# Combined SMRT and Illumina RNA Sequencing Reveals the Alternative Splicing-Mediated Regulation of Anthocyanin Accumulation in Potato (Solanum tuberosum L.)

## TL;DR

This study shows how alternative splicing of a specific gene affects anthocyanin production in potatoes.

## Contribution

The study reveals how alternative splicing of the StB3-like gene regulates anthocyanin accumulation in potatoes.

## Key findings

- Alternative splicing events were identified in anthocyanin-related genes and transcription factors in potato.
- StB3-like gene produces three spliced variants, two of which enhance anthocyanin accumulation when co-expressed with StAN1.
- The regulatory role of alternative splicing in anthocyanin biosynthesis is confirmed in a StAN1-dependent manner.

## Abstract

Anthocyanins, as natural pigments with high nutritional value, have been extensively studied in terms of their biosynthetic pathways. However, the specific impact of alternative splicing (AS) on anthocyanin biosynthesis in potatoes and its potential regulatory mechanisms remain unclear. This study analyzed full-length transcriptome (SMRT) and Illumina RNA-seq datasets from three developmental stages of multiple potato (Solanum tuberosum L.) cultivars. After RNA-seq correction, numerous AS events were identified in both white and colored samples. To dissect the regulatory role of AS in anthocyanin biosynthesis, we further analyzed the AS patterns of anthocyanin biosynthesis-related structural genes and transcription factors (TFs). Through this targeted analysis, we found that a subset of these structural genes and TFs exhibited AS, generating functionally diverse transcript variants. Among these, the candidate gene StB3-like (Soltu.DM.04G010530), a B3 family TF associated with anthocyanin synthesis, was selected for preliminary functional validation. This gene produced three alternatively spliced transcripts (StB3-like-1, -2, -3). Transient co-expression with StAN1 (Soltu.DM.10G020850) in tobacco showed that StB3-like-2 and StB3-like-3 significantly increased anthocyanin accumulation, whereas StB3-like-1 had no effect. These results confirm that different transcripts produced by the alternative splicing of the same gene differentially regulate anthocyanin accumulation in a StAN1-dependent manner, laying the groundwork for further investigation into the role of alternative splicing in potato anthocyanin accumulation.