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AbstractLycium ruthenicumis a characteristic plant resource in Northwest China. The fruit with high anthocyanin content is the main reason for its high nutritional value and economic value, while the regulation of key genes regulating anthocyanin biosynthesis in black fruit is not clear. In this study, high-throughput sequencing technology was used to compare the expression differences of all genes in black and purple fruits. The purple samples obtained 45.13 and 51.49 M Raw reads, while the black fruit samples measured 53.85 and 47.38 M Raw Reads. After splicing and assembly, 162,842 transcripts were obtained after removing redundant and duplicated results. Homology comparison of all predicted proteins found that 36,978 (22.43%) genes in Lycium ruthenicumwere homologous to Solanum tuberosum, followed by 24,459 (14.84%) genes were homologous to Capsicum annuum. Overall, 3685 genes were up-regulated in purple fruit, 2837 genes were down-regulated in purple fruit, and the remaining 156320 genes were not differentially expressed in black and purple fruit. Through homology comparison of anthocyanin biosynthesis and metabolism structure genes in Lycium ruthenicum, 63 homologous genes, 4 MYB transcription factors and 5 bHLH transcription factors were screened. LrAN2p(909 bp) in purple fruit was 136 bp more upstream of ‘ATG’ than LrAN2(774 bp) in black fruit. Three highly expressed copies of bHLH transcription factors AN1bwith different lengths were screened in black fruit, but they were almost not expressed in purple fruit. This study possibly identified the major LrAN1bgenes regulating the black fruit trait.