Populus euphratica Oliv. is a key desert tree, blocking sand, stabilizing dunes, ameliorating microclimates, and providing wildlife habitats, vital for ecological stability. In addition, its outstanding drought, cold, and salinity tolerance, and features like heterophylly, make it valuable for studying plant adaptation and growth. However, incompatibility of hybridization, poor rooting, and limited genetic research platforms hinder conservation and utilisation. Consequently, the development of robust biotechnology platforms is urgently needed in P. euphratica. Complete genomic information facilitates the elucidation of gene function and the efficient application of genome editing, but the available genome assemblies of P. euphratica remain discontinuous and incomplete due to gaps and missing telomeres (Zhang et al. 2022, 2020). Here, we assembled the telomere-to-telomere (T2T) haplotype-resolved genome of P. euphratica ‘ZH1’ (Figure 1A, Table S1, and Data S1). The gapless T2T genome, composed of 38 chromosomes and 76 telomeres, encompassed two haploids of 506.85 and 501.23 Mb (Figures S1 and S2, Table S2). The contig N50 values of two haploids were 24.99 and 24.72 Mb, respectively, and the BUSCO assessment showed 98.9% completeness, significantly improving the continuity and completeness (Table S3). The mapping rates for HiFi, ONT, and NGS reads were 99.92%, 99.35%, and 98.94%, respectively. The genome continuity inspector (GCI) assessments for two haploids were 93.07 and 96.26, respectively. The consensus quality value (QV) assessments were 72.33 and 73.41, respectively, higher than other T2T assemblies of Populus species (Table S4). The repeat element was 58.37% and 58% of the genome assembly (Table S5). The rDNAs were especially enriched on Chr9 (Tables S6 and S7). A total of 38 257 and 38 176 protein-coding genes were identified, 98.56% of which were confirmed entirely using 113 RNA-seq samples, and 97.18% were annotated by public databases (Tables S8 and S9). Comparative genomic analysis showed that the T2T assembly filled gaps in the previous genome and annotated novel genes (Figure S3). These results suggest the availability of a complete genome assembly of P. euphratica. Despite the well-established transformation systems for Populus, their application is currently limited to only a few species and cultivars. Notably, the regeneration and transformation systems for P. euphratica have remained elusive, significantly hindering functional characterisation of genes and the genetic application. Here, we developed indirect and direct strategies for regenerating P. euphratica. In an indirect way, sterilised leaves and petioles were transferred into callus-induction media to initiate callus formation. The healthy callus was transferred to shoot-induction media to initiate adventitious buds and elongated shoots. The elongated shoots were transferred to root-induction media to form complete plants. In a direct manner, sterilised explants were to initiate buds directly (Figure 1B). Both indirect and direct processes exhibited high regeneration efficiency and could be used for transgenic applications of P. euphratica (Table S10 and Data S2). The variation of leaf shape constitutes one of the distinctive characteristics of P. euphratica in response to environmental change. MiR319 plays a significant role in regulating leaf morphology (Cheng et al. 2021). We cloned the PemiR319 gene from P. euphratica, constructed an overexpressing vector pXHKFG-PemiR319, and transformed it into P. euphratica (Data S3 and File S1). The green fluorescence signal was observed across the transgenic P. euphratica (Figure 1C). The transgenic P. euphratica exhibited serrated leaves (Figure 1D), a phenotype consistent with PagmiR319-overexpressing 84 K poplar (Figure S4). The RUBY reporter has been widely used as a marker in plant transgenics. The RUBY reaction necessitates sequential catalysis by three enzymes. Thus, we constructed a multiple-gene coexpression system pXHKFG-RUBY (Data S3). Transgenic callus and shoots with RUBY exhibited a distinct red colour, markedly differentiated from its negative counterparts (Figure S5), and the rooting P. euphratica plants also exhibited red (Figure 1E). Efficient gene editing requires precise selection of editing targets, which necessitates reliance on high-quality genome information of the recipient. Based on the CRISPR/Cas9 principle, we employed CRISPR-Local (Sun et al. 2019) for genome-wide target selection by integrating the T2T genome of P. euphratica, thereby enhancing the specificity of target sites across the genome and reducing off-target events. We selected two target sites of the phytoene desaturase (PDS) gene in P. euphratica (PePDS) and constructed a two-site editing system pXHGCK-PePDS (Data S3 and File S2). The positive PePDS-edited pink shoots were successfully achieved (Figure 1F). The sequencing results showed that different editing events had occurred at both target sites of the PePDS gene (Figure 1G), whereas the potential top-off-target sites had no edits (Figure S5). The editing efficiency of the direct regeneration way was lower than that of the indirect way. In the direct way, there were fewer homozygous mutations, and most were chimeric or unmodified. In the indirect way, biallelic and homozygous edits were predominant (Table S11). To our knowledge, this was the first report of genome editing in P. euphratica. In summary, this work first provides a high-quality T2T gap-free genome assembly of P. euphratica (ZH1-T2T), serving as a critical foundation for gene exploration and enabling efficient target selection for genome editing. Secondly, we have developed an efficient regeneration system for P. euphratica, providing an essential method for propagating and proliferating superior clonal lines. Thirdly, we have established an efficient transformation system for P. euphratica, offering significant support for gene characterisation and genetic improvement. We have also successfully conducted genome editing in P. euphratica. These studies collectively provide robust technical support for the conservation and utilisation of P. euphratica. X.H. supervised the project. X.H. and Y.A. wrote the paper and performed the regeneration. R.Y. and S.Y. performed the transgenics. R.Y. and X.G. cultivated the samples. X.H. and Y.L. performed genome assembly. Y.D. and Y.Z. collected samples. We thank Prof. Weilun Yin and Xinli Xia (Beijing Forestry University, China), and Prof. Mengzhu Lu and Zaikang Tong (Zhejiang A&F University, China) for the technical assistance. This work was supported by the Science and Technology Innovation 2030-Major Project (2023ZD0405702-05) and the National Natural Science Foundation of China (32371902). The authors declare no conflicts of interest. All raw sequencing data and the genome assembly have been deposited in the CNSA under accession number CNP0008260. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
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