EuHDZ25 positively affects rubber biosynthesis by targeting EuFPS1 in Eucommia leaves.

Eucommia ulmoides is a temperate gum source plant that produces trans-polyisoprene (TPI), also known as Eucommia rubber. The structural configuration and function of TPI offer a new material with important potential for industrial development. In this study, we detected the TPI content in the leaves of diploid and triploid E. ulmoides plants. The average TPI content in the leaves of triploid E. ulmoides was significantly higher than that of diploid. Transcriptome data and weighted gene co-expression network analyses identified a significant positive correlation between the EuFPS1 gene and TPI content. Overexpression of EuFPS1 increased the density of rubber particles and TPI content, indicating its crucial role in TPI biosynthesis. In addition, the expression of EuHDZ25 in E. ulmoides was significantly positively correlated with EuFPS1 expression. Yeast one-hybrid and dual-luciferase assays demonstrated that EuHDZ25 mainly promotes TPI biosynthesis through positive regulation of EuFPS1 expression. The significantly up-regulated expression of EuHDZ25 and its consequent upregulation of EuFPS1 during the biosynthesis of TPI may partially explain the increased TPI content of triploids. This study provides an important theoretical foundation for further exploring the molecular mechanism of secondary metabolites content variation in polyploids and can help to promote the development and utilization of rubber resources.

MYC2 regulates stomatal density and water use efficiency via targeting EPF2/EPFL4/EPFL9 in poplar.

Although polyploid plants have lower stomatal density than their diploid counterparts, the molecular mechanisms underlying this difference remain elusive. Here, we constructed a network based on the triploid poplar transcriptome data and triple-gene mutual interaction algorithm and found that PpnMYC2 was related to stomatal development-related genes PpnEPF2, PpnEPFL4, and PpnEPFL9. The interactions between PpnMYC2 and PagJAZs were experimentally validated. PpnMYC2-overexpressing poplar and Arabidopsis thaliana had reduced stomatal density. Poplar overexpressing PpnMYC2 had higher water use efficiency and drought resistance. RNA-sequencing data of poplars overexpressing PpnMYC2 showed that PpnMYC2 promotes the expression of stomatal density inhibitors PagEPF2 and PagEPFL4 and inhibits the expression of the stomatal density-positive regulator PagEPFL9. Yeast one-hybrid system, electrophoretic mobility shift assay, ChIP-qPCR, and dual-luciferase assay were employed to substantiate that PpnMYC2 directly regulated PagEPF2, PagEPFL4, and PagEPFL9. PpnMYC2, PpnEPF2, and PpnEPFL4 were significantly upregulated, whereas PpnEPFL9 was downregulated during stomatal formation in triploid poplar. Our results are of great significance for revealing the regulation mechanism of plant stomatal occurrence and polyploid stomatal density, as well as reducing stomatal density and improving plant water use efficiency by overexpressing MYC2.

Integrated transcriptomic and metabolomic analysis reveals the effects of polyploidization on the lignin content and metabolic pathway in Eucalyptus.

BACKGROUND: Lignin is a major restriction factor for the industrial production of biomass resources, such as pulp and bioenergy. Eucalyptus is one of the most important sources of pulp and bioenergy. After polyploidization, the lignin content of forest trees is generally reduced, which is considered a beneficial genetic improvement. However, the differences in the lignin content between triploid and diploid Eucalyptus and the underlying regulatory mechanism are still unclear. RESULTS: We conducted a comprehensive analysis at the phenotypic, transcriptional and metabolite levels between Eucalyptus urophylla triploids and diploids to reveal the effects of polyploidization on the lignin content and lignin metabolic pathway. The results showed that the lignin content of Eucalyptus urophylla triploid stems was significantly lower than that of diploids. Lignin-related metabolites were differentially accumulated between triploids and diploids, among which coniferaldehyde, p-coumaryl alcohol, sinapaldehyde and coniferyl alcohol had significant positive correlations with lignin content, indicating that they might be primarily contributing metabolites. Most lignin biosynthetic genes were significantly downregulated, among which 11 genes were significantly positively correlated with the lignin content and above metabolites. Furthermore, we constructed a co-expression network between lignin biosynthetic genes and transcription factors based on weighted gene co-expression network analysis. The network identified some putative orthologues of secondary cell wall (SCW)-related transcription factors, among which MYB52, MYB42, NAC076, and LBD15 were significantly downregulated in Eucalyptus urophylla triploids. In addition, potential important transcription factors, including HSL1, BEE3, HHO3, and NAC046, also had high degrees of connectivity and high edge weights with lignin biosynthetic genes, indicating that they might also be involved in the variation of lignin accumulation between triploid and diploid Eucalyptus urophylla. CONCLUSIONS: The results demonstrated that some lignin-related metabolites, lignin biosynthetic genes and transcription factors in Eucalyptus urophylla triploids may be relatively sensitive in response to the polyploidization effect, significantly changing their expression levels, which ultimately correlated with the varied lignin content. The analysis of the underlying formation mechanism could provide beneficial information for the development and utilization of polyploid biomass resources, which will be also valuable for genetic improvement in other bioenergy plants.

