Article https://doi.org/10.1038/s41467-023-40838-1 Comparative genomic analyses reveal the genetic basis of the yellow-seed trait in Brassica napus Cunmin Qu 1,2,3,12 , Meichen Zhu 1,2,3,12 , Ran Hu 1,2,3,12 , Yongchao Niu 4,12 , Si Chen 1,2,3,12 , Huiyan Zhao 1,2,3,12 , Chengxiang Li 5 , Zhen Wang 1,2,3 , Nengwen Yin 1,2,3 , Fujun Sun 1,2,3 , Zhiyou Chen 1,2,3 , Shulin Shen 1,2,3 , Guoxia Shang 6 , Yan Zhou 1,2,3 , Xingying Yan 1,2,3 , Lijuan Wei 1,2,3 , Liezhao Liu 1,2,3 , Bin Yi 7 , Jinmin Lian 8 , Jiang Li 8 , Zhanglin Tang 1,2,3 , Ying Liang 1,2,3 , Xinfu Xu 1,2,3 , Rui Wang 1,2,3 , Jiaming Yin 1,2,3 , Huafang Wan 1,2,3 , Hai Du 1,2,3 , Wei Qian 1,2,3 , Yourong Chai 1,2,3 , Qingyuan Zhou 1,2,3 , Yajun He 1,2,3 , Silin Zhong 4 , Xiao Qiu 9 , Hao Yu 5,10 , Hon-Ming Lam 4 , Kun Lu 1,2,3 , Fuyou Fu 11 & Jiana Li 1,2,3 Yellow-seed trait is a desirable breeding characteristic of rapeseed (Brassica napus) that could greatly improve seed oil yield and quality. However, the underlying mechanisms controlling this phenotype in B. napus plants are difficult to discern because of their complexity. Here, we assemble high-quality genomes of yellow-seeded (GH06) and black-seeded (ZY821). Combining in- depth fine mapping of a quantitative trait locus (QTL) for seed color with other omics data reveal BnA09MYB47a, encoding an R2R3-MYB-type transcription factor, as the causal gene of a major QTL controlling the yellow-seed trait. Functional studies show that sequence variation of BnA09MYB47a underlies the functional divergence between the yellow- and black-seeded B. napus. The black-seed allele BnA09MYB47a ZY821 , but not the yellow-seed allele BnA09MYB47a GH06 , promotes flavonoid biosynthesis by directly activating the expression of BnTT18. Our discovery suggests a possible approach to breeding B. napus for improved commercial value and facilitates flavonoid biosynthesis studies in Brassica crops. Rapeseed (Brassica napus L.; AACC, 2n = 38), a relatively recent allo- tetraploid (<7500 years ago), originated from natural hybridization between its diploid progenitors B. rapa (AA, 2n = 20) and B. oleracea (CC, 2n = 18) 1 . B. napus is an important oilseed crop that supplies vegetable oil for human consumption and biodiesel production, and is a source of protein-rich feed for livestock 2 . The yellow-seed trait is particularly desirable in B. napus, as it is associated with higher oil and protein yields and improved seed quality 3,4 . Furthermore, this trait is associated with more transparent oil, lower fiber content, desirable aroma, and higher nutrients content 5,6 (Supplementary Fig. 1), and facilitates industrial processing, as yellow seeds lack the pigment deposits found in black/dark B. napus that interfere with processing. Although B. napus is the most important Brassica oilseed species, natural rapeseed germplasms with the yellow-seed trait have not been identified. Yellow-seeded B. napus lines were created by interspecific hybridization with related species, for example, between B. rapa and B. oleracea, Brassica juncea and Brassica carinata, B. napus, and Sinapis alba, and Brassica alboglabra and B. rapa 7–10 . However, as the yellow- seed trait is a quantitative trait that is also affected by environmental factors, obtaining stable commercial B. napus varieties with yellow Received: 25 May 2022 Accepted: 12 August 2023 Check for updates A full list of affiliations appears at the end of the paper. e-mail: drlukun@swu.edu.cn; fufuyou@gmail.com; ljn1950@swu.edu.cn Nature Communications | (2023)14:5194 1 1234567890():,; 1234567890():,;