Unveiling the Complexity of Red Clover (<i>Trifolium pratense</i> L.) Transcriptome and Transcriptional Regulation of Isoflavonoid Biosynthesis Using Integrated Long- and Short-Read RNAseq

Red clover (Trifolium pratense L.) is used as forage and contains a high level of isoflavonoids. Although isoflavonoids in red clover were discovered a long time ago, the transcriptional regulation of isoflavonoid biosynthesis is virtually unknown because of the lack of accurate and comprehensive characterization of the transcriptome. Here, we used a combination of long-read (PacBio Iso-Seq) and short-read (Illumina) RNAseq sequencing to develop a more comprehensive full-length transcriptome in four tissues (root, stem, leaf, and flower) and to identify transcription factors possibly involved in isoflavonoid biosynthesis in red clover. Overall, we obtained 50,922 isoforms, including 19,860 known genes and 2817 novel isoforms based on the annotation of RefGen Tp_v2.0. We also found 1843 long non-coding RNAs, 1625 fusion genes, and 34,612 alternatively spliced events, with some transcript isoforms validated experimentally. A total of 16,734 differentially expressed genes were identified in the four tissues, including 43 isoflavonoid-biosynthesis-related genes, such as stem-specific expressed TpPAL, TpC4H, and Tp4CL and root-specific expressed TpCHS, TpCHI1, and TpIFS. Further, weighted gene co-expression network analysis and a targeted compound assay were combined to investigate the association between the isoflavonoid content and the transcription factors expression in the four tissues. Twelve transcription factors were identified as key genes for isoflavonoid biosynthesis. Among these transcription factors, the overexpression of TpMYB30 or TpRSM1-2 significantly increased the isoflavonoid content in tobacco. In particular, the glycitin was increased by 50–100 times in the plants overexpressing TpRSM1-2, in comparison to that in the WT plants. Our study provides a comprehensive and accurate annotation of the red clover transcriptome and candidate genes to improve isoflavonoid biosynthesis and accelerate research into molecular breeding in red clover or other crops