Upland cotton (Gossypium hirsutum), an allotetraploid, contains At- and Dt- subgenome and most genes have multiple homologous copies, which pose a huge challenge to investigate genes’ function due to the functional redundancy. Therefore, it is of great significance to establish effective techniques for the functional genomics in cotton. In this study, we tested two novel genome editing vectors and compared them with the CRISPR/Cas9 system (pRGEB32-GhU6.7) developed in our laboratory previously. In the first new vector, the sgRNA transcription unite was constructed into the replicon (LIR-Donor-SIR-Rep-LIR) of the bean yellow dwarf virus (BeYDV) and named as pBeYDV-Cas9-KO and in the second vector, the ubiquitin promoter that drives Cas9 protein was replaced with a constitutive CaMV 35S promoter and defined as pRGEB32-35S. The results from transgenic cotton calli/plants revealed that pBeYDV-Cas9-KO vector showed the highest editing efficiency of GhCLA1 in At and Dt subgenomes edited simultaneously up to 73.3% compared to the 44.6% of pRGEB32-GhU6.7 and 51.2% of pRGEB32-35S. The editing efficiency of GhCLA1 in At and Dt subgenome by pBeYDV-Cas9-KO was 85.7% and 97.2%, respectively, whereas the efficiency by pRGEB32-GhU6.7 and pRGEB32-35S vectors was 67.7%, 86.5%, 84%, and 87.2%, respectively. The editing profile of pBeYDV-Cas9-KO was mainly composed of fragment deletion, accounting for 84.0% and ranging 1–10 bp in length. The main editing sites are located at positions 11–17 upstream of PAM site. The off-target effects were not detected in all potential off-target sites. Taken together, the pBeYDV-Cas9-KO system has high editing efficiency and specificity with wide editing range than the traditional CRISPR/Cas9 system, which provides a powerful tool for cotton functional genomics research and molecular breeding.

Apocynum pictum Schrenk is a semishrub of the Apocynaceae family with a wide distribution throughout the Tarim Basin that holds significant ecological, medicinal, and economic values. Here, we report the assembly of its chromosome-level reference genome using Nanopore long-read, Illumina HiSeq paired-end, and high-throughput chromosome conformation capture sequencing. The final assembly is 225.32 Mb in length with a scaffold N50 of 19.64 Mb. It contains 23,147 protein-coding genes across 11 chromosomes, 21,148 of which (91.36%) have protein functional annotations. Comparative genomics analysis revealed that A. pictum diverged from the closely related species Apocynum venetum approximately 2.2 million years ago and has not undergone additional polyploidizations after the core eudicot WGT-γ event. Karyotype evolution analysis was used to characterize interchromosomal rearrangements in representative Apocynaceae species and revealed that several A. pictum chromosomes were derived entirely from single chromosomes of the ancestral eudicot karyotype. Finally, we identified 50 members of the well-known stress-responsive WRKY transcription factor family and used transcriptomic data to document changes in their expression at 2 stages of drought stress, identifying a number of promising candidate genes. Overall, this study provides high-quality genomic resources for evolutionary and comparative genomics of the Apocynaceae, as well as initial molecular insights into the drought adaptation of this valuable desert plant.

Background: Upland cotton (Gossypium hirsutum) is a major natural fiber crop and an important model for studying genome evolution and gene function in polyploid plants. However, its large and highly redundant genome presents substantial challenges for efficient and coordinated multiplex genome editing. Methods: Here, we developed a high-efficiency CRISPR/Cas12a-based multiplex genome editing system in cotton by integrating a tRNA–crRNA polycistronic expression strategy with a Bean yellow dwarf virus (BeYDV)-derived replicon. Results: This platform enabled coordinated expression of multiple crRNAs and simultaneous targeting of 16 loci within a centromere-proximal region of chromosome D03 (18.65–24.47 Mb). In individual transgenic lines, up to 10 target sites were edited concurrently, with nine targets exhibiting editing efficiencies above 56% and the highest efficiency reaching 96.46%. High-density multiplex editing predominantly induced small insertions and deletions at target loci. Notably, edited plants exhibited reduced growth and pronounced cytological abnormalities, including chromosome bridges, lagging chromosomes, and abnormal meiotic products. Transcriptome analysis revealed widespread dysregulation of genes involved in chromosome segregation and cell cycle regulation. Despite these functional perturbations, HiFi long-read sequencing detected no large-scale chromosomal rearrangements, indicating that genome instability arises from cumulative local perturbations rather than global structural alterations. Conclusions: Together, our results establish an efficient multiplex genome editing platform in cotton and highlight potential constraints of high-density editing on genome stability in complex plant genomes.

Introduction: Calvin Cycle Protein 12 (CP12) is a key regulator of the Calvin-Benson-Bassham (CBB) cycle that mediates CO₂ assimilation through dark/light modulation. Beyond its canonical role, emerging evidence indicates that CP12 may also function as a molecular chaperone and participate in plant stress responses. However, its gene family characteristics and roles under heat stress remain unclear in cotton. Methods: We performed a genome-wide identification and characterization of the CP12 gene family in four cotton species ( Gossypium hirsutum, G. barbadense, G. arboreum, and G. raimondii ). Phylogenetic classification, conserved motif analysis, gene structure, synteny, and promoter cis-element analyses were conducted. Transcriptome datasets from flowers, leaves, and buds under heat stress were analyzed to determine expression patterns, and these were further correlated with physiological indicators. Results: A total of 11, 10, 5, and 4 CP12 genes were identified in G. hirsutum, G. barbadense, G. arboreum , and G. raimondii , respectively. Phylogenetic analysis grouped them into three clades (I–III), supported by conserved motif and structural features. Synteny analysis indicated that whole-genome and segmental duplications were the primary drivers of expansion. Promoter analysis revealed enrichment of stress-responsive elements. Expression profiling showed clade-specific divergence: Clade I genes were strongly induced by heat stress, with Ghir_CP12_10 displaying ~10-fold upregulation in flowers, while Clade II genes were generally downregulated. These expression trends were associated with physiological changes, including reduced net photosynthetic rate and elevated malondialdehyde, catalase, and peroxidase levels. Discussion: Our findings demonstrate that the cotton CP12 gene family has undergone functional divergence. Clade I members act as positive regulators of thermotolerance, potentially stabilizing photosynthetic complexes and protecting enzymes from oxidative damage under heat stress. This study provides new insights into the evolution and function of CP12 genes and establishes a foundation for future functional validation and breeding of heat-tolerant cotton varieties.