High-quality genome assembly enables prediction of allele-specific gene expression in hybrid poplar.

Poplar (Populus) is a well-established model system for tree genomics and molecular breeding, and hybrid poplar is widely used in forest plantations. However, distinguishing its diploid homologous chromosomes is difficult, complicating advanced functional studies on specific alleles. In this study, we applied a trio-binning design and PacBio High-Fidelity long-read sequencing to obtain haplotype-phased telomere-to-telomere genome assemblies for the two parents of the well-studied F1 hybrid "84K" (Populus alba × P. tremula var. glandulosa). Almost all chromosomes, including the telomeres and centromeres, were completely assembled for each haplotype subgenome apart from two small gaps on one chromosome. By incorporating information from these haplotype assemblies and extensive RNA-seq data, we analyzed gene expression patterns between the two subgenomes and alleles. Transcription bias at the subgenome level was not uncovered, but extensive expression differences were detected between alleles. We developed machine-learning (ML) models to predict allele-specific expression (ASE) with high accuracy and identified underlying genome features most highly influencing ASE. One of our models with 15 predictor variables achieved 77% accuracy on the training set and 74% accuracy on the testing set. ML models identified gene body CHG methylation, sequence divergence, and transposon occupancy both upstream and downstream of alleles as important factors for ASE. Our haplotype-phased genome assemblies and ML strategy highlight an avenue for functional studies in Populus and provide additional tools for studying ASE and heterosis in hybrids.

Morphology, sucrose metabolism and gene network reveal the molecular mechanism of seed fiber development in poplar.

Poplar is an important tree species for ecological protection, wood production, bioenergy and urban greening; it has been widely planted worldwide. However, the catkin fibers produced by female poplars can cause environmental pollution and safety hazards during spring. This study focused on Populus tomentosa, and revealed the sucrose metabolism regulatory mechanism of catkin fibers development from morphological, physiological and molecular aspects. Paraffin section suggested that poplar catkin fibers were not seed hairs and produced from the epidermal cells of funicle and placenta. Sucrose degradation via invertase and sucrose synthase played the dominant role during poplar catkin fibers development. The expression patterns revealed that sucrose metabolism-related genes played important roles during catkin fibers development. Y1H analysis indicated that there was a potential interaction between sucrose synthase 2 (PtoSUS2)/vacuolar invertase 3 (PtoVIN3) and trichome-regulating MYB transcription factors in poplar. Finally, the two key genes, PtoSUS2 and PtoVIN3, had roles in Arabidopsis trichome density, indicating that sucrose metabolism is important in poplar catkin fibers development. This study is not only helpful for clarifying the mechanism of sucrose regulation during trichome development in perennial woody plants, but also establishes a foundation to solve poplar catkin fibers pollution through genetic engineering methods.

Identification and Validation of Reliable Reference Genes for Gene Expression Studies in Koelreuteria paniculata.

RT-qPCR is considered a rapid and reliable technique for analyzing gene expression. This technique is commonly used to analyze the expression of various genes at diverse transcriptional levels in different samples. However, few studies have characterized ornamental Koelreuteria species for reliable reference genes. In this study, eight reference genes were evaluated as controls in RT-qPCR with SYBR green to quantify gene expression in different Koelreuteria paniculata samples. All selected reference genes showed a broad range of Ct values in all samples, which was supportive of their variable expression. Our results showed significant variation in the stable expression of K. paniculata genes. Sample data, analyzed using geNorm, NormFinder, and BestKeeper, showed that phospholipase (PLA2) and ß-actin (ACT) were the most suitable and statistically reliable reference genes, whereas ribosomal protein L13 (RPL13) and elongation factor 1-α (EF1α) were less stable and unsuitable for use as internal controls. To compare gene expression levels, two or more reference genes should be used for data normalization. Thus, the stability and expression of both PLA2 and ACT were believed to provide better normalization and quantification of the transcript levels for gene expression studies in K. paniculata.

Adaptation by Type III CRISPR-Cas Systems: Breakthrough Findings and Open Questions.

CRISPR-Cas systems acquire heritable defense memory against invading nucleic acids through adaptation. Type III CRISPR-Cas systems have unique and intriguing features of defense and are important in method development for Genetics research. We started to understand the common and unique properties of type III CRISPR-Cas adaptation in recent years. This review summarizes our knowledge regarding CRISPR-Cas adaptation with the emphasis on type III systems and discusses open questions for type III adaptation studies.

