Abstract: 【Objective】To identify the key genes controlling anthocyanin synthesis and accumulation, to uncover changes in anthocyanin content of the seed coat during seed development, and the primary anthocyanin components responsible for the red seed coat of Taixingaijiaohong (TXAJH); and to lay the groundwork for a thorough understanding of the regulatory mechanism of red seed coat formation.【Method】Using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-ESI-MS/MS), the anthocyanin composition and concentration of the yellow seed coat of soybean Suinong 14 (SN14) and the red seed coat of soybean TXAJH at various developmental stages were identified. The potential areas of red testa-related genes were first identified using bulked segregant analysis (BSA) on the recombinant inbred lines (RILs) made by crossing SN14 and TXAJH.  Based on this discovery, we performed marker linkage analysis to restrict the candidate intervals and predict the candidate genes, and qRT-PCR to confirm the expression of the anticipated candidate genes.【Result】When seed coats from the four developmental phases of SN14 and TXAJH were analyzed, a total of 12 anthocyanins were discovered. Cluster analysis of total anthocyanins revealed substantial changes in the seed coat's anthocyanin composition between TXAJH and SN14 as well as between TXAJH before and after color development. The anthocyanin content of the SN14 seed coat gradually decreased as the seed developed, whereas the TXAJH seed coat's content increased quickly and remained stable. After the development of the seed coat's color, the anthocyanin contents of SN14 and TXAJH showed highly significant differences, and at the mature stage, the TXAJH seed coat's anthocyanin content was more than 200 times that of SN14. The crimson coloring of the TXAJH seed coat was largely due to cyanidin-3-O-glucoside (Cy-3-glu), peonidin-3-O-glucoside (Pn-3-glu), and petunidin-3-O-glucoside (Pt-3-glu). The candidate interval for the red seed coat gene on chromosome 8 was discovered at 8.66 Mb by BSA-seq association analysis. 27 polymorphic markers were used in the marker linkage analysis, which produced 10 haplotypes and reduced the candidate interval to 702 kb. Nonsynonymous variations in 37 genes between the parents were found during this interval, These include the genes for encode the anthocyanin reductase 1 (Glyma.08g062000), the bHLH transcription factor (Glyma.08g061300 and Glyma.08g063900), and the MYB transcript factor (Glyma.08g059900). These genes may be involved in regulating the biosynthesis of anthocyanins, and anthocyanin reductase 1 can convert anthocyanins to proanthocyanidins (PA). The results of gene expression analysis revealed that candidate genes and genes related to the anthocyanin biosynthesis pathway had comparable expression patterns in SN14 and TXAJH, and both were expressed at lower levels in SN14 and at higher levels in TXAJH. It was discovered that there was a significant link between the principal constituents of seed coat anthocyanins and the level of candidate gene expression.【Conclusion】The anthocyanin makeup of SN14 and TXAJH's seed coats differed, and Cy-3-glu, Pn-3-glu, and Pt-3-glu may be to blame for the TXAJH's seed coat's red hue. According to predictions, Glyma.08g059900, Glyma.08g061300, Glyma.08g062000, and Glyma.08g063900 will likely be a candidate gene for the red seed coat, in which Glyma.08g059900, Glyma.08g061300, and Glyma.08g063900 may control a number of anthocyanin biosynthesis pathway genes.

Key words: seed coat color, anthocyanin, BSA-seq, gene mapping, transcription factors