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. 2015 Sep;27(9):2401-14.
doi: 10.1105/tpc.15.00310. Epub 2015 Sep 11.

The Birth of a Black Rice Gene and Its Local Spread by Introgression

Tetsuo Oikawa  1 , Hiroaki Maeda  2 , Taichi Oguchi  1 , Takuya Yamaguchi  2 , Noriko Tanabe  1 , Kaworu Ebana  3 , Masahiro Yano  3 , Takeshi Ebitani  2 , Takeshi Izawa  4
Affiliations

The Birth of a Black Rice Gene and Its Local Spread by Introgression

Tetsuo Oikawa et al. Plant Cell. 2015 Sep.

Abstract

The origin and spread of novel agronomic traits during crop domestication are complex events in plant evolution. Wild rice (Oryza rufipogon) has red grains due to the accumulation of proanthocyanidins, whereas most cultivated rice (Oryza sativa) varieties have white grains induced by a defective allele in the Rc basic helix-loop-helix (bHLH) gene. Although the events surrounding the origin and spread of black rice traits remain unknown, varieties with black grains due to anthocyanin accumulation are distributed in various locations throughout Asia. Here, we show that the black grain trait originated from ectopic expression of the Kala4 bHLH gene due to rearrangement in the promoter region. Both the Rc and Kala4 genes activate upstream flavonol biosynthesis genes, such as chalcone synthase and dihydroflavonol-4-reductase, and downstream genes, such as leucoanthocyanidin reductase and leucoanthocyanidin dioxygenase, to produce the respective specific pigments. Genome analysis of 21 black rice varieties as well as red- and white-grained landraces demonstrated that black rice arose in tropical japonica and its subsequent spread to the indica subspecies can be attributed to the causal alleles of Kala4. The relatively small size of genomic fragments of tropical japonica origin in some indica varieties indicates that refined introgression must have occurred by natural crossbreeding in the course of evolution of the black trait in rice.

