Heterosis is critical for agricultural productivity, yet the fixation of hybrid vigor in subsequent generations is prevented by meiotic segregation. Synthetic apomixis—the engineering of clonal seed formation—offers a transformative solution, but current systems often suffer from low efficiency or compromised fertility. Here, we report the development of a highly efficient synthetic apomixis system in hybrid rice based on the heterologous expression of PsBBML, a parthenogenesis gene derived from the apomictic wild species Pennisetum squamulatum. When combined with the MiMe genetic background, the full-length genomic sequence of PsBBML (gPsBBML) confers robust parthenogenetic activity, achieving up to 100% clonal seed production across generations while fully retaining parental heterozygosity, albeit with moderately reduced seed set. Critically, we demonstrate that the native intron-containing genomic structure of PsBBML consistently outperforms its coding sequence, and that expression driven by the rice egg cell-specific promoter OsECA1 maximizes clonal efficiency. These findings establish PsBBML as a potent trigger of parthenogenesis and illuminate previously unrecognized roles for intronic architecture and promoter selection in optimizing apomictic reproduction. This work provides a practical framework for fixing heterosis in rice and offers valuable design principles for engineering synthetic apomixis in a broader range of agricultural crops.

Although microRNA1447 (miR1447) regulates poplar responses to abiotic stress and pest infestation, how miR1447 regulates poplar immunity against pathogens and its underlying molecular mechanisms remain to be elucidated. Here, we revealed that miR1447 functioned as a negative regulator of poplar disease resistance against fungal and bacterial pathogens using overexpression (OE) and short tandem target mimic (STTM) poplar lines of miR1447. Moreover, we demonstrated that PopTCTP contributed to poplar immunity as a target of miR1447 through integrative analysis of overexpression and RNAi lines, degradomes, transient co-expression assay and GFP fluorescence report system, and found that PopTCTP interacted with dnaJ A6. Further molecular and genetic analyses revealed that the promoters of miR1447 and PopTCTP were responsive to exogenous salicylic acid (SA) treatment. We showed that the negative regulatory role of miR1447 in SA signalling and poplar resistance was weakened with exogenous SA treatment. Notably, the miR1447-PopTCTP module contributed to PTI in poplar triggered by flg22 and associated with crosstalk between PTI and ETI via regulating MAPK signalling and scavenging ROS. Taken together, these findings unveil a novel pathway by which the miR1447-PopTCTP-SA signalling mediates disease resistance to diverse pathogens in poplar, offering promising genetic targets for tree breeding of disease resistance.

Meiosis is an indispensable process in sexual reproduction, involving the recombination of genetic information and the production of haploid gamete cells through the segregation of sister chromatids. In crop breeding, elucidating the molecular mechanisms of meiosis is fundamental for manipulating recombination frequency and distribution, as well as for generating polyploid plants. In this review, we summarize current knowledge on the processes and genes involved in genetic recombination during Meiosis I, and the regulatory mechanisms of the second meiotic division during Meiosis II. Furthermore, we have outlined the breeding innovations achieved through the manipulation of meiosis, including the enhancement of genetic recombination frequency, alteration of recombination distribution, construction of artificial apomixis systems, and implementation of autopolyploid progressive heterosis (APH). This knowledge forms the cornerstone for further crop breeding applications, ultimately contributing to the optimization of crop yield and quality.

