Red raspberry (Rubus idaeus L.) is a high-value crop, acclaimed for its fruit quality characteristics and putative health benefits. Among fruit quality characteristics, color is a critical trait in determining market acceptability and consumer preference. Red raspberry fruit encompasses a wide array of colors, notably yellow, orange, and red. Red fruited cultivars are the most common in commercial settings and market selection is dependent on the color intensity: while the processing industry needs dark berries for most applications, the fresh market requires bright red and non-darkening fruit. Anthocyanins, a group of water-soluble phenolic compounds, are regarded as the major contributors to raspberry red fruit color. Knowledge on the genetics of raspberry fruit pigmentation would be valuable for breeding programs, but to date the genetic control of the different red intensities of raspberries remains elusive. This research aims to map the genetic regions underlying the red shades of raspberry fruit through a genome-wide association study (GWAS). Fruits from 765 red raspberry cultivars and selections – including red-, orange-, and yellow-fruited genotypes – were harvested over four seasons (2018-2021) and analyzed for total anthocyanin content and color. Total anthocyanins were measured through the pH differential methods and color was assessed using a high-throughput digital phenotyping protocol. Leaves from all genotypes were harvested in summer 2022 and used for genomic DNA extraction. Whole-genome sequencing of DNA samples was achieved through Illumina NovaSeq6000, with an average coverage of 30×. Sequences were aligned to the ‘Malling Jewel’ reference genome using BWA-MEM and single nucleotide polymorphisms (SNPs) were identified following the GATK pipeline. Total anthocyanin content ranged between zero (yellow genotypes) and 113.21 mg/100 g fresh weight (FW) of peonidin-3-O-glucoside equivalents (P3OG eq.) and averaged 42.09 mg/100 g FW P3OG eq. Color coordinates L* (lightness) varied between 11.99 and 48.21 and averaged 21.21, a* (red-green) spanned -1.18 and 38.38 and averaged 26.85, b* (yellow-blue) ranged between 4.57 and 30.04 and averaged 18.05. The association between the detected genetic variants and the phenotypic data (fruit color and total anthocyanins) will enable the identification of SNP markers that explain the variation in observed red shades of berries. Such markers will be used in raspberry breeding programs to facilitate the development of cultivars with desired fruit color.
