Pangenomics as a framework for discovering structural variants underlying cold tolerance in chilli pepper

Pangenomics-based structural variant discovery offers a transformative framework to resolve missing cold-tolerance heritability in chilli pepper and accelerate climate-resilient cultivar development. Chilli pepper (Capsicum annuum L.) is a globally important crop severely restricted by cold sensitivity. SNP-based GWAS explain only 20-30% of the variance in cold tolerance, leaving a "missing heritability" gap. The GanZi/XianSheng paradox cultivars sharing >99% coding sequence identity yet exhibiting 5-10-fold differences in cold tolerance strongly implicates regulatory and dosage-based structural variants (SVs) as the primary drivers of phenotypic variation, a gap that pangenomics is positioned to address. This review synthesizes pangenomics, structural variant discovery, QTL mapping, and genome editing as an integrated framework for developing cold-tolerant chilli pepper cultivars. Based on cross-species evidence from rice, barley, and tomato pangenome studies, we propose four SV mechanisms predicted to underlie the GanZi cold-tolerance advantage: (1) regulatory SVs in promoters of CaMYB80, CBF/DREB (DREB, Dehydration-Responsive Element-Binding protein), and CaICE1; (2) copy number variations (CNVs) in antioxidant gene families (CaSOD (Superoxide Dismutase), CaCAT (Catalase), CaAPX (Ascorbate Peroxidase)) and the chlorophyll biosynthesis gene CaALAD (δ-Aminolevulinic Acid Dehydratase); (3) transposable element insertions creating novel regulatory elements in osmolyte biosynthesis genes (P5CS (Δ1-Pyrroline-5-Carboxylate Synthase), BADH (Betaine Aldehyde Dehydrogenase)); and (4) chromosomal inversions coordinating master cold-regulator gene clusters. A multi-SV haplotype in GanZi is predicted to explain 60-80% of phenotypic variance in cold tolerance, more than three times the explanatory power of SNP-based approaches. We outline a pangenome construction strategy (15-17 genomes, PacBio/Nanopore long-read sequencing, 18-24 months, $650K-$980K) followed by SV-GWAS on 200-500 accessions to identify 10-25 high-impact SVs. A four-phase breeding roadmap integrates QTL identification (1-2 years), candidate gene discovery via fine mapping and VIGS (1.5-2 years), CRISPR/Cas9 genome editing via TSWV/TRV viral vectors achieving 6.3-77.9% heritable editing efficiency (2-3 years), and multi-environment field validation across 4-6 locations (2-3 years).This integrated pangenomics-to-breeding strategy is projected to deliver climate-resilient chilli pepper cultivars with 2-5°C improvements in freezing tolerance within 7-10 years, substantially outpacing the 10-12-year timeline of traditional breeding. The proposed structural variant mechanisms remain to be experimentally confirmed in pepper and represent a testable research agenda for the chilli pepper genomics community.