Effect of a suitable treatment period on the genetic transformation efficiency of the plant leaf disc method.

BACKGROUND: Agrobacterium tumefaciens-mediated leaf disc genetic transformation is an important way to achieve transgenics or gene editing. Ensuring stable and efficient genetic transformation is still an important problem in modern biology. It is assumed that the difference in the development status of genetic transformation cells of receptor materials is the main reason for the difference and instability of genetic transformation efficiency; the stable and efficient genetic transformation rate can be obtained by defining the appropriate treatment period of the receptor material and applying genetic transformation in a timely manner. RESULTS: Based on these assumptions, we studied and established an efficient and stable Agrobacterium-mediated plant transformation system with hybrid poplar (Populus alba × Populus glandulosa, 84 K) leaves, stem segments and tobacco leaves as the research objects. There were differences in the development process of leaf bud primordial cells from different explants, and the genetic transformation efficiency was significantly related to the cell development stage of the in vitro cultured materials. Among them, the genetic transformation rate of poplar and tobacco leaves was the highest on the 3rd and 2nd day of culture, reaching 86.6% and 57.3%, respectively. The genetic transformation rate of poplar stem segments was the highest on the 4th day of culture, reaching 77.8%. The best treatment period was from the development of leaf bud primordial cells to the S phase of the cell cycle. The number of cells detected using flow cytometry and 5-ethynyl-2'-deoxyuridine (EdU) staining, the expression of cell cycle-related protein CDKB1; 2, CDKD1; 1, CYCA3; 4, CYCD1; 1, CYCD3; 2, CYCD6; 1, and CYCH; 1 of explants, and morphological changes of explants can be used as indicators to determine the appropriate treatment period for genetic transformation. CONCLUSIONS: Our study provides a new and universal set of methods and characteristics to identify the S phase of the cell cycle and apply genetic transformation treatments at the appropriate time. Our results are of great significance for improving the efficiency and stability of plant leaf disc genetic transformation.

Morphological, Transcriptome, and Hormone Analysis of Dwarfism in Tetraploids of Populus alba × P. glandulosa.

Breeding for dwarfism is an important approach to improve lodging resistance. Here, we performed comparative analysis of the phenotype, transcriptome, and hormone contents between diploids and tetraploids of poplar 84K (Populus alba × P. glandulosa). Compared with diploids, the indole-3-acetic acid (IAA) and gibberellin (GA3) contents were increased, whereas the jasmonic acid (JA) and abscisic acid (ABA) contents were decreased in tetraploids. RNA-sequencing revealed that differentially expressed genes (DEGs) in leaves of tetraploids were mainly involved in plant hormone pathways. Most DEGs associated with IAA and GA promotion of plant growth and development were downregulated, whereas most DEGs associated with ABA and JA promotion of plant senescence were upregulated. Weighted gene co-expression network analysis indicated that certain transcription factors may be involved in the regulation of genes involved in plant hormone pathways. Thus, the altered expression of some genes in the plant hormone pathways may lead to a reduction in IAA and GA contents, as well as an elevation in ABA and JA contents, resulting in the dwarfing of tetraploids. The results show that polyploidization is a complex biological process affected by multiple plant hormone signals, and it provides a foundation for further exploration of the mechanism of tetraploids dwarfing in forest trees.

Genome-wide characterization and analysis of Golden 2-Like transcription factors related to leaf chlorophyll synthesis in diploid and triploid Eucalyptus urophylla.

