Haplotype‐resolved genome of a heterozygous wild peach reveals the PdaWRKY4‐PdaCYP716A1 module mediates resistance to aphids by regulating betulin biosynthesis.

ABSTRACT Wild species of domesticated crops provide valuable genetic resources for resistance breeding. <italic>Prunus davidiana</italic>, a wild relative of peach with high heterozygosity and diverse stress tolerance, exhibits high resistance against aphids. However, the highly heterozygous genome of <italic>P. davidiana</italic> makes determining the underlying factors influencing resistance traits challenging. Here, we present the 501.7 Mb haplotype‐resolved genome assembly of <italic>P. davidiana</italic>. Genomic comparisons of the two haplotypes revealed 18,152 structural variations, 2,699 Pda_hap1‐specific and 2,702 Pda_hap2‐specific genes, and 1,118 allele‐specific expressed genes. Genome composition indicated 4.1% of the <italic>P. davidiana</italic> genome was non‐peach origin, out of which 94.5% was derived from almond. Based on the haplotype genome, the aphid resistance quantitative trait locus (QTL) was mapped at the end of Pda03. From the aphid resistance QTL, <italic>PdaWRKY4</italic> was identified as the major dominant gene, with a 9‐bp deletion in its promoter of the resistant phenotype. Specifically, <italic>PdaWRKY4</italic> regulates aphid resistance by promoting <italic>PdaCYP716A1</italic>‐mediated anti‐aphid metabolite betulin biosynthesis. Moreover, we employed a genome design to develop a breeding workflow for rapidly and precisely producing aphid‐resistant peaches. In conclusion, this study identifies a novel aphid resistance gene and provides insights into genome design for the development of resistant fruit cultivars. [ABSTRACT FROM AUTHOR]