Citrus production is challenged by multiple biotic and abiotic stresses, with Huanglongbing (HLB) disease being the most damaging to global citrus production. Long-term field evaluations have revealed that certain citrus relatives have varying HLB tolerance. This study investigates the genetic relationships among citrus genotypes with differing HLB tolerance levels to inform future breeding strategies for citrus improvement. The genome sequencing data from 39 citrus species and hybrids were analyzed: 23 tolerant, 8 moderately tolerant, and 8 susceptible to HLB. Variant calling identified 445,222 high-quality SNPs, with chromosome 5 showing the highest contribution of unique variants. Polymorphic information content (PIC) ranged from 0.27 to 0.38, with the highest SNP frequency observed at a PIC value of 0.27 (21,500 loci). The mean heterozygosity across genotypes was 0.136, with Citrus aurantium showing the highest and Citrus polyandra the lowest heterozygosity. Phylogenetic analysis grouped the genotypes into six distinct clusters. Admixture analysis (K=5) was performed to explore ancestral genetic components, while PCA accounted for 62.76% of total genetic variation. These findings provide critical insights for developing HLB-tolerant citrus hybrids and highlight the underlying genetic diversity present in citrus germplasm.