Golden 2-Like (GLK) transcription factors play a crucial role in chloroplast development and chlorophyll synthesis in many plant taxa. To date, no systematic analysis of GLK transcription factors in tree species has been conducted. In this study, 40 EgrGLK genes in the Eucalyptus grandis genome were identified and divided into seven groups based on the gene structure and motif composition. The EgrGLK genes were mapped to 11 chromosomes and the distribution of genes on chromosome was uneven. Phylogenetic analysis of GLK proteins between E. grandis and other species provided information for the high evolutionary conservation of GLK genes among different species. Prediction of cis-regulatory elements indicated that the EgrGLK genes were involved in development, light response, and hormone response. Based on the finding that the content of chlorophyll in mature leaves was the highest, and leaf chlorophyll content of triploid Eucalyptus urophylla was higher than that of the diploid control, EgrGLK expression pattern in leaves of triploid and diploid E. urophylla was examined by means of transcriptome analysis. Differential expression of EgrGLK genes in leaves of E. urophylla of different ploidies was consistent with the trend in chlorophyll content. To further explore the relationship between EgrGLK expression and chlorophyll synthesis, co-expression networks were generated, which indicated that EgrGLK genes may have a positive regulatory relationship with chlorophyll synthesis. In addition, three EgrGLK genes that may play an important role in chlorophyll synthesis were identified in the co-expression networks. And the prediction of miRNAs targeting EgrGLK genes showed that miRNAs might play an important role in the regulation of EgrGLK gene expression. This research provides valuable information for further functional characterization of GLK genes in Eucalyptus.

Induction and Characterization of Tetraploid Through Zygotic Chromosome Doubling in Eucalyptus urophylla.

Improvements in plant growth can bring great benefits to the forest industry. Eucalyptus urophylla is an important plantation species worldwide, and given that ploidy increases are often associated with plant phenotype changes, it was reasoned that its polyploidization may have good prospects and great significance toward its cultivation. In this study, the zygotic development period of E. urophylla was observed through paraffin sections, and a correlation between the development time of flower buds after pollination and the zygotic development period was established. On this basis, it was determined that the 25th day after pollination was the appropriate time for a high temperature to induce zygotic chromosome doubling. Then tetraploid E. urophylla was successfully obtained for the first time through zygotic chromosome doubling induced by high temperature, and the appropriate conditions were treating flower branches at 44°C for 6 h. The characterization of tetraploid E. urophylla was performed. Chromosome duplication brought about slower growing trees with thicker leaves, larger cells, higher net photosynthetic rates, and a higher content of certain secondary metabolites. Additionally, the molecular mechanisms for the variation in the tetraploid's characteristics were studied. The qRT-PCR results showed that genes mediating the tetraploid characteristics showed the same change trend as those of the characteristics, which verified that tetraploid trait variation was mainly caused by gene expression changes. Furthermore, although the tetraploid had no growth advantage compared with the diploid, it can provide important germplasm resources for future breeding, especially for the creation of triploids.

Insights into the Molecular Regulation of Lignin Content in Triploid Poplar Leaves.

After polyploidization, plants usually undergo some morphological and physiological changes, including the lignin content of polyploids usually becoming lower than that of diploids. However, the regulatory mechanism of the variation of lignin content in polyploid plants remains unclear. Therefore, in this research, we used full-sib poplar triploids and diploids to explore the molecular regulatory basis of lignin content in poplar triploid leaves through the determination of lignin content, the observation of xylem cells, and transcriptome sequencing. The results showed that the lignin content of triploid leaves was significantly lower than that of diploid leaves. The xylem cells of triploid leaves were significantly larger than those of diploids. Transcriptome sequencing data show that most lignin biosynthesis genes were significantly downregulated, and genes related to cell growth were mostly upregulated in triploid leaves compared with diploid leaves. In addition, co-expression network analysis showed that several transcription factors might be involved in the regulation of lignin biosynthesis. Consequently, the altered expression of genes related to lignin might lead to the reduced lignin content in triploids. These results provide a theoretical basis for further exploring the molecular mechanism of the variation of polyploid lignin content and the utilization of polyploid lignocellulosic resources.

Integrated transcriptome and miRNA sequencing approaches provide insights into salt tolerance in allotriploid Populus cathayana.