Comprehensive Analyses of Four PtoNF-YC Genes from Populus tomentosa and Impacts on Flowering Timing.

Flowering is an important link in the life process of angiosperms, and it is also an important sign of the transformation of plants from vegetative to reproductive growth. Although the flowering regulation network of Arabidopsis is well-understood, there has been little research on the molecular mechanisms of perennial woody plant flower development regulation. Populus tomentosa is a unique Chinese poplar species with fast growth, strong ecological adaptability, and a long lifecycle. However, it has a long juvenile phase, which seriously affects its breeding process. Nuclear factor-Y (NF-Y) is an important type of transcription factor involved in the regulation of plant flowering. However, there are few reports on PtoNF-Y gene flowering regulation, and the members of the PtNF-YC subfamily are unknown. In this study, four key genes were cloned and analyzed for sequence characteristics, gene structure, genetic evolution, expression patterns, and subcellular localization. The plant expression vector was further constructed, and transgenic Arabidopsis and P. tomentosa plants were obtained through genetic transformation and a series of molecular tests. The flowering time and other growth characteristics were analyzed. Finally, the expression level of flowering genes was detected by quantitative PCR, the interaction between PtoNF-YC and PtoCOL proteins was measured using the yeast two-hybrid system to further explain the flowering regulation mechanism, and the molecular mechanisms by which PtNF-YC6 and PtNF-YC8 regulate poplar flowering were discussed. These results lay the foundation for elucidating the molecular regulation mechanism of PtoNF-YC in flowering and furthering the molecular design and breeding of poplar, while providing a reference for other flowering woody plants.

Transcriptome and miRNAome analysis reveals components regulating tissue differentiation of bamboo shoots.

Primary thickening determines bamboo yield and wood property. However, little is known about the regulatory networks involved in this process. This study identified a total of 58,652 genes and 150 miRNAs via transcriptome and small RNA sequencing using the underground thickening shoot samples of wild-type (WT) Moso bamboo (Phyllostachys edulis) and a thick wall (TW) variant (P. edulis "Pachyloen") at five developmental stages (WTS1/TWS1-WTS5/TWS5). A total of 14,029 (65.17%) differentially expressed genes and 68 (45.33%) differentially expressed miRNAs were identified from the WT, TW, and WTTW groups. The first two groups were composed of four pairwise combinations, each between two successive stages (WTS2/TWS2_versus_WTS1/TWS1, WTS3/TWS3_versus_WTS2/TWS2, WTS4/TWS4_versus_WTS3/TWS3, and WTS5/TWS5_versus_WTS4/TWS4), and the WTTW group was composed of five combinations, each between two relative stages (TWS1-5_versus_WTS1-5). Additionally, among the phytohormones, zeatin showed more remarkable changes in concentrations than indole-3-acetic acid, gibberellic acid, and abscisic acid throughout the five stages in the WT and the TW groups. Moreover, 125 cleavage sites were identified for 387 miRNA-mRNA pairs via degradome sequencing (P < 0.05). The dual-luciferase reporter assay confirmed that 13 miRNAs bound to 12 targets. Fluorescence in situ hybridization localized miR166 and miR160 in the shoot apical meristem and the procambium of Moso bamboo shoots at the S1 stage. Thus, primary thickening is a complex process regulated by miRNA-gene-phytohormone networks, and the miRNAome and transcriptome dynamics regulate phenotypic plasticity. These findings provide insights into the molecular mechanisms underlying wood formation and properties and propose targets for bamboo breeding.

High quality haplotype-resolved genome assemblies of Populus tomentosa Carr., a stabilized interspecific hybrid species widespread in Asia.

Populus has a wide ecogeographical range spanning the Northern Hemisphere, and interspecific hybrids are common. Populus tomentosa Carr. is widely distributed and cultivated in the eastern region of Asia, where it plays multiple important roles in forestry, agriculture, conservation, and urban horticulture. Reference genomes are available for several Populus species, however, our goals were to produce a very high quality de novo chromosome-level genome assembly in P. tomentosa genome that could serve as a reference for evolutionary and ecological studies of hybrid speciation throughout the genus. Here, combining long-read sequencing and Hi-C scaffolding, we present a high-quality, haplotype-resolved genome assembly. The genome size was 740.2 Mb, with a contig N50 size of 5.47 Mb and a scaffold N50 size of 46.68 Mb, consisting of 38 chromosomes, as expected with the known diploid chromosome number (2n = 2x = 38). A total of 59,124 protein-coding genes were identified. Phylogenomic analyses revealed that P. tomentosa is comprised of two distinct subgenomes, which we deomonstrate is likely to have resulted from hybridization between Populus adenopoda as the female parent and Populus alba var. pyramidalis as the male parent, with an origin of approximately 3.93 Ma. Although highly colinear, significant structural variation was found between the two subgenomes. Our study provides a valuable resource for ecological genetics and forest biotechnology.