Foxtail millet (Setaria italica L.) is widely cultivated in arid and semi-arid regions. Its exceptional drought resistance and tolerance to infertile soils makes it an ideal crop for extreme climates and marginal lands. Recent innovations in foxtail millet cultivation-such as the development of ultra-early maturing mini mutants, the establishment of a robust genetic transformation system, the release of multi-omics databases, the construction of pan-genome maps, and the assembly of T2T genomes-have positioned it as a model organism in C4 cereal crop research. The polyploid potential of foxtail millet (such as larger fruits, higher yields, and enhanced stress resistance) remains unrealized. Currently, polyploidy is primarily induced using low concentrations of colchicine, a method limited by its toxicity, low induction rates, and adverse effects on plants. This underscores the urgent requirement for developing new, safer, and more efficient chromosome duplication technologies. Here, we demonstrate that the Siosd1-1D mutant in foxtail millet effectively produces tetraploid progeny, thus laying the groundwork for establishing a apomixis system in this crop. This study marks the first successful cloning of the SiOSD1 gene in foxtail millet, followed by precise gene editing using CRISPR/Cas9 technology to create the frameshift mutant siosd1-1D. siosd1-1D mutant's self-cross progeny completely transitioned to tetraploidy, like that observed with the AtOSD1 and OsOSD1 genes. Mutations in the OSD1 gene permit reproductive cells to bypass the second meiotic division, directly forming diploid gametes and subsequently producing tetraploid offspring through self-crossing. Our study highlights the highly conserved function of the OSD1 gene across monocots and dicots, providing an important theoretical basis for further research in other species. Furthermore, OSD1 gene mutations are crucial in achieving a apomixis system, particularly under the MiMe (Mitosis instead of Meiosis) strategy. Phenotypic evaluation of the tetraploid foxtail millet progeny derived from the siosd1-1D mutant revealed significant enhancements in grain size, stomatal dimensions, flag leaf length, and leaf sheath angle compared to that in the diploid wild-type; thus it displays typical polyploid traits. Further comparison of phenotypic differences between tetraploid millet and diploid wild-type millet, along with transcriptome sequencing, revealed that gene expression regulates millet ploidy changes. Tetraploid millet exhibits advantages in fruit size and potential yield. However, it also exhibits a general decline in seed setting rate and viability. These features could significantly limit its practical applications. Moreover, homozygous mutations in SiOSD1 gene could lead to continuous doubling in foxtail millet. To address these issues and obtain stable polyploids, future polyploid breeding strategies should integrate SiOSD1 cloning and haploid induction strategies. Overall, in this study, we confirm that editing meiosis-related endogenous genes is an effective strategy to create tetraploids with larger organs. Furthermore, we provide important scientific insights into the molecular mechanisms behind ploidy changes in foxtail millet.

In molecular design breeding, the simultaneous introduction of desired functional genes through specific nucleotide modifications and the elimination of genes regulating undesired phenotypic traits or agronomic components require advanced gene editing tools. Due to limited editing efficiency, even with the use of highly precise editing tools, such as prime editing (PE), simultaneous editing of multiple mutation types poses a challenge. Here, we replaced Cas9 nickase (nCas9) with Cas9 to construct a Cas9-mediated PE (Cas9-PE) system in rice. This system not only enables precise editing, but also allows for site-specific random mutation. Moreover, leveraging the precision of Cas9-PE, we established a transgene-free multiplex gene editing system using a co-editing strategy. This strategy involved the Agrobacterium-mediated transient expression of the precise editing rice endogenous acetolactate synthase gene ALSS627I to confer herbicide bispyribac-sodium (BS) resistance as a selection marker. This study provides a versatile and efficient multiplex gene editing tool for molecular design breeding.

CRISPR/Cas-based genome editing has been extensively employed in the breeding and genetic improvement of trees, yet precise editing remains challenging in these species. Prime editing (PE), a revolutionary technology for precise editing, allows for arbitrary base substitutions and the insertion/deletion of small fragments. In this study, we focused on the model tree poplar 84K ( Populus alba × P. glandulosa ). We used the 2 × 35S promoter to express a fusion protein of spCas9 nickase (nCas9) and engineered Moloney murine leukemia virus (MMLV), and the Arabidopsis thaliana AtU6 promoter to express an engineered PE guide RNA (epegRNA) and Nick gRNA, pioneering the establishment of the Prime Editor 3 (PE3) system in dicot poplar. Single-base substitutions, multiple-base substitutions, and small-fragment insertions/deletions were edited into three endogenous target genes. The desired edits were identified in hygromycin-resistant (transformed) calli at seven out of nine target sites, with an average editing efficiency ranging from 0.1 to 3.6%. Furthermore, stable T 0 plants contained the desired edits at four out of nine targets, with editing efficiencies ranging from 3.6 to 22.2%. Establishment of the PE3 system provides a powerful tool for the precise modification of the poplar genome.

Application of disease-resistant varieties is the most effective and environmentally friendly way to control crop diseases. However, there is often a trade-off between disease resistance and yield. Several recent studies have demonstrated that genome-editing technology brings a new strategy for generating disease-resistant crops without yield penalties.

In hybrid plants, heterosis often produces large, vigorous plants with high yields; however, hybrid seeds are generated by costly and laborious crosses of inbred parents. Apomixis, in which a plant produces a clone of itself via asexual reproduction through seeds, may produce another revolution in plant biology. Recently, synthetic apomixis enabled clonal reproduction of F1 hybrids through seeds in rice (Oryza sativa), but the inheritance of the synthetic apomixis trait and superior heterotic phenotypes across generations remained unclear. Here, we propagated clonal plants to the T4 generation and investigated their genetic and molecular stability at each generation. By analyzing agronomic traits, as well as the genome, methylome, transcriptome, and allele-specific transcriptome, we showed that the descendant clonal plants remained stable. Unexpectedly, in addition to normal clonal seeds, the plants also produced a few aneuploids that had eliminated large genomic segments in each generation. Despite the identification of rare aneuploids, the observation that the synthetic apomixis trait is stably transmitted through multiple generations helps confirm the feasibility of using apomixis in the future.

