Blackberries (Rubus spp.) are globally consumed and well known for their rich anthocyanin and antioxidant content and distinct flavors. Improving blackberries has been challenging due to genetic complexity of traits and limited genomic resources. The blackberry genome has been particularly challenging to assemble due to its polyploid nature. Here, we present the first chromosome-scale and haplotype-phased assembly for the primocane-fruiting, thornless tetraploid blackberry selection BL1 (Rubus L. subgenus Rubus Watson). The genome assembly was generated using Oxford Nanopore Technology and Hi-C scaffolding, resulting in a 919 Mb genome distributed across 27 pseudochromosomes, with an N50 of 35.73 Mb. This assembly covers >92% of the genome length and contains over 98% of complete BUSCOs. Approximately, 58% of the assembly consists of repetitive sequences, with long terminal repeats being the most abundant class. A total of 87,968 protein-coding genes were predicted, of which, 82% were functionally annotated. Genome mining and RNA-Seq analyses identified possible candidate genes and transcription factors related to thornlessness and the key structural genes and transcription factors for anthocyanin biosynthesis. Activator genes including PAP1 and TTG1 and repressor genes such as ANL2 and MYBPA1 play an important role in the fine tuning of anthocyanin production during blackberry development. Resequencing of seven tetraploid blackberry cultivars/selections with different horticultural characteristics revealed candidate genes that could impact fruiting habit and disease resistance/susceptibility. This tetraploid reference genome should provide a valuable resource for accelerating genetic analysis of blackberries and facilitating the development of new improved cultivars with enhanced horticultural and nutritional traits.

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