Investigation of PtSGT1 and PtSGT4 Function in Cellulose Biosynthesis in Populus tomentosa Using CRISPR/Cas9 Technology.

Cellulose synthesis is a complex process in plant cells that is important for wood processing, pulping, and papermaking. Cellulose synthesis begins with the glycosylation of sitosterol by sitosterol glycosyltransferase (SGT) to produce sitosterol-glucoside (SG), which acts as the guiding primer for cellulose production. However, the biological functions of SGTs in Populus tomentosa(P. tomentosa) remain largely unknown. Two full-length PtSGT genes (PtSGT1 and PtSGT4) were previously isolated from P. tomentosa and characterized. In the present study, CRISPR/Cas9 gene-editing technology was used to construct PtSGT1-sgRNA and PtSGT4-sgRNA expression vectors, which were genetically transformed into P. tomentosa using the Agrobacterium-mediated method to obtain transgenic lines. Nucleic acid and amino acid sequencing analysis revealed both base insertions and deletions, in addition to reading frame shifts and early termination of translation in the transgenic lines. Sugar metabolism analysis indicated that sucrose and fructose were significantly downregulated in stems and leaves of mutant PtSGT1-1 and PtSGT4-1. Glucose levels did not change significantly in roots and stems of PtSGT1-1 mutants; however, glucose was significantly upregulated in stems and downregulated in leaves of the PtSGT4-1 mutants. Dissection of the plants revealed disordered and loosely arranged xylem cells in the PtSGT4-1 mutant, which were larger and thinner than those of the wild-type. This work will enhance our understanding of cellulose synthesis in the cell walls of woody plants.

Genome-wide analysis of the MYB-related transcription factor family and associated responses to abiotic stressors in Populus.

MYB proteins are one of the most abundant transcription factor families in the plant kingdom. Evidence has increasingly revealed that MYB-related proteins function in diverse plant biological processes. However, little is known about the genome-wide characterization and functions of MYB-related proteins in Populus, an important model and commercial tree species. In this study, 152 PtrMYBRs were identified and unevenly located on 19 Populus chromosomes. A phylogenetic analysis divided them into six major subgroups, supported by conserved gene organization, consensus motifs, and protein domain architecture. Promoter assessment and gene ontology classification results indicated that the MYB-related family is likely involved in plant development and responses to various environmental stressors. The Populus MYB-related family members showed various expression patterns in different tissues and stress conditions, implying their crucial roles in the development and stress responses in Populus. Co-expression analyses revealed that Populus MYB-related genes might participate in the regulation of antioxidant defense system and various signaling pathways in response to stress. The three-dimensional structures of different subgroup of Populus MYB-related proteins further provided functional information at the proteomic level. These findings provide valuable information for a prospective functional dissection of MYB-related proteins and genetic improvement of Populus.

Characterization and expression pattern of the trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase gene families in Populus.

Trehalose plays an important role in plant metabolism, growth development, and stress tolerance. Trehalose-6-phosphate synthase gene (TPS) and trehalose-6-phosphate phosphatase gene (TPP) are vital for the synthesis of trehalose. Populus is a prominent perennial woody plant, in which systematic genome-wide analysis of the TPS and TPP family is limited. In this study, 13 PtTPS and 10 PtTPP genes were identified in the Populus genome. Phylogenetic analysis indicated PtTPS and PtTPP genes were both divided into two subfamilies, and gene members of each subfamily have highly conserved intron structures. Analysis of cis-acting elements showed that PtTPS and PtTPP genes were involved in plant hormones and environmental stress responses. Expression profiles also found PtTPSs and PtTPPs expressed differently in response to salt stress, cold, mechanical damage, salicylic acid, and methyl jasmonate treatment. Furthermore, reverse transcription quantitative real-time PCR results found PtTPSs and PtTPPs displayed a specific expression pattern in the seven developmental stages of Populus male and female floral buds. This work will not only lead a foundation on reveal the functions of PtTPS and PtTPP gene families in trehalose regulation of poplar but also provide references to related trehalose research in other perennial plants.