MAIN CONCLUSION: Some salt-stress responsive DEGs, mainly involved in ion transmembrane transport, hormone regulation, antioxidant system, osmotic regulation, and some miRNA jointly regulated the salt response process in allotriploid Populus cathayana. The molecular mechanism of plant polyploid stress resistance has been a hot topic in biological research. In this study, Populus diploids and first division restitution (FDR) and second division restitution (SDR) triploids were selected as research materials. All materials were treated with 70 mM NaCl solutions for 30 days in the same pot environment. We observed the growth state of triploids and diploids and determined the ratio of potassium and sodium ions, peroxidase (POD) activity, proline content, and ABA and jasmonic acid (JA) hormone content in leaves in the same culture environment with the same concentration of NaCl solution treatment. In addition, RNA-seq technology was used to study the differential expression of mRNA and miRNA. The results showed that triploid Populus grew well and the K+ content and the K+/Na+ ratio in the salt treatment were significantly lower than those in the control. The contents of ABA, JA, POD, and proline were increased compared with contents in diploid under salt stress. The salt-stress responsive DEGs were mainly involved in ion transport, cell homeostasis, the MAPK signaling pathway, peroxisome, citric acid cycle, and other salt response and growth pathways. The transcription factors mainly included NAC, MYB, MYB_related and AP2/ERF. Moreover, the differentially expressed miRNAs involved 32 families, including 743 miRNAs related to predicted target genes, among which 22 miRNAs were significantly correlated with salt-stress response genes and related to the regulation of hormones, ion transport, reactive oxygen species (ROS) and other biological processes. Our results provided insights into the physiological and molecular aspects for further research into the response mechanisms of allotriploid Populus cathayana to salt stress. This study provided valuable information for the salt tolerance mechanism of allopolyploids.

Growth-regulating factor 5 (GRF5)-mediated gene regulatory network promotes leaf growth and expansion in poplar.

Although polyploid plants have larger leaves than their diploid counterparts, the molecular mechanisms underlying this difference (or trait) remain elusive. Differentially expressed genes (DEGs) between triploid and full-sib diploid poplar trees were identified from two transcriptomic data sets followed by a gene association study among DEGs to identify key leaf growth regulators. Yeast one-hybrid system, electrophoretic mobility shift assay, and dual-luciferase assay were employed to substantiate that PpnGRF5-1 directly regulated PpnCKX1. The interactions between PpnGRF5-1 and growth-regulating factor (GRF)-interacting factors (GIFs) were experimentally validated and a multilayered hierarchical regulatory network (ML-hGRN)-mediated by PpnGRF5-1 was constructed with top-down graphic Gaussian model (GGM) algorithm by combining RNA-sequencing data from its overexpression lines and DAP-sequencing data. PpnGRF5-1 is a negative regulator of PpnCKX1. Overexpression of PpnGRF5-1 in diploid transgenic lines resulted in larger leaves resembling those of triploids, and significantly increased zeatin and isopentenyladenine in the apical buds and third leaves. PpnGRF5-1 also interacted with GIFs to increase its regulatory diversity and capacity. An ML-hGRN-mediated by PpnGRF5-1 was obtained and could largely elucidate larger leaves. PpnGRF5-1 and the ML-hGRN-mediated by PpnGRF5-1 were underlying the leaf growth and development.

Genetic analysis of admixture and hybrid patterns of Populus hopeiensis and P. tomentosa.

Hybridization and introgression have resulted in reticulate evolution within the genus Populus. Consequently, the origin and evolutionary history of some hybrids has become blurred. P. hopeiensis and P. tomentosa are endemic to China, and there is still controversy about their origin. We employ phylogeny, Bayesian estimation of admixture, and approximate Bayesian computation to investigate their origin with 10 nuclear DNA and 6 cpDNA regions. The combined evidences firmly support the hypothesis that they are hybrids and dominated by F1s. P. hopeiensis was generated via hybridization between the paternal species P. alba and maternal species P. davidiana. Surprisingly, P. tomentosa was divided into two genetic types with different maternal parents. P. adenopoda hybridized with P. alba directly to generate the first genetic type (mb1) and hybridized with P. davidiana followed by P. alba to generate the second (mb2). In both genetic types, P. alba acted as the male parent. The maternal parent was P. adenopoda and P. davidiana for mb1 and mb2, respectively. Hybridization not only generated these hybrids but also resulted in a unidirectional gene flow from P. davidiana to P. adenopoda. The Populus species have maintained a delicate balance between their genetic integrity and gene exchange.

