Populus species, particularly P. trichocarpa, have long served as model trees for genomics research, owing to fully sequenced genomes. However, the high heterozygosity, and the presence of repetitive regions, including centromeres and ribosomal RNA gene clusters, have left 59 unresolved gaps, accounting for approximately 3.32% of the P. trichocarpa genome. In this study, the callus induction method was improved to derive a doubled haploid (DH) callus line from P. ussuriensis anthers. Leveraging long-read sequencing, we successfully assembled a nearly gap-free, telomere-to-telomere (T2T) P. ussuriensis genome spanning 412.13 Mb. This genome assembly contains only seven gaps and has a contig N50 length of 19.50 Mb. Annotation revealed 34,953 protein-coding genes in this genome, which is 465 more than that of P. trichocarpa. Notably, centromeric regions are characterized by higher-order repeats, we identified and annotated centromere regions in all DH genome chromosomes, a first for poplars. The derived DH genome exhibits high collinearity with P. trichocarpa and significantly fills gaps present in the latter's genome. This T2T P. ussuriensis reference genome will not only enhance our understanding of genome structure, and functions within the poplar genus but also provides valuable resources for poplar genomic and evolutionary studies.

As a plant-specific transcription factor, the SPL gene family plays a critical role in plant growth and development. Although the SPL gene family has been identified in diverse plant species, there have been no genome-wide identification or systematic study reports on the SPL gene family in Catalpa bungei. In this study, we identified 19 putative SPL gene family members in the C. bungei genome. According to the phylogenetic relationship, they can be divided into eight groups, and the genes in the same group have a similar gene structure and conserved motifs. Synteny analysis showed that fragment duplication played an important role in the expansion of the CbuSPL gene family. At the same time, CbuSPL genes have cis-acting elements and functions related to light response, hormone response, growth and development, and stress response. Tissue-specific expression and developmental period-specific expression analysis showed that CbuSPL may be involved in flowering initiation and development, flowering transition, and leaf development. In addition, the ectopic expression of CbuSPL4 in Arabidopsis confirmed that it can promote early flowering and induce the expression of related flowering genes. These systematic research results will lay a foundation for further study on the functional analysis of SPL genes in C. bungei.

The molecular karyotype could represent the basic genetic make-up in a cell nucleus of an organism or species. A doubled haploid (DH) is a genotype formed from the chromosome doubling of haploid cells. In the present study, molecular karyotype analysis of the poplar hybrid Populus simonii × P. nigra (P. xiaohei) and the derived doubled haploids was carried out with labeled telomeres, rDNA, and two newly repetitive sequences as probes by fluorescence in situ hybridization (FISH). The tandem repeats, pPC349_XHY and pPD284_XHY, with high-sequence homology were used, and the results showed that they presented the colocalized distribution signal in chromosomes. For P. xiaohei, pPD284_XHY produced hybridizations in chromosomes 1, 5, 8, and 9 in the hybrid. The combination of pPD284_XHY, 45S rDNA, and 5S rDNA distinctly distinguished six pairs of chromosomes, and the three pairs of chromosomes showed a significant difference in the hybridization between homologous chromosomes. The repeat probes used produced similar FISH hybridizations in the DH; nevertheless, pPD284_XHY generated an additional hybridization site in the telomere region of chromosome 14. Moreover, two pairs of chromosomes showed differential hybridization distributions between homologous chromosomes. Comparisons of the distinguished chromosomes between hybrid and DH poplar showed that three pairs of chromosomes in the DH presented hybridization patterns that varied from those of the hybrid. The No. 8 chromosome in DH and one of the homologous chromosomes in P. xiaohei shared highly similar FISH patterns, which suggested the possibility of intact or mostly partial transfer of the chromosome between the hybrid and DH. Our study will contribute to understanding the genetic mechanism of chromosomal variation in P. xiaohei and derived DH plants.