Unraveling the genetic diversity and structure of Quercus liaotungensis population through analysis of microsatellite markers.

BACKGROUND: Quercus liaotungensis Koidz. is an ecologically and economically important tree species widely distributed in Northern China. However, the effective assessment, utilization, and protection of Q. liaotungensis resources remain unexplored. METHODS: In total, 120 samples obtained from 12 Q. liaotungensis populations of Northern China were investigated for genetic diversity and structure using 19 simple sequence repeat (SSR) primer pairs. RESULTS: The total number of alleles detected was 293, the average number of effective allele (Ne) was 6.084, the genetic differentiation coefficient (Fst) was 0.033, and the mean observed heterozygosity (Ho) and expected heterozygosity (He) were 0.690 and 0.801, respectively. Moreover, analysis of molecular variance (AMOVA) showed a 5.5% genetic variation among 12 Q. liaotungensis populations, indicating that a high level of genetic diversity and a low degree of genetic differentiation among Q. liaotungensis populations. STRUCTURE and cluster analysis divided the 12 Q. liaotungensis populations into the following three subpopulations: Bashang Plateau subpopulation (SH), Liaodong Peninsula subpopulation (NC), and Loess Plateau subpopulation (other 10 populations). The cluster analysis based on 19 climatic factors was consistent with the genetic structure. A positive correlation was found between genetic distance and geographical distance (r = 0.638, p = 0.028) by the Mantel test, and two boundaries were found among the 12 Q. liaotungensis populations by the Barrier analysis, indicating that Q. liaotungensis populations existed isolated by geographical distance and physical barrier. CONCLUSION: This study suggests that geographical isolation, physical barrier, climatic types, and natural hybridization promote the formation of genetic structures, which can contribute to future protection and genetic improvement of Q. liaotungensis.

Genetic containment in vegetatively propagated forest trees: CRISPR disruption of LEAFY function in Eucalyptus gives sterile indeterminate inflorescences and normal juvenile development.

Eucalyptus is among the most widely planted taxa of forest trees worldwide. However, its spread as an exotic or genetically engineered form can create ecological and social problems. To mitigate gene flow via pollen and seeds, we mutated the Eucalyptus orthologue of LEAFY (LFY) by transforming a Eucalyptus grandis × urophylla wild-type hybrid and two Flowering Locus T (FT) overexpressing (and flowering) lines with CRISPR Cas9 targeting its LFY orthologue, ELFY. We achieved high rates of elfy biallelic knockouts, often approaching 100% of transgene insertion events. Frameshift mutations and deletions removing conserved amino acids caused strong floral alterations, including indeterminacy in floral development and an absence of male and female gametes. These mutants were otherwise visibly normal and did not differ statistically from transgenic controls in juvenile vegetative growth rate or leaf morphology in greenhouse trials. Genes upstream or near to ELFY in the floral development pathway were overexpressed, whereas floral organ identity genes downstream of ELFY were severely depressed. We conclude that disruption of ELFY function appears to be a useful tool for sexual containment, without causing statistically significant or large adverse effects on juvenile vegetative growth or leaf morphology.

Characterization of the complete chloroplast genome of Calophaca sinica Rehd.

Calophaca sinica Rehd. is a tree species with high economic value, whose resource has been declining due to unreasonable exploitation. In this study, we sequenced, assembled, and annotated the complete chloroplast genome of C. sinica. The whole chloroplast genome size is 129,345 bp, it lacks an inverted repeat (IR) region. The GC content of the whole chloroplast genome is 34.51%. The chloroplast genome comprises 112 unique genes, including 77 protein-coding genes (PCGs), 30 transfer RNA (tRNA) genes, and 5 ribosomal RNA (rRNA) genes. Phylogenetic analyses of chloroplast genomes derived from 15 species indicated that C. sinica is close to Caragana and Tibetia species in Papilionoideae.

Identification and characterization of the CONSTANS-like gene family and its expression profiling under light treatment in Populus.