Analysis of genetic composition and transmitted parental heterozygosity of natural 2n gametes in Populus tomentosa based on SSR markers.

MAIN CONCLUSION: Natural 2n female gametes and transmission of parental heterozygosity by natural 2n gametes in Populus tomentosa are reported for the first time, which provides a new approach to polyploid breeding. Naturally occurring 2n pollen is widespread in Populus tomentosa and plays an important role in polyploid breeding. However, the competitiveness of 2n pollen is lower than that of haploid pollen during pollination and fertilization, so 2n pollen is less efficient at fertilizing haploid female gametes to produce polyploids. In theory, polyploids can also be obtained when 2n female gametes are fertilized by haploid pollen. Thus, the question becomes whether natural 2n female gametes exist in P. tomentosa, which can be answered by examining the genetic composition of natural 2n gametes. In this study, the origin of 87 triploids from the hybrid combination "X-2 × Z-5" was identified by SSR markers and 21% of natural 2n gametes were found to originate from female parents. Four SSR loci with low recombination rates were used to identify the genetic composition of natural 2n gametes. The results showed that the genetic composition of 2n female gametes was mainly characterized by SDR, while 2n male gametes were mainly produced by FDR. Moreover, the transmission of parental heterozygosity by natural 2n gametes, which is significantly different between female and male parents in FDR and SDR types, was analysed using 42 SSR primers. Here, we report naturally occurring 2n female gametes for the first time in P. tomentosa and reveal the genetic constitution and transmitted parental heterozygosity of these gametes. Our results provide a foundation for theoretical research into 2n gametes and their application in new polyploid breeding strategies.

Proteomic Changes Between Populus Allotriploids and Diploids Revealed Using an iTRAQ-based Quantitative Approach.

BACKGROUND: Polyploid breeding is a powerful approach for Populus genetic improve-ment because polyploid trees have valuable characteristics, including better timber quality and a higher degree of stress resistance compared with their full-sib diploids. However, the genetic mech-anism underlying this phenomenon remains unknown. OBJECTIVE: To better understand the proteomic changes between Populus allotriploids and diploids, we examined the proteomic profiles of allotriploid and diploid Populus by iTRAQ labeling coupled with two-dimensional liquid chromatography and MALDI-TOF/TOF mass spectrometry (MS). METHOD: iTRAQ labeling coupled with two-dimensional liquid chromatography and MALDI-TOF/TOF mass spectrometry (MS). RESULTS: Between the Populus allotriploid and the full-sib diploid, 932 differentially expressed proteins (DEPs) were identified. These DEPs were primarily involved in stress, defense, transportation, transcriptional and/or translational modification, and energy production. The pathway analysis indi-cated that most of the DEPs were implicated in carbohydrate transport and metabolism, nitrogen me-tabolism and glycolysis, and the ribosome assembly pathway. These data suggest high protein di-vergence between Populus allotriploids and diploids, and rapid changes during hybridization. CONCLUSION: The results provide new data for further understanding of the mechanisms of polyploid trees that generally display increased height growth compared with their full-sib diploids.

MicroRNA expression changes following synthesis of three full-sib Populus triploid populations with different heterozygosities.

KEY MESSAGE: Through high-throughput sequencing, we compared the relative expression levels of miRNA in three full-sib Populus triploid populations with that in their parents and one diploid hybrid population. We found similar numbers of miRNAs differentially expressed between the parents and the four progeny hybrid populations. In addition, unbalanced parental expression level dominance of miRNAs were found in the three allotriploid and interspecific hybrid populations, which may reprogram gene expression networks and contribute to the growth of Populus hybrids. These results indicated that hybridization has a great impact on the miRNA expression variation in the newly synthesized Populus triploid and diploid hybrid populations. However, we also found no significant differences in miRNA expression among one diploid and three triploid hybrid populations, hinting that miRNA abundances do not increase with the genome content. No dosage effect of miRNA expression could lead to dosage-dependent negative effects on target genes and their downstream pathway in polyploids. We speculate that polyploids may gain advantages from the slight decrease in miRNA regulation, suggesting an important molecular mechanism of polyploid advantage. Hybridization with three types of induced 2n gametes transmitted different parental heterozygosities has been proven as an efficient method for Populus triploid production. Several researches have shown that miRNA could be non-additively expressed in allopolyploids. However, it is still unclear whether the non-additively expressed miRNAs result from the effect of hybridization or polyploidization, and whether a dose response to the additional genomic content exists for the expression of miRNA. Toward this end, through high-throughput sequencing, we compared the expression levels of miRNA in three full-sib Populus triploid populations with that in their parents and one interspecific hybrid population. We found similar numbers of miRNAs differentially expressed between the parents and the four progeny hybrid populations. Unbalanced parental expression level dominance of miRNAs were found in the three triploid and diploid hybrid populations, which may reprogram gene expression networks and affect the growth of Populus hybrids. These results indicated that hybridization has a great impact on the miRNA expression variation in the newly synthesized Populus triploid and diploid hybrid populations. However, we also found no significant differences in miRNA expression among the three triploid populations and the diploid hybrid population. No dosage effect of miRNA expression could lead to dosage-dependent negative effects on target genes and their downstream pathway in polyploids. We speculate that polyploids may gain advantages from the decrease in miRNA negative regulation, suggesting an important molecular mechanism of polyploid advantage.

