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ABSTRACT Understanding genotype–phenotype relationship is fundamental in biology. With the benefit from next-generation sequencing and high-throughput phenotyping methodologies, there have been generated much genome and phenome data for Saccharomyces cerevisiae. This makes it an excellent model system to understand the genotype–phenotype relationship. In this paper, we presented the reconstruction and application of the yeast pan-genome in resolving genotype–phenotype relationship by a machine learning-assisted approach.