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Figures

Figure 1.
Figure 1.
Identification of Kala4. (A) Fine mapping. The Kala4 locus was previously localized to chromosome 4 between two SSR markers, RM3092 and RM2441. This region contains three bHLH protein-encoding genes. Fine mapping narrowed the Kala4 locus to an ∼25.6-kb region between two SNP markers (+23843 and −1707). Six SNPs and one in/del between ‘Koshihikari’ and ‘Hong Xie Nuo’ in this region were mapped onto the reference ‘Nipponbare’ genome. The number in parentheses indicates the number of recombinant plants. (B) Grain color of transgenic plants overexpressing Os04g0557500. The transgenic lines (O/E#1 and O/E#5) show black seed color in contrast to the white seeds of the parental ‘BBA-type NIL.’ (C) Quantification of mRNAs by RT-qPCR analysis. Total RNAs were extracted from leaves of three independent hygromycin-resistant plants of the T1 generation for O/E. The y axis is shown in a logarithmic scale.
Figure 2.
Figure 2.
Analysis of DNA Methylation in Kala4. (A) McrBC-CGH analysis. The DNA methylation patterns in pericarps of ‘Koshihikari’ (upper panel) and ‘Hong Xie Nuo’ (lower panel) differ around the 85-kb region at the Kala4 locus. The relative positions of the three bHLH genes are shown below the lower panel. Red box indicates a region where difference of DNA methylation pattern between ‘Koshihikari’ and ‘Hong Xie Nuo’ was observed. (B) Analysis of the intron 2 region of Os04g0557500. The DNA methylation patterns upstream of intron 2 in ‘Koshihikari’ (upper panel) and ‘Hong Xie Nuo’ (lower panel) were analyzed by bisulfite sequencing of the region between markers +507 and +2967. (C) Diagram of the DNA methylation-variable region in the 25.6-kb candidate region. Black boxes and lines indicate exons and introns of Os04g0557500. The LINE1 retrotransposon insertion is located in intron 2 of Os04g0557500 and the DNA methylation-variable region is located in 5′-upstream of intron 2 (shown as red bidirectional arrow). The numbers indicate the position from the start site of Kala4 transcription.
Figure 3.
Figure 3.
Relationship of the Os04g0557500 Promoter Structure and Black Rice Trait. (A) Structures of the Os04g0557500 promoter regions in ‘Koshihikari’ and ‘Hong Xie Nuo.’ Upper bar indicates genomic structure of Os04g0557500 gene in ‘Koshihikari’. Lower bar indicates that in ‘Hong Xie Nuo.’ Blue lines and boxes indicate the ORF region. The promoter region in ‘Hong Xie Nuo’ possesses a duplicated region of Os04g0557500 (4679 bp) including exon 1, intron 1, exon 2, and the part of intron 2 (red boxes and lines). In addition to this duplication, the Os04g0557500 promoter region in ‘Hong Xie Nuo’ has an ∼11.02-kb insertion of a genome fragment originating from ∼83 kb upstream of Os04g0557500 (orange bar). Bar = 5.0 kb. (B) Promoter sequence constructs of Os04g0557500. Schematic diagram of the three types of promoter:Os04g0557500 cDNA constructs with the white rice-type promoter sequence from ‘Koshihikari’ (green box), black rice-type promoter sequence from ‘Black rice NIL’ (orange box), and the 1.4-kb common promoter sequence shared between ‘Koshihikari’ and ‘Hong Xie Nuo’ (black box). (C) Grain color of transgenic plants harboring the three promoter:Os04g0557500 constructs. Grains of three representative lines for each promoter:Os04g0557500 construct (T1) are shown. ‘Koshihikari,’ ‘BBA-type NIL,’ and ‘Black rice NIL’ are shown as white and black color controls.
Figure 4.
Figure 4.
Characterization of Black Rice Suppressor Mutants. (A) Grain color of the suppressor mutants. The seven suppressor mutants showed variation in grain color from the white grain of ‘Koshihikari’ and the black grain of ‘Black rice NIL.’ (B) Expression of Os04g0557500 mRNA in the suppressor mutants. Total RNAs were extracted from three to five 10-d-old seedlings. Three independent total RNAs extractions and RT-qPCRs were performed. The y axis is shown in a logarithmic scale. Means + se of three independent experiments are shown. (C) McrBC-DNA gel blot hybridization. Genomic DNAs were extracted from 10 to 15 12-d-old seedlings. The extracted DNAs were digested with NdeI with (+) or without (−) subsequent McrBC digestion. Hybridization was performed with the exon1N probe. In the case of digestion with McrBC, the distinct band sizes were detected according to the DNA methylation status of intron 2 in Os04g0557500
Figure 5.
Figure 5.
Structural Variations of the Kala4 Promoters in 21 Black Rice Varieties. The Kala4 promoters of 21 black rice varieties were classified into three types based on the length of inserted fragments. Type I promoters have the longest insertion (11.02 kb) with the same size as ‘Hong Xie Nuo.’ Type II promoters have a deletion of ∼2.7 kb at the 5′ junction. Type III promoters have the 2.7-kb deletion at the 5′ junction and a 2.4-kb deletion at the 3′ junction. The original Kala4 ORF sequence (blue region), insertion from ∼83 kb upstream of Kala4 (orange), and the duplicated segment of Kala4 (red) for each type are shown.
Figure 6.
Figure 6.
Clustering Analysis of White, Red, and Black Rice Landraces. Golden Gate assay results revealed clustering of the white, red, and black rice landraces into four subspecies. The heat map shows genome similarity between varieties based on Pearson’s correlation coefficient of genetic distance of the genome-wide Golden Gate genotyping data. The 21 black rice landraces (Supplemental Table 5) were classified into subspecies indica, tropical japonica, and temperate japonica. Yellow and blue colors indicate higher and lower correlation coefficients, respectively. The black rice varieties are highlighted in gray.
Figure 7.
Figure 7.
Haplotype Analysis of Black Rice Varieties. (A) Introgression of the japonica allele of Kala4 into indica subspecies. Six sequence markers were constructed at ∼0.2-Mb intervals in the 28.0 to 29.0 Mb region of chromosome 4. Seven indica black rice lines were sequenced at these markers with ‘Nipponbare’ (japonica), ‘Koshihikari’ (japonica), and ‘93-11’ (indica) as reference sequences. Yellow and blue bars indicate japonica and indica type genotypes, respectively. The marker of 28.40 Mb was the closest to Kala4. (B) DNA polymorphisms between temperate japonica and tropical japonica in the proximal region of Kala4. Four SNP markers were examined in the two white temperate japonica, seven black indica, five black temperate japonica, and six white (or red) tropical japonica varieties.
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