Multiplex genome editing (MGE) technologies constitute essential tools for rapid genome modification of multiple targets in one gene or multiple genes simultaneously. However, the vector construction process is complicated, and the number of mutation targets is constrained using the conventional binary vectors. Here, we describe a simple CRISPR/Cas9 MGE system based on classical isocaudomer technique in rice, which is comprised of only two simple vectors, and can theoretically be used to edit an unlimited number of genes simultaneously.

Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)-mediated genome editing has greatly accelerated progress in plant genetic research and agricultural breeding by enabling targeted genomic modifications. Moreover, the prime editing system, derived from the CRISPR/Cas system, has opened the door for even more precise genome editing. Prime editing has the capability to facilitate all 12 types of base-to-base conversions, as well as desired insertions or deletions of fragments, without inducing double-strand breaks and requiring donor DNA templet. In a short time, prime editing has been rapidly verified as functional in various plants, and can be used in plant genome functional analysis as well as precision breeding of crops. In this review, we summarize the emergence and development of prime editing, highlight recent advances in improving its efficiency in plants, introduce the current applications of prime editing in plants, and look forward to future prospects for utilizing prime editing in genetic improvement and precision molecular breeding.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 is the third generation of novel targeted genome editing technology after zinc finger nucleases (ZFNs) and transcription activator like effector nucleases (TALENs). It is also one of the most promising techniques for mutating and modifying genes. The CRISPR-Cas9 system has the advantages of simplicity, high efficiency, high specificity, and low production cost, thus greatly promoting the study of gene function. Meanwhile, it has attracted the attention of biologists. After the development and improvement in recent years, CRISPR-Cas9 system has become increasingly mature and has been widely used in crop improvement. Firstly, this review systematically summarizes the generation and advantages of CRISPR-Cas9 system. Secondly, three derivative technologies of the CRISPR-Cas9 system are introduced. Thirdly, this review focuses on the application of CRISPR-Cas9 system in gene knockout, gene knock-in, and gene regulation, as well as the improvement of yield, quality, and biological resistance of important crops such as rice, wheat, soybean, corn, and potato. Finally, this review proposes the potential challenges of CRISPR-Cas9 system, and discusses the future development of CRISPR-Cas9 system.

>Dear Editor,Prime editing(PE) systems are important genome-editing tools developed for mediating arbitrary small DNA insertions, deletions and all 12 base-to-base conversions using a CRISPR-nCas9 fusion of an engineered M-MLV and PE guide RNAs(peg RNAs)(Anzalone et al., 2019). At present,

Cytosine methylation is one of the major types of DNA epigenetic modifications and plays an important role in maintaining normal cell function and regulating gene expression. Bisulfite sequencing PCR (BSP) based cloning and sequencing is a general method for detecting DNA methylation at specific sites, which can clarify the methylation status of each CpG site in the target fragment. However, this method requires large amounts of single-clonal sequencing, which is complicated to operate, time consuming and expensive. Therefore, the development of an accurate, efficient and convenient DNA methylation detection technology is of great significance to improve the efficiency of epigenetic research. Based on the high-throughput mutation detection platform Hi-TOM (high-throughput tracking of mutations) developed by our group, we further established a site-specific DNA methylation high-throughput detection platform Hi-Meth (High-throughput Detection of DNA Methylation). After bisulfite treatment of DNA samples, the specific site-specific DNA methylation analysis results could be obtained through the Hi-Meth platform by performing only one round of PCR amplification. Using the Hi-Meth platform, the DNA methylation status of two promoter regions of rice were detected. The DNA methylation results from Hi-Meth were consistent with the results from BSP-based method. Thus, site-specific DNA methylation analysis results could be obtained accurately and conveniently through the Hi-Meth platform. In conclusion, Hi-Meth provides an important methylation detection platform for specific DNA regions, which has important significance for epigenetic research.

