
NBS-LRR resistance genes cluster in tandem arrays embedded in repetitive sequence, a genomic architecture that systematically undermines their detection by single-reference approaches. This limitation is especially severe in tea (Camellia sinensis), whose 3.1 Gb genome is approximately 80% repetitive and concentrates immune gene diversity in structurally complex regions. The study constructed a HiFi haplotype-resolved variation graph pangenome of C. sinensis from 16 phased assemblies encompassing eight accessions spanning C. sinensis var. sinensis (CSS) and C. sinensis var. assamica (CSA) cultivars, as well as wild Camellia taliensis relatives. This pangenome constitutes the first graph-based framework dedicated to systematic characterization of NBS-LRR presence–absence variation (PAV) in tea. Graph-based analysis identified 976 high-confidence NBS-LRR genes pangenome-wide—21.5% more than detectable from the single Longjing 43 linear reference (Wang et al., 2020)—with 40.8% showing PAV across accessions. Variable genes were disproportionately concentrated in tandem gene clusters, consistent with non-allelic homologous recombination as a structural diversification mechanism observed across other crop pangenomes. Wild C. taliensis haplotypes harbored substantially more NBS-LRR genes per haplotype (mean 831) than CSS (753) or CSA (782) cultivars, a pattern consistent with domestication-associated gene loss. Population-scale PAV genotyping of 487 resequenced accessions, integrated with infection time-series transcriptomics, identified 11 candidate PAV loci associated with anthracnose resistance. The top-ranked candidate, CsNBS-chr7-0431 (CNL subclass), exhibited population-level differentiation (FST = 0.46), was present in 87.2% of resistant versus 24.7% of susceptible accessions, and showed peak infection-induced upregulation (log2FC = 2.83 at 48 hpi). These findings reveal PAV in NBS-LRR clusters as a major, previously under-characterized dimension of tea immune diversity and provide a graph pangenome resource and a PAV marker panel with direct utility for anthracnose resistance breeding.
