS) gene was used to detect transformation events at early stages of plant regeneration and to optimize parameters affecting poplar transformation. The use of zeatin riboside in shoot-induction medium, regeneration of shoots via indirect organogenesis, and early selection pressure were the major modifications that drastically improved the efficiency of poplar transformation and minimized the number of untransformed regenerants. Transgenic shoots were routinely obtained 4–10 weeks after co-culture with A. tumefaciens, with a greater than 90% rate of plant recovery. Stable transgene integration, ranging from a single insertion to ten copies per genome, was confirmed by Southern blot analysis. The mean transformation frequency was 36.3% and about two-thirds of the lines had 1–2 transgene copies. Among the explants, petioles and leaves had a higher transformation frequency than did stem segments. Growth characteristics and the morphology of transgenic poplar plants were identical to untransformed controls. These findings will accelerate the development of P. nigra × P. maximowiczii plants with novel traits, and may also be useful to improve transformation procedures for other Populus species.     

New resources for genetic studies in Populus nigra: genome‐wide SNP discovery and development of a 12k Infinium array

Whole genome resequencing of 51 Populus nigra (L.) individuals from across Western Europe was performed using Illumina platforms. A total number of 1 878 727 SNPs distributed along the P. nigra reference sequence were identified. The SNP calling accuracy was validated with Sanger sequencing. SNPs were selected within 14 previously identified QTL regions, 2916 expressional candidate genes related to rust resistance, wood properties, water‐use efficiency and bud phenology and 1732 genes randomly spread across the genome. Over 10 000 SNPs were selected for the construction of a 12k Infinium Bead‐Chip array dedicated to association mapping. The SNP genotyping assay was performed with 888 P. nigra individuals. The genotyping success rate was 91%. Our high success rate was due to the discovery panel design and the stringent parameters applied for SNP calling and selection. In the same set of P. nigra genotypes, linkage disequilibrium throughout the genome decayed on average within 5–7 kb to half of its maximum value. As an application test, ADMIXTURE analysis was performed with a selection of 600 SNPs spread throughout the genome and 706 individuals collected along 12 river basins. The admixture pattern was consistent with genetic diversity revealed by neutral markers and the geographical distribution of the populations. These newly developed SNP resources and genotyping array provide a valuable tool for population genetic studies and identification of QTLs through natural‐population based genetic association studies in P. nigra.     

Development of a high-throughput SNP array for sea cucumber (Apostichopus japonicus) and its application in genomic selection with MCP regularized deep neural networks

High-throughput single nucleotide polymorphism (SNP) genotyping assays are powerful tools for genetic studies and genomic breeding applications for many species. Though large numbers of SNPs have been identified in sea cucumber (Apostichopus japonicus), but, as yet, no high-throughput genotyping platform is available for this species. In this study, we designed and developed a high-throughput 24 K SNP genotyping array named HaishenSNP24K for A. japonicus, based on the multi-objective-local optimization (MOLO) algorithm and HD-Marker genotyping method. The SNP array exhibited a relatively high genotyping call rate (> 96%), genotyping accuracy (>95%) and exhibited highly polymorphic in sea cucumber populations. In addition, we also assessed its application in genomic selection (GS). Deep neural networks (DNN) that can capture the complicated interactions of genes have been proposed as a promising tool in GS for SNP-based genomic prediction of complex traits in animal breeding. To overcome the problem of over-fitting when using the HaishenSNP24K array as high-dimensional DNN input, we developed minmax concave penalty (MCP) regularization for sparse deep neural networks (DNN-MCP) that finds an optimal sparse structure of a DNN by minimizing the square error subject to the non-convex penalty MCP on the parameters (weights and biases). Compared to two linear models, namely RR-GBLUP and Bayes B, and the nonlinear model DNN, DNN-MCP has greatly improved the genomic prediction ability for three quantitative traits (e.g., wet weight, dry weight and survival time) in the sea cucumber population. To the best of our knowledge, this is the first work to develop a high-throughput SNP array for A. japonicus and a new model DNN-MCP for genomic prediction of complex traits in GS. The present results provide evidence that supports the HaishenSNP24K array with DNN-MCP will be valuable for genetic studies and molecular breeding in A. japonicus.     

Early growth,dry matter allocation and water use efficiency of two sympatric Populus species as affected by water stress

We exposed cuttings of two sympatric species of Sect. Tacamahaca Spach, Populus cathayana Rehder and Populus przewalskii Maximowicz, to two watering regimes in a greenhouse. In the semi-controlled environmental study, two watering treatments which were watered to 100 and 25% of field capacity were used, respectively. The effects of water deficit on early growth, biomass allocation and water use efficiency (WUE) were investigated. We found that there were significant interspecific differences in early growth, dry matter allocation and water use efficiency between two sympatric Populus species. Compared with P. cathayana, P. przewalskii showed higher shoot height, dry matter accumulation, number of leaves, total leaf area, fine root mass, fine root/total root ratio and water use efficiency under both well-watered and water-stressed treatments. On the other hand, P. przewalskii also showed higher root mass/foliage area ratio, root/shoot ratio and carbon isotope composition than P. cathayana under water-stressed treatment. The results suggested that there were different water-use strategies between two sympatric Populus species, P. przewalskii with higher drought tolerance may employ a conservative water-use strategy, whereas P. cathayana with lower drought tolerance may employ a prodigal water-use strategy. The findings confirm the existence of interspecific genetic differences in early growth, dry matter allocation and water use efficiency as affected by water stress, these variations in drought responses may be used as criteria for species selection and tree improvement.     

