As an important noncereal food crop grown worldwide, the genetic improvement of potato in tuber yield and quality is largely constrained due to the lacking of a high-quality reference genome and understanding of the regulatory mechanism underlying the formation of superior alleles. Here, a chromosome-scale haplotype-resolved genome assembled from an anther-cultured progeny of ‘Ningshu 15’, a tetraploid variety featured by its high starch content and drought resistance was presented. The assembled genome size was 1.653 Gb, with a contig N50 of approximately 1.4 Mb and a scaffold N50 of 61 Mb. The long terminal repeat assembly index score of the two identified haplotypes of ‘Ningshu 15’ was 11.62 and 11.94, respectively. Comparative genomic analysis revealed that positive selection occurred in gene families related to starch, sucrose, fructose and mannose metabolism, and carotenoid biosynthesis. Further genome-wide association study in 141 accessions identified a total number of 53 quantitative trait loci related to fructose, glucose, and sucrose content. Among them, a tonoplast sugar transporter encoding gene, StTST2, closely associated with glucose content was identified. Constitutive expression of StTST2 in potato and Arabidopsis increased the photosynthetic rate, chlorophyll and sugar content, biomass tuber and seed production in transgenic plants. In addition, co-immunoprecipitation assays demonstrated that StTST2 directly interacted with SUT2. Our study provides a high-quality genome assembly and new genetic locus of potato for molecular breeding.

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