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.

Thesium chinense Turcz. (T. chinense), a perennial herb in the Santalaceae family, exhibits potent antibacterial and anti-inflammatory properties. Transcriptome sequencing was performed on one- and two-year-old T. chinense plants across seedling, flowering, and fruiting stages (all sampled from the same location) using the illumina NovaSeq 6000 platform. A total of 58,706 unigenes were identified, including 1656 transcription factors (TFs). Further analysis classified these TFs into seven functional categories, enabling the reconstruction of a representative TF regulatory network. Differential expression analysis revealed that the number of differentially expressed genes (DEGs) ranged from 2000 to 5000 during different developmental stages in first-year plants, while varying between 1000 and 2000 in second-year plants. Comparative analysis of DEGs between one- and two-year-old plants showed that they were primarily associated with sesquiterpene, triterpene, and terpene skeleton biosynthesis, as well as other metabolic pathways. Additionally, analysis of key genes involved in flavonoid biosynthesis—the major bioactive compounds in T. chinense—revealed their predominant accumulation during the first year of growth. This study provides valuable insights into the developmental biology of T. chinense and establishes a foundation for future research on flavonoid biosynthesis pathway genes and their therapeutic applications.