Genetic breeding and molecular identification in varieties depend on high-performance genotyping tools. The high heterozygosity of the litchi genome contributes to increased resequencing costs and elevated error rates in hybridization-based genotyping methods. In this study, a liquid chip named Litchi40K v1.0 was developed with high-depth resequencing data from 875 litchi samples, and its efficacy was validated across three different populations. In the L. chinensis var. fulvosus population, three subpopulations characterized by spatial distribution, and a total of 1110 genes were identified in the genomic regions with subpopulation differentiation. Additionally, a total of 30 significant signals associated with diverse agronomic traits were identified. The H002 haplotype of LITCHI02696, dominant in the Sub2 subgroup, significantly increased the soluble solid content in the L. chinensis var. fulvosus population. In a hybrid F₁ population, a high-density genetic map was constructed and 79 dwarfing-related QTLs were identified with the liquid chip. An NAC transcription factor was identified as a candidate gene with a heterozygous frameshift variant in the male parent. To facilitate the digitization of germplasm resources, 384 SNPs were selected, and the DNA fingerprint map revealed clear genetic relationships and a total of 10 potential synonym groups or instances of bud mutations were identified in 164 main cultivated litchi varieties. This study provides cost-effective, flexible, and versatile liquid chip for genetic analysis and digitalization of germplasm resources in litchi.

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