**Background:** Long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) are two major classes of non-coding RNAs that regulate gene expression at pre- and post-transcriptional levels. Once considered 'junk,' these ncRNAs are now recognized as key regulators in plant development and stress responses. Black pepper (Piper nigrum L.), the 'king of spices,' is economically important but lacks any prior study on lncRNAs or circRNAs. This study aims to fill that gap by performing genome-wide identification and characterization of these ncRNAs and their regulatory networks.
**Methods:** A total of 53 raw RNA-Seq datasets (>1.2 billion reads) were downloaded from NCBI, covering eight BioProjects, six tissues (flower, fruit, leaf, panicle, root, and stem), and six black pepper cultivars (Reyin-1, Bragantina, Thottumuriyan, IPN No. LK-0-WU-0014181, panniyur-1, and Genotype 4226) from four countries. Raw reads were quality-checked using FastQC v0.11.8 and trimmed with Trimmomatic v0.39 (Phred score <30 removed). Clean reads (1,251,005,293; 97.88% of raw reads) were aligned to the black pepper reference genome using HISAT2 v2.2.0, achieving 70%–94% mapping across samples. Transcriptome assembly was performed with StringTie v2.1.4, yielding 294,777 transcripts. For lncRNA identification, transcripts shorter than 200 bp were removed, ORFs longer than 300 nucleotides were discarded, and coding potential was assessed using CPC2 v1.0.1 and PLEK. Housekeeping RNAs were removed by BlastN against SILVA, GtRNAdb, and RNACentral (≥95% identity cutoff). Transcripts matching NCBI-nr or Pfam proteins were removed (BlastX, e-value 10^-3). For circRNA identification, clean reads were aligned using BWA v0.7.17 and circRNAs were predicted using CIRI2 v2.0 with ≥2 backspliced reads. miRNA targets were identified using psRNATarget (expectation ≤2, UPE ≤25) and TargetFinder. GO and KEGG enrichment analyses were performed using OmicsBox and REVIGO.
**Key Results:** A total of 6,406 novel lncRNAs were identified, with tissue-wise distribution: 1,115 (flower), 2,621 (fruit), 2,727 (leaf), 828 (panicle), 1,214 (root), and 1,003 (stem). Approximately 80% of lncRNAs were intergenic. Length ranged from 200 to 10,667 nucleotides, with >80% shorter than 1,500 nt. Average exon count was 1.88, with 84.85% having 1–2 exons. Conservation analysis against 38 plant species in CANTATAdb v2.0 found only 45 high-confidence matches (42 lncRNAs matching 27 database lncRNAs from 13 species), confirming poor cross-species conservation. A total of 36 lncRNAs were identified as potential precursors for 14 pre-miRNAs (≥90% match). ceRNA analysis revealed 1,702 lncRNA–miRNA interactions involving 1,054 unique miRNAs and 396 unique lncRNAs, and 3,274 miRNA–mRNA interactions. For circRNAs, 4,621 distinct circRNAs were identified (3,871 novel), with tissue distribution: 333 (flower), 2,939 (fruit), 1,004 (leaf), 156 (panicle), 400 (root), and 386 (stem). Most circRNAs were ≤1,000 nt (median ~400 nt). Genomic origins: 47.98% intergenic, 43.37% exonic, 8.66% intronic. TargetFinder identified 2,591 circRNA–miRNA interactions (690 unique miRNAs targeting 432 circRNAs), and 6,213 miRNA–mRNA interactions (619 miRNAs targeting 744 mRNAs). GO and KEGG enrichment of target genes revealed pathways including protein modification, transmembrane transport, signaling, diterpenoid biosynthesis, fatty acid biosynthesis, RAS signaling, and plant–pathogen interaction. A web resource, BPncRDB, was developed and is freely accessible at http://backlin.cabgrid.res.in/bpncrdb/index.php.
**Clinical Implications:** This study has no direct clinical implications as it is a plant genomics study. However, the findings may indirectly support the development of black pepper varieties with improved yield and stress tolerance, which could benefit nutritional and functional food applications given black pepper's bioactive compounds (e.g., piperine) with reported roles in tumor suppression, chemoprevention, and metabolic health.