Transcriptome and Association Analyses of Candidate Genes Associated with Black Rice Phenotype

Red rice accumulates tannins in pericarp/testa. The loss-of-function of two genes, Rd and Rc, changed the color to white during rice domestication. Black rice is likely to be related to the red rice but accumulates anthocyanins. Molecular genetic background of black rice, however, is not well identified. In this study, we performed transcriptome and association analyses to elucidate molecular mechanisms of black rice. We used a new black rice cultivar, Toyamakuro75, contains three introgressed genomic segments from a traditional black rice cultivar, Kouketsumochi, to a white rice japonica cultivar Koshihikari background. Transcriptome analysis using pericarp/testa in early stage of developing seeds revealed that expression of LDOX and a bHLH transcription factor gene significantly increased specifically in black rice. Since this bHLH gene was located on the integration of chromosome 4 we consider that this gene is one of the genes make black rice and named kala4. Association analysis using genome-wide hundreds of single nucleotide polymorphisms (SNPs) of 70 accessions (i.e. Toyamakuro75, 14 aus, 15 indica, 12 tropical japonica, 5 template japonica, 23 black rice accessions), demonstrated that the genomic regions of including kala4 in chromosome 4 have similar SNP patterns in all black rice accessions and common to tropical japonica rice accessions as well as black rice accessions. These results strongly suggested that up-regulation of LDOX in pericarp/testa due to a gain-of-function event in kala4 played a pivotal role for the creation of black rice, and the origin of the functional allele of kala4 occurred in tropical japonica rice.