Caught red-handed: Rc encodes a basic helix-loop-helix protein conditioning red pericarp in rice

Abstract

Rc is a domestication-related gene required for red pericarp in rice (Oryza sativa). The red grain color is ubiquitous among the wild ancestors of O. sativa, in which it is closely associated with seed shattering and dormancy. Rc encodes a basic helix-loop-helix (bHLH) protein that was fine-mapped to an 18.5-kb region on rice chromosome 7 using a cross between Oryza rufipogon (red pericarp) and O. sativa cv Jefferson (white pericarp). Sequencing of the alleles from both mapping parents as well as from two independent genetic stocks of Rc revealed that the dominant red allele differed from the recessive white allele by a 14-bp deletion within exon 6 that knocked out the bHLH domain of the protein. A premature stop codon was identified in the second mutant stock that had a light red pericarp. RT-PCR experiments confirmed that the Rc gene was expressed in both red- and white-grained rice but that a shortened transcript was present in white varieties. Phylogenetic analysis, supported by comparative mapping in rice and maize (Zea mays), showed that Rc, a positive regulator of proanthocyanidin, is orthologous with INTENSIFIER1, a negative regulator of anthocyanin production in maize, and is not in the same clade as rice bHLH anthocyanin regulators.

Figure 1.

Phenotypes and Fine-Mapping of Rc. (A) Rc allele phenotypes. Top row, left to right: seeds from cv Jefferson and O. rufipogon; bottom row, left to right: seeds from Surjamkuhi and H75. (B) Fine mapping. (i) QTL log of the odds (LOD) plot (y axis) and marker order (x axis). The black oval indicates the position of the centromere; Rc indicates the map position of Rc. (ii) Progress made narrowing the QTL by generation. (iii) Scheme of genotypes for three recombinant classes. Black bars represent DNA from the O. rufipogon parent, white bars represent DNA from the cv Jefferson parent, and gray bars represent an interval containing a break point between cv Jefferson and O. rufipogon DNA. White or red indicates the color of the pericarp conditioned by each class. The ruler shows where the markers and break points are positioned along the psuedomolecule for chromosome 7, and the numbers indicate megabase pairs. Markers that bracket or are included in the 18.5-kb target region are shown in red. The Institute for Genomic Research (TIGR) gene models are shown below the ruler, and numbers indicate the last digits of the gene identifiers, where all begin with LOC_Os07g11_. Transposable elements (TEs) are shown in green. Alternative splice variants are indicated by gene names ending with .1 and .2. The last four genes are staggered for clarity of presentation. (iv) The bHLH gene model, predicted from the O. rufipogon sequence, is enlarged to show the location of intragenic markers (indicated with arrows). The bHLH domain is indicated by black boxes at the end of exon 6 and the beginning of exon 7.

Figure 2.

Physical/Genetic Distance around the Rc Locus. Graph showing recombination rates across the 410-kb region examined in the BC2F6 generation of fine-mapping. The x axis is positioned along the psuedomolecule, and the y axis shows the number of recombination events per 100 plants. Physical/genetic distance is given in kb/cM above each interval. TIGR gene models are shown above the marker designations, with TEs in gray. Gene models are ordered as in Figure 1. Intervals indicated by asterisks have recombination rates significantly different from the average at the 5% level after Bonferroni correction; those indicated by double asterisks are significant at 0.1%.

Figure 3.

Expression Analysis of Rc. (A) Transcripts of Rc and actin detected by RT-PCR in leaves, panicles before fertilization, pericarp from grains in the milk or dough stage of filling, and pericarp from mature seeds from both cv Jefferson (J; white seeds) and O. rufipogon (R; red seeds). (B) Short transcripts of Rc detected by RT-PCR from the milk and dough stages of filling from cv Jefferson (J) and O. rufipogon (R) run out on polyacrylamide. (C) Gene models from TIGR version 3 using the cv Nipponbare sequence and FgenesH prediction using the O. rufipogon sequence. The regions of mRNA amplified are indicated by horizontal lines, the longer one used in (A) and the shorter one used in (B). The arrow indicates the location of the 14-bp deletion.

Figure 4.

Coding Sequence Differences between Rc Alleles. (A) Graphic representation of coding sequence differences in LOC_Os07g11020.1 between several pairs of genotypes. The mRNA is represented by rectangles, and the beginning and end of the exons are indicated by vertical lines. Sequence changes are annotated as follows: closed circles, nonsynonymous substitution; lines, synonymous substitution; closed triangles, in-frame indel; open triangles, frame-shift indel; point-up triangles, deletion from O. rufipogon or H75; point-down triangle, insertion into O. rufipogon. (B) Table showing polymorphic sites within the coding region of LOC_Os07g11020.1 for several different alleles of Rc. Functional nucleotide polymorphisms are highlighted in gray.

Figure 5.

Phylogenetic Analysis of Rc and Other bHLH Proteins. Topology derived from Bayesian analysis using the GTR+G model after 5 million generations (saving 5001 trees), discarding the first 100 trees as burnin. It was compared with a single most parsimonious tree of 3629 steps found after a heuristic search using 100 random addition sequence replicates holding a maximum of 100,000 trees (maximum not reached). The parsimony tree differed from the Bayesian analysis. The parsimony-preferred positions of taxa are shown by arrows where they connect to other branches; for example, in the parsimony tree, JAF13 and Delila are grouped together to the exclusion of MYC1. Parsimony bootstrap percentages (from 1000 pseudoreplicates) are shown above the branches, whereas posterior probabilities from the HKY model and the GTR+G model are shown, left and right, below the branches (after 100 burnin trees of 5001 trees were discarded). Thicker branches have >80% bootstrap and >0.95 posterior probability in each analysis. The topology should be considered unrooted. Genes that have been shown to function as regulators of the anthocyanin or proanthocyanidin pathways are named; additional sequences were retrieved from BLAST searches using Rc as the query and align across more than just the bHLH domain of ∼60 amino acids. The pound sign indicates a known negative regulator.