The CONSTANS-like (COL) genes play an important role in the photoperiodic flowering pathway. Poplar is a perennial woody plant with a long juvenile phase, but the molecular characterization of COL genes in Populus is limited. In this study, 14 COL genes were identified in the Populus genome. Phylogenetic analysis indicated the PtCOL proteins were divided into three subgroups, and the members of each subgroup had similar gene structure and motif composition. Chromosome distribution analysis showed that 14 PtCOL genes were distributed on 10 chromosomes. Multiple sequence alignment indicated that these proteins contained a highly conserved B-box1 and a conserved CCT domain, but the B-box2 structure was divided into three different types. Promoter analysis found that there were several light-responsive cis-elements in the PtCOL genes. Furthermore, tissue-specific expression showed that all nine PtCOL genes were widely expressed in various tissues and organs of Populus, and were preferentially expressed in the leaves. Additionally, the transcription level of PtCOL exhibited a diurnal oscillation pattern in different light conditions. This study not only provided comprehensive information for further analysis of the function of the PtCOL gene family, but also revealed the biological roles of PtCOL genes in the photoperiod-dependent flowering process of Populus.

Altered sucrose metabolism and plant growth in transgenic Populus tomentosa with altered sucrose synthase PtSS3.

Improvement of wood quality is an important focus of forest genetics and breeding research. Sucrose synthase (SS) catalyzes the reaction of sucrose and uridine diphosphate into uridine diphosphate glucose and fructose. It is a key enzyme involved in cell wall formation during secondary growth by providing the UDP-Glucose substrate for cellulose biosynthesis. In this study, we isolated the single-copy gene PtSS3 from the SS gene family of Populus tomentosa and analyzed its structure. To identify its function in secondary growth, we generated 19 transgenic lines of P. tomentosa using PtSS3 overexpression (OE) and artificial microRNA (amiRNA) constructs. We also performed comprehensive analyses of the transgenic P. tomentosa plants, including phenotypic analyses, quantitative real-time PCR, enzyme activity assays and sugar metabolism. We found significantly higher PtSS3 enzyme activity, fructose, and glucose levels and significantly lower sucrose levels in the stems and leaves of OE-PtSS3 plants. The opposite trend was observed in the amiRNA-PtSS3 lines. Gene expression analyses showed that PtSS3 transcript levels in stems and leaves were up-regulated in the OE-PtSS3 lines and down-regulated in the amiRNA-PtSS3 lines, and the OE-PtSS3 plants grew taller than the wild-type and amiRNA-PtSS3 plants. These findings indicate that PtSS3 plays an important role in sucrose metabolism and growth of trees.

RNA interference suppression of AGAMOUS and SEEDSTICK alters floral organ identity and impairs floral organ determinacy, ovule differentiation, and seed-hair development in Populus.

The role of the floral homeotic gene AGAMOUS (AG) and its close homologues in development of anemophilous, unisexual catkins has not previously been studied. We transformed two RNA interference (RNAi) constructs, PTG and its matrix-attachment-region flanked version MPG, into the early-flowering female poplar clone 6K10 (Populus alba) to suppress the expression of its two duplicate AG orthologues. By early 2018, six out of 22 flowering PTG events and 11 out of 12 flowering MPG events showed modified floral phenotypes in a field trial in Oregon, USA. Flowers in catkins from modified events had 'carpel-inside-carpel' phenotypes. Complete disruption of seed production was observed in seven events, and sterile anther-like organs in 10 events. Events with strong co-suppression of both the two AG and two SEEDSTICK (STK) paralogues lacked both seeds and associated seed hairs. Alterations in all of the modified floral phenotypes were stable over 4 yr of study. Trees from floral-modified events did not differ significantly (P < 0.05) from nonmodified transgenic or nontransgenic controls in biomass growth or leaf morphology. AG and STK genes show strong conservation of gene function during poplar catkin development and are promising targets for genetic containment of exotic or genetically engineered trees.

A Global View of Transcriptome Dynamics During Male Floral Bud Development in Populus tomentosa.