Due to a production error, a pdf file containing Supplemental Figures S1 through S13 and Supplemental Tables S1 through S5 was mistakenly omitted from other supplemental data published online with this article. The pdf file has been restored. The description in the abstract “Surprisingly, the simultaneous loss of DRM2, CHROMOMETHYLASE3 (CMT2), and CMT3 functions, which completely erases all non-CG methylation in Arabidopsis, only partially reduced it in rice.” should be corrected to “Surprisingly, the simultaneous loss of DRM2, CHROMOMETHYLASE2 (CMT2), and CMT3 functions, which completely erases all non-CG methylation in Arabidopsis, only partially reduced it in rice.” Er ra tu m

>Dear Editor,The clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9) system, since it was excavated, has been rapidly developed and sparked a revolution in the genome editing field. In principle,CRISPR/Cas9 system relies on the recognition of specific loci on the genome, which is titled the protospacer adjacent motif (PAM). However, the canonical Streptococcus pyogenes Cas9 (SpCas9) nuclease only recognizes NGG or NAG PAMs, rendering an inherent obstacle in amplifying the application of CRISPR/Cas9 technology.

Clustered regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 (CRISPR/Cas9) has not been completely established in Hevea brasiliensis. In the present study, firstly, five endogenous U6 promoters from H. brasiliensis were identified and exploited to drive single guide RNA (sgRNA) transcription for establishing CRISPR/Cas9 transient editing system in H. brasiliensis protoplast. All five promoters were functional, however, their corresponding editing efficiencies were different and varied from 8.47 % to 24.92 %. Secondly, mutation profiles were characterized using 10 sgRNAs targeted five flowering time related genes (HbFT1, HbFT2 and HbTFL1-1, HbTFL1-2, HbTFL1-3) in H. brasiliensis protoplast. Three mutation patterns i. e. deletion, insertion and base substitution were detected. Among these mutations, deletion was the most prominent one and the insertions were observed only in half of 10 target sites, in which the highest insertion frequency (15 %) occurred at TS6 target site. Base substitutions including transition and transversion were detected in 8 out of 10 target sites by deep sequencing. Lastly, stable transformation editing vector targeting phytoene desaturase gene (HbPDS) was constructed and transformed into rubber tree callus, and the ?+1? bp homozygous insertions were detected for 3 out of 16 calli at the target site, which turned to expected albino phenotype in the following subculture. This study pronounced the establishment of genome editing in H. brasiliensis by CRISPR/Cas9 plasmid system.

Gene editing technology in woody plants has great potential for understanding gene function, and altering traits affecting economically and ecologically important traits. Gene editing applications in woody species require a high genome editing efficiency due to the difficulty during transformation and complexities resulting from gene redundancy. In this study, we used poplar 84K (Populus alba × P. glandulosa), which is a model hybrid for studying wood formation and growth. We developed a new CRISPR/Cas9 system to edit multiple genes simultaneously. Using this system, we successfully knocked out multiple targets of the PHYTOENE DESATURASE 8 in poplar. We found the mutation rate of our CRISPR/Cas9 system is higher (67.5%) than existing reports in woody trees. We further improved the mutation rate up to 75% at editing sites through the usage of the mannopine synthase (MAS) promoter to drive Cas9. The MAS-CRISPR/Cas9 is an improved genome-editing tool for woody plants with a higher efficiency and a higher mutation rate than currently available technologies.

DNA methylation in the non-CG context is widespread in the plant kingdom and abundant in mammalian tissues such as the brain and pluripotent cells. Non-CG methylation in Arabidopsis thaliana is coordinately regulated by DOMAINS REARRANGED METHYLTRANSFERASE (DRM) and CHROMOMETHYLASE (CMT) proteins but has yet to be systematically studied in major crops due to difficulties in obtaining genetic materials. Here, utilizing the highly efficient multiplex CRISPR-Cas9 genome-editing system, we created single- and multiple-knockout mutants for all the nine DNA methyltransferases in rice (Oryza sativa) and profiled their whole-genome methylation status at single-nucleotide resolution. Surprisingly, the simultaneous loss of DRM2, CHROMOMETHYLASE3 (CMT2), and CMT3 functions, which completely erases all non-CG methylation in Arabidopsis, only partially reduced it in rice. The regions that remained heavily methylated in non-CG contexts in the rice Os-dcc (Osdrm2/cmt2/cmt3a) triple mutant had high GC contents. Furthermore, the residual non-CG methylation in the Os-dcc mutant was eliminated in the Os-ddccc (Osdrm2/drm3/cmt2/cmt3a/cmt3b) quintuple mutant but retained in the Os-ddcc (Osdrm2/drm3/cmt2/cmt3a) quadruple mutant, demonstrating that OsCMT3b maintains non-CG methylation in the absence of other major methyltransferases. Our results showed that OsCMT3b is subfunctionalized to accommodate a distinct cluster of non-CG-methylated sites at highly GC-rich regions in the rice genome.