High temperature-induced production of unreduced pollen and its cytological effects in Populus.

Temperature change is of potential to trigger the formation of unreduced gametes. In this study, we showed that short periods of high temperature treatment can induce the production of 2n pollen in Populus pseudo-simonii Kitag. The meiotic stage, duration of treatment, and temperature have significant effects on the induction of 2n pollen. Heat stress resulted in meiotic abnormalities, including failure of chromosome separation, chromosome stickiness, laggards and micronuclei. Spindle disorientations in the second meiotic division, such as parallel, fused, and tripolar spindles, either increased in frequency or were induced de novo by high temperature treatment. We found that the high temperature treatment induced depolymerisation of meiotic microtubular cytoskeleton, resulting in the failure of chromosome segregation. New microtubular cytoskeletons were able to repolymerise in some heat-treated cells after transferring them to normal conditions. However, aberrant cytokinesis occurred owing to defects of new radial microtubule systems, leading to production of monads, dyads, triads, and polyads. This suggested that depolymerisation and incomplete restoration of microtubules may be important for high temperature-induction of unreduced gametes. These findings might help us understand how polyploidisation is induced by temperature-related stress and support the potential effects of global climate change on reproductive development of plants.

Transcriptomic changes following synthesis of a Populus full-sib diploid and allotriploid population with different heterozygosities driven by three types of 2n female gamete.

Diploid gametes are usually applied to produce triploids of Populus [originating from first-division restitution (FDR), second-division restitution (SDR), and postmeiotic restitution (PMR) 2n eggs]. Three types of 2n gametes transmitted different parental heterozygosities in Populus. Failed spindle formation and no chromosomal separation to opposite poles during meiosis I mean that FDR 2n gametes carry nonsister chromatids that are potentially heterozygous. By contrast, SDR 2n gametes result from failed sister chromatid separation in meiosis II, and therefore, they carry sister chromatid that are potentially homozygous. Completely homozygous 2n gametes can arise from the PMR mechanism. The alteration of gene expression resulting from allopolyploidization is a prominent feature in plants. We compared gene expression in the full-sib progeny of three allotriploid Populus populations (triploid-F, triploid-S, and triploid-P) with that in its parent species, and their full-sib diploid F1 hybrid. Genome-wide expression level dominance was biased toward the maternal in the diploid F1 hybrid and three allotriploid populations, whereas our data indicated important, but different, effects of the transmission of different heterozygosity by 2n female gametes in the expression patterns of allopolyploids. Because of the higher level of heterozygosity, the triploids had higher rates of non-additive and transgressive expression patterns in the triploid-F than in triploid-S and triploid-P. Compared with diploid F1, about 30-fold more genes (251) were differently expressed in the triploid-F than in the triploid-S (9) and triploid-P (8), respectively. These findings indicate that hybridization and polyploidization have immediate and distinct effects on the large-scale patterns of gene expression, and different effects on the transmission of heterozygosity by three 2n female gametes.

Inheritance and variation of Cytosine methylation in three populus allotriploid populations with different heterozygosity.

