Heterosis is a fundamental biological phenomenon characterized by the superior performance of hybrids over their parents. Although tremendous progress has been reported in seed crops, the molecular mechanisms underlying heterosis in clonally propagated crops are largely unknown. Potato (Solanum tuberosum L.) is the most important tuber crop and an ongoing revolution is transforming potato from a clonally propagated tetraploid crop into a seed-propagated diploid hybrid potato. In our previous study, we developed the first generation of highly homozygous inbred lines of potato and hybrids with strong heterosis. Here, we integrated transcriptome, metabolome, and DNA methylation data to explore the genetic and molecular basis of potato heterosis at three developmental stages. We found that the initial establishment of heterosis in diploid potato was mainly due to dominant complementation. Flower color, male fertility and starch and sucrose metabolism showed obvious gene dominant complementation in hybrids, and hybrids devoted more energy to primary metabolism for rapid growth. In addition, we identified ~2,700 allele-specific expression genes at each stage, which likely function in potato heterosis and might be regulated by CHH allele-specific methylation level. Our multi-omics analysis provides insight into heterosis in potato and facilitates the exploitation of heterosis in potato breeding.

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