Optimization of a real-time PCR assay for the detection of the quarantine pathogen Melampsora medusae f. sp. deltoidae

Melampsora medusae (Mm), one of the causal agents of poplar rust, is classified as an A2 quarantine pest for European Plant Protection Organization (EPPO) and its presence in Europe is strictly controlled. Two formae speciales have been described within Mm, Melampsora medusae f. sp. deltoidae (Mmd), and Melampsora medusae f. sp. tremuloidae (Mmt) on the basis of their pathogenicity on Populus species from the section Aigeiros (e.g. Populus deltoides) or Populus (e.g. Populus tremuloides), respectively. In this study, a real-time polymerase chain reaction (PCR) assay was developed allowing the detection of Mmd, the forma specialis that is economically harmful. A set of primers and hydrolysis probe were designed based on sequence polymorphisms in the large ribosomal RNA subunit (28S). The real-time PCR assay was optimized and performance criteria of the detection method, i.e. sensitivity, specificity, repeatability, reproducibility, and robustness, were assessed. The real-time PCR method was highly specific and sensitive and allowed the detection of one single urediniospore of Mmd in a mixture of 2 mg of urediniospores of other Melampsora species. This test offers improved specificity over currently existing conventional PCR tests and can be used for specific surveys in European nurseries and phytosanitary controls, in order to avoid introduction and spread of this pathogen in Europe.     

SNP genotyping in melons: genetic variation, population structure, and linkage disequilibrium

Novel sequencing technologies were recently used to generate sequences from multiple melon (Cucumis melo L.) genotypes, enabling the in silico identification of large single nucleotide polymorphism (SNP) collections. In order to optimize the use of these markers, SNP validation and large-scale genotyping are necessary. In this paper, we present the first validated design for a genotyping array with 768 SNPs that are evenly distributed throughout the melon genome. This customized Illumina GoldenGate assay was used to genotype a collection of 74 accessions, representing most of the botanical groups of the species. Of the assayed loci, 91 % were successfully genotyped. The array provided a large number of polymorphic SNPs within and across accessions. This set of SNPs detected high levels of variation in accessions from this crop’s center of origin as well as from several other areas of melon diversification. Allele distribution throughout the genome revealed regions that distinguished between the two main groups of cultivated accessions (inodorus and cantalupensis). Population structure analysis showed a subdivision into five subpopulations, reflecting the history of the crop. A considerably low level of LD was detected, which decayed rapidly within a few kilobases. Our results show that the GoldenGate assay can be used successfully for high-throughput SNP genotyping in melon. Since many of the genotyped accessions are currently being used as the parents of breeding populations in various programs, this set of mapped markers could be used for future mapping and breeding efforts.     

Identification of Pyrus Single Nucleotide Polymorphisms (SNPs) and Evaluation for Genetic Mapping in European Pear and Interspecific Pyrus Hybrids

We have used new generation sequencing (NGS) technologies to identify single nucleotide polymorphism (SNP) markers from three European pear (Pyrus communis L.) cultivars and subsequently developed a subset of 1096 pear SNPs into high throughput markers by combining them with the set of 7692 apple SNPs on the IRSC apple Infinium® II 8K array. We then evaluated this apple and pear Infinium® II 9K SNP array for large-scale genotyping in pear across several species, using both pear and apple SNPs. The segregating populations employed for array validation included a segregating population of European pear (‘Old Home’בLouise Bon Jersey’) and four interspecific breeding families derived from Asian (P. pyrifolia Nakai and P. bretschneideri Rehd.) and European pear pedigrees. In total, we mapped 857 polymorphic pear markers to construct the first SNP-based genetic maps for pear, comprising 78% of the total pear SNPs included in the array. In addition, 1031 SNP markers derived from apple (13% of the total apple SNPs included in the array) were polymorphic and were mapped in one or more of the pear populations. These results are the first to demonstrate SNP transferability across the genera Malus and Pyrus. Our construction of high density SNP-based and gene-based genetic maps in pear represents an important step towards the identification of chromosomal regions associated with a range of horticultural characters, such as pest and disease resistance, orchard yield and fruit quality.     

Transient expression for functional gene analysis using Populus protoplasts

Despite the availability of the Populus genome sequence and the development of genetic, genomic, and transgenic approaches for its improvement, the lengthy life span of Populus and the cumbersome process required for its transformation have impeded rapid characterization of gene functions in Populus. Protoplasts provide a versatile and physiologically relevant cell system for high-throughput analysis and functional characterization of plant genes. Here, a highly efficient transient expression system using Populus mesophyll protoplasts was developed based on the following three steps. The first step involved formulating a new enzyme cocktail containing 2 % Cellulase C2605 and 0.5 % Pectinase P2611, which was shown to enable efficient large-scale isolation of homogenous Populus mesophyll protoplasts. The second step involved optimization of transfection conditions, such as the polyethylene glycol concentration and amount of plasmid DNA to ensure a >80 % transfection efficiency for Populus protoplasts. The third step involved using the Populus protoplast transient expression system to successfully determine the subcellular localizations of proteins, emulate signaling events during pathogen infection, and prepare protein extracts for Western blotting and protein–protein interaction assays. This rapid and highly efficient transient gene expression system in Populus mesophyll protoplasts will facilitate the rapid identification of gene functions and elucidation of signaling pathways in Populus.