To obtain a comprehensive overview of the dynamic transcriptome during male floral bud development in Populus tomentosa, high-throughput RNA-seq was conducted during eight flowering-related stages. Among the 109,212 de novo assembled unigenes, 6,959 were differentially expressed during the eight stages. The overrepresented classed of genes identified by Gene Ontology (GO) enrichment included 'response to environmental stimuli' and 'plant-type spore development'. One-third of the differentially expressed genes were transcription factors (TFs). Several genes and gene families were analyzed in depth, including MADS-box TFs, Squamosa promoter binding protein-like family, receptor-like kinases, FLOWERING LOCUS T/TERMINAL-FLOWER-LIKE 1 family, key genes involved in anther and tapetum development, as well as LEAFY, WUSCHEL and CONSTANS. The results provided new insights into the roles of these and other well known gene families during the annual flowering cycle. To explore the mechanisms regulating poplar flowering, a weighted gene co-expression network was constructed using 98 floral-related genes involved in flower meristem identity and flower development. Many modules of co-expressed genes and hub genes were identified, such as APETALA1 and HUA1. This work provides many new insights on the annual flowering cycle in a perennial plant, and a major new resource for plant biology and biotechnology.

Dynamic transcriptomic analysis of the early response of female flowers of Populus alba × P. glandulosa to pollination.

Pollination is an important event in plant sexual reproduction, and post-pollination response is an essential process for reproduction. Populus alba × P. glandulosa is used widely in scientific research, especially in cross breeding as parents. Adult female P. alba × P. glandulosa flowers are highly compatible with pollen from male P. tomentosa, but the early post-pollination response of flowers at the molecular levels is unclear. In this study, RNA-seq was employed to comprehensively understand the response of female P. alba × P. glandulosa flowers to pollination. Enrichment analysis reveals that the 'plant hormone signal transduction' pathway is enhanced during pollen-pistil interaction. Moreover, genes related to auxin, gibberellin and ethylene biosynthesis were significantly up-regulated. Ca2+ and H+-related genes and cell wall-related genes are interrelated, and all of them are essential for pollen tube elongation in pistil, especially, free Ca2+ providing a concentration gradient for pollen tube guidance and involved in signal transduction. Furthermore, RNA-seq results indicate that genes involved in the adhesion and guidance for pollen germination and pollen tube growth are abundantly present in the extracellular matrix. Our study provides an overview and detailed information for understanding the molecular mechanism of early post-pollination response in this hybrid poplar reproduction.

Dynamic changes in the transcriptome of Populus hopeiensis in response to abscisic acid.

Abscisic acid (ABA) plays a fundamental role in plant response and adaptation to abiotic stresses, such as drought, high salinity and low temperature. Populus hopeiensis exhibits exceptional tolerance to water-deficit environments and is therefore an excellent choice for studying drought tolerance in trees. This study provides a global view of transcriptome dynamics in P. hopeiensis in response to exogenous ABA using Illumina RNA-sequencing. Endogenous ABA content increased and reached a peak at 8 h after ABA treatment and then significantly decreased at latter time points. Differential expression analysis and Gene ontology enrichment revealed that the number of transcripts exhibited significant increase during the first 8 hours after ABA treatment, which then significantly decreased at 12 and 24 h. Transcription factors (TFs) analysis showed that six different patterns were observed based on the expression of the six TFs families (AP2/ERF, NAC, MYB, MYB-related, bZIP and WRKY) and the majority of differentially expressed TFs increased rapidly after ABA treatment. This study provides a robust resource for investigating the functions of genes induced by ABA and will help to develop a better understanding of the molecular regulatory mechanism in response to drought in poplar.

Genome-Wide Identification of the Invertase Gene Family in Populus.

Invertase plays a crucial role in carbohydrate partitioning and plant development as it catalyses the irreversible hydrolysis of sucrose into glucose and fructose. The invertase family in plants is composed of two sub-families: acid invertases, which are targeted to the cell wall and vacuole; and neutral/alkaline invertases, which function in the cytosol. In this study, 5 cell wall invertase genes (PtCWINV1-5), 3 vacuolar invertase genes (PtVINV1-3) and 16 neutral/alkaline invertase genes (PtNINV1-16) were identified in the Populus genome and found to be distributed on 14 chromosomes. A comprehensive analysis of poplar invertase genes was performed, including structures, chromosome location, phylogeny, evolutionary pattern and expression profiles. Phylogenetic analysis indicated that the two sub-families were both divided into two clades. Segmental duplication is contributed to neutral/alkaline sub-family expansion. Furthermore, the Populus invertase genes displayed differential expression in roots, stems, leaves, leaf buds and in response to salt/cold stress and pathogen infection. In addition, the analysis of enzyme activity and sugar content revealed that invertase genes play key roles in the sucrose metabolism of various tissues and organs in poplar. This work lays the foundation for future functional analysis of the invertase genes in Populus and other woody perennials.