Integrated Short‐and Long‐Read Single‐Cell Sequencing Reveals Cellular Heterogeneity and Isoform Diversity During Ovarian Development in Baoshan Pigs

Ovarian development is a dynamic process essential for female reproductive function and requires coordinated interactions among diverse ovarian cell populations. In this study, we integrated Illumina short-read and Oxford Nanopore long-read single-cell RNA sequencing approaches to profile ovarian tissues from Baoshan pigs at 1, 3, 5, and 8 months of age. We performed integrated analyses of cell clustering and annotation, cell-cell communication, full-length transcript identification, and alternative splicing to characterize cellular and transcriptomic dynamics during ovarian development. A total of 24 646 high-quality single cells were obtained, identifying 10 major ovarian cell types, whose spatial distribution patterns were further validated by immunofluorescence staining. Ovarian cellular composition underwent dynamic changes across developmental stages, with distinct functional states and transcriptional regulatory networks among cell types. Cell-cell communication analysis revealed a shift in the dominant signaling sender cell type from SC to SMC, while key pathways, including COLLAGEN and NOTCH, showed sustained activity throughout ovarian development. Long-read single-cell sequencing identified 71 352 non-redundant isoforms from 11 942 genes, with FSM isoforms predominating and exhibiting higher expression levels across developmental stages. The M3 stage exhibited the highest diversity of stage-specific isoforms and alternative splicing events, with exon skipping being predominant. Our study establishes a comprehensive transcriptomic atlas of ovarian development in Baoshan pigs, providing insights into cellular dynamics, intercellular communication, and transcript isoform diversity.