DNA methylation is an epigenetic mechanism with the potential to regulate gene expression and affect plant phenotypes. Both hybridization and genome doubling may affect the DNA methylation status of newly formed allopolyploid plants. Previous studies demonstrated that changes in cytosine methylation levels and patterns were different among individual hybrid plant, therefore, studies investigating the characteristics of variation in cytosine methylation status must be conducted at the population level to avoid sampling error. In the present study, an F1 hybrid diploid population and three allotriploid populations with different heterozygosity [originating from first-division restitution (FDR), second-division restitution (SDR), and post-meiotic restitution (PMR) 2n eggs of the same female parent] were used to investigate cytosine methylation inheritance and variation relative to their common parents using methylation-sensitive amplification polymorphism (MSAP). The variation in cytosine methylation in individuals in each population exhibited substantial differences, confirming the necessity of population epigenetics. The total methylation levels of the diploid population were significantly higher than in the parents, but those of the three allotriploid populations were significantly lower than in the parents, indicating that both hybridization and polyploidization contributed to cytosine methylation variation. The vast majority of methylated status could be inherited from the parents, and the average percentages of non-additive variation were 6.29, 3.27, 5.49 and 5.07% in the diploid, FDR, SDR and PMR progeny populations, respectively. This study lays a foundation for further research on population epigenetics in allopolyploids.

Differential transcriptome analysis between Populus and its synthesized allotriploids driven by second-division restitution.

In this report, we compared transcriptomic differences between a synthetic Populus section Tacamahaca triploid driven by second-division restitution and its parents using a high-throughput RNA-seq method. A total of 4,080 genes were differentially expressed between the high-growth vigor allotriploids (SDR-H) and their parents, and 719 genes were non-additively expressed in SDR-H. Differences in gene expression between the allotriploid and male parent were more significant than those between the allotriploid and female parent, which may be caused by maternal effects. We observed 3,559 differentially expressed genes (DEGs) between the SDR-H and male parent. Notably, the genes were mainly involved in metabolic process, cell proliferation, DNA methylation, cell division, and meristem and developmental growth. Among the 1,056 DEGs between SDR-H and female parent, many genes were associated with metabolic process and carbon utilization. In addition, 1,789 DEGs between high- and low-growth vigor allotriploid were mainly associated with metabolic process, auxin poplar transport, and regulation of meristem growth. Our results indicated that the higher poplar ploidy level can generate extensive transcriptomic diversity compared with its parents. Overall, these results increased our understanding of the driving force for phenotypic variation and adaptation in allopolyploids driven by second-division restitution.

Induction of 2n female gametes in Populus adenopoda Maxim by high temperature exposure during female gametophyte development.

In order to produce triploid plants, 2n female gametes were induced by treating female buds and developing embryo sacs of Populus adenopoda Maxim with high temperature exposure. During megasporogenesis, tests were conducted on the relationship between female gametophyte development and morphological changes of female catkins. In the resulting progeny, 12 triploids were produced, and the highest rate of triploid production was 40%. Cytological observation revealed that the pachytene to diakinesis phase of meiotic stages may be a suitable period for inducing megaspore chromosome doubling through high temperature exposure. On the other hand, catkins of 6-72 h after pollination were treated for inducing embryo sac chromosome doubling. In the offspring seedlings, 51 triploids were detected and the highest efficiency of triploid production was 83.33%. Correlation analysis between the proportion of each embryo sac's developmental stage and the percentage of triploid production indicated that the second mitotic division may be the most effective stage for 2n female gamete induction. Our findings showed that high temperature exposure is an ideal method for 2n female gamete induction. Heterozygous offspring are valuable for breeding programs of P. adenopoda.

In vitro tetraploid induction from leaf explants of Populus pseudo-simonii Kitag.

Tetraploid plants were produced from leaf explants of diploid Populus pseudo-simonii by treating the leaves with colchicine. Leaf explants were cultured on MS basal medium containing 1.78 µM BA and 1.08 µM NAA for 0, 6 and 12 days, and then transferred to the same MS liquid medium with colchicine at concentrations of 25, 50 and 75 µM for 1, 2 and 3 days. The highest efficiency of tetraploid induction was 14.6% by treating leaf explants that were pre-cultured for 6 days and then cultured in liquid MS with 50 µM colchicine for 3 days. Flow cytometric analysis was used to screen the tetraploids out from the regenerated plants and chromosome number counting was employed to confirm the polyploidy level. Size and frequency of leaf stomata between diploid and tetraploid plants were demonstrated to have significant differences.