Ethylene (ET) is an important phytohormone that regulates plant growth, development and stress responses. The ethylene-insensitive3/ethylene-insensitive3-like (EIN3/EIL) transcription factor family, as a key regulator of the ET signal transduction pathway, plays an important role in regulating the expression of ET-responsive genes. Although studies of EIN3/EIL family members have been completed in many species, their role in doubled haploid (DH) poplar derived from another culture of diploid Populus simonii × P. nigra (donor tree, DT) remains ambiguous. In this study, a total of seven EIN3/EIL gene family members in the DH poplar genome were identified. Basic physical and chemical property analyses of these genes were performed, and these proteins were predicted to be localized to the nucleus. According to the phylogenetic relationship, EIN3/EIL genes were divided into two groups, and the genes in the same group had a similar gene structure and conserved motifs. The expression patterns of EIN3/EIL genes in the apical buds of different DH poplar plants were analyzed based on transcriptome data. At the same time, the expression patterns of PsnEIL1, PsnEIN3, PsnEIL4 and PsnEIL5 genes in different tissues of different DH plants were detected via RT-qPCR, including the apical buds, young leaves, functional leaves, xylem, cambium and roots. The findings presented above indicate notable variations in the expression levels of PsnEIL genes across various tissues of distinct DH plants. Finally, the PsnEIL1 gene was overexpressed in DT, and the transgenic plants showed a dwarf phenotype, indicating that the PsnEIL1 gene was involved in regulating the growth and development of poplar. In this study, the EIN3/EIL gene family of DH poplar was analyzed and functionally characterized, which provides a theoretical basis for the future exploration of the EIN3/EIL gene function.

In this study, the poplar doubled haploid (DH) plants were used as the experimental material to explore the huge phenotypic differences between homozygous DH plants and the paternal plants, and the molecular regulation mechanism of the differential phenotypes. In this experiment, through morphological and histological observation and statistics, we found that the double haploid plants had significantly reduced plant height and ground diameter, increased leaf aspect ratio, premature senescence phenotype of top bud, and significant changes in the shape and cell area of the shoot apical meristem. Significantly differentially expressed genes were obtained using RNA-seq transcriptome sequencing. They were subjected to GO enrichment and KEGG analysis. Transcription factors with key functions were screened out for qRT-PCR to verify gene expression changes to predict gene function. The results showed that after the IAA and ABA treatment, the expression levels of some hormone-responsive genes in wild type plants were significantly changed with different treatment time. In the dihaploid plants, the corresponding genes also changed to different degrees, which reflected the changes in the response of the dihaploid plants to hormones. Compared to in WT, the differential expressed genes in the double haploids were involved in multiple physiological process such as response to oxidative stress, response to salicylic acid, plant pathogen interaction, and plant hormone signal transduction. A TF–centered gene regulatory network for phytohormone synthesis and plant senescence was constructed with the expression patterns of differentially expressed transcription factors (TFs). This study increases researchers’ understanding of the regulation of poplar growth and development and provides new research ideas for the creation of new species of poplar.

Plant suspension homogeneous cells are invaluable materials for investigating molecular mechanisms underlying various biological processes. In this study, we established and characterized a doubled haploid cell line from Populus simonii × P. nigra, designated as Qu-1. This cell line exhibited high viability and dispersibility under suspension culture conditions and retained the ability to regenerate into whole plants. K-mer analysis confirmed the homozygous genome of Qu-1, and a chromosome-level genome assembly was subsequently achieved by using PacBio sequencing. Additionally, we established an efficient transient transformation protocol using PEG-mediated protoplasts and a stable Agrobacterium-mediated transformation system for Qu-1. To explore the mutagenesis potential of this cell line, we treated the cell line with ethyl methanesulfonate (EMS) and performed genome resequencing to identify mutation sites. Overall, Qu-1 represents the first poplar cell line with a homozygous genome and is as a powerful tool for molecular biology research in woody plants.