Whole-genome resequencing and transcriptomic analysis of genes regulating anthocyanin biosynthesis in black rice plants

Anthocyanins are involved in many diverse functions in rice, but their benefits have yet to be clearly demonstrated. Our objective in this study was to identify anthocyanin-related genes in black rice plants. We identified anthocyanin-related genes in black rice plants using a combination of whole-genome resequencing, RNA-sequencing (RNA-seq), microarray experiments, and reverse-transcriptase polymerase chain reaction (RT-PCR). Using multi-layer screening from 30 rice accessions, we identified 172,922 single-nucleotide polymorphisms (SNPs) and 1276 differentially expressed genes that appear to be related to anthocyanin biosynthesis. We identified 18 putative genes from 172,922 SNPs using intensive selective sweeps. The 18 candidate genes identified from SNPs were not significantly correlated with the RNA-seq expression pattern or other well-known anthocyanin biosynthesis/metabolism genes. We also identified nine putative genes from 1276 differentially expressed genes using RNA-seq transcriptome analysis. In addition, we identified four phylogenetic groups from these nine candidate genes and 51 pathway-network genes. Finally, we verified nine anthocyanin-related genes using a newly designed microarray and semi-quantitative RT-PCR. We suggest that these nine identified genes appear to be related to the regulation of anthocyanin biosynthesis and/or metabolism.