Ehd3, encoding a plant homeodomain finger-containing protein, is a critical promoter of rice flowering.

Oryza sativa (rice) flowers in response to photoperiod, and is a facultative short-day (SD) plant. Under SD conditions, flowering is promoted through the activation of FT-like genes (rice florigens) by Heading date 1 (Hd1, a rice CONSTANS homolog) and Early heading date 1 (Ehd1, with no ortholog in the Arabidopsis genome). On the other hand, under long-day (LD) conditions, flowering is delayed by the repressive function of Hd1 on FT-like genes and by downregulation of Ehd1 by the flowering repressor Ghd7 - a unique pathway in rice. We report here that an early heading date 3 (ehd3) mutant flowered later than wild-type plants, particularly under LD conditions, regardless of the Hd1-deficient background. Map-based cloning revealed that Ehd3 encodes a nuclear protein that contains a putative transcriptional regulator with two plant homeodomain (PHD) finger motifs. To identify the role of Ehd3 within the gene regulatory network for rice flowering, we compared the transcript levels of genes related to rice flowering in wild-type plants and ehd3 mutants. Increased transcription of Ghd7 under LD conditions and reduced transcription of downstream Ehd1 and FT-like genes in the ehd3 mutants suggested that Ehd3 normally functions as an LD downregulator of Ghd7 in floral induction. Furthermore, Ehd3 ghd7 plants flowered earlier and show higher Ehd1 transcript levels than ehd3 ghd7 plants, suggesting a Ghd7-independent role of Ehd3 in the upregulation of Ehd1. Our results demonstrate that the PHD-finger gene Ehd3 acts as a promoter in the unique genetic pathway responsible for photoperiodic flowering in rice.

Genetic control of a transition from black to straw-white seed hull in rice domestication.

The genetic mechanism involved in a transition from the black-colored seed hull of the ancestral wild rice (Oryza rufipogon and Oryza nivara) to the straw-white seed hull of cultivated rice (Oryza sativa) during grain ripening remains unknown. We report that the black hull of O. rufipogon was controlled by the Black hull4 (Bh4) gene, which was fine-mapped to an 8.8-kb region on rice chromosome 4 using a cross between O. rufipogon W1943 (black hull) and O. sativa indica cv Guangluai 4 (straw-white hull). Bh4 encodes an amino acid transporter. A 22-bp deletion within exon 3 of the bh4 variant disrupted the Bh4 function, leading to the straw-white hull in cultivated rice. Transgenic study indicated that Bh4 could restore the black pigment on hulls in cv Guangluai 4 and Kasalath. Bh4 sequence alignment of all taxa with the outgroup Oryza barthii showed that the wild rice maintained comparable levels of nucleotide diversity that were about 70 times higher than those in the cultivated rice. The results from the maximum likelihood Hudson-Kreitman-Aguade test suggested that the significant reduction in nucleotide diversity in rice cultivars could be caused by artificial selection. We propose that the straw-white hull was selected as an important visual phenotype of nonshattered grains during rice domestication.

Identification and fine mapping of a major quantitative trait locus originating from wild rice, controlling cold tolerance at the seedling stage.

Cold tolerance at the seedling stage (CTSS) is an important trait affecting stable rice production in temperate climates and areas of high elevation. In this study, 331 single nucleotide polymorphism (SNP) markers were developed and used along with phenotypic evaluation to identify quantitative trait loci (QTLs) associated with CTSS from a mapping population of 184 F(2) plants derived from a cold tolerant wild rice, W1943 (Oryza rufipogon), and a sensitive indica cultivar, Guang-lu-ai 4 (GLA4). Three QTLs were detected on chromosomes 3, 10 and 11. A major locus, qCtss11 (QTL for cold tolerance at seedling stage), was located on the long arm of chromosome 11 explaining about 40% of the phenotypic variation. Introduction of the W1943 allele of qCtss11 to the GLA4 genetic background increased CTSS. Based on the phenotypic and genotypic assessment of advanced backcross progenies, qCtss11 was dissected as a single Mendelian factor. A high-resolution genetic map was constructed using 23 markers across the qCtss11 locus. As a result, qCtss11 was fine mapped to a 60-kb candidate region defined by marker AK24 and GP0030 on chromosome 11, in which six genes were annotated. Expression and resequence analyses of the six candidates supported the hypothesis that Os11g0615600 and/or Os11g0615900 are causal gene(s) of the CTSS.

Ehd2, a rice ortholog of the maize INDETERMINATE1 gene, promotes flowering by up-regulating Ehd1.

Recent research into the flowering of rice (Oryza sativa) has revealed both unique and conserved genetic pathways in the photoperiodic control of flowering compared with those in Arabidopsis (Arabidopsis thaliana). We discovered an early heading date2 (ehd2) mutant that shows extremely late flowering under both short- and long-day conditions in line with a background deficient in Heading date1 (Hd1), a rice CONSTANS ortholog that belongs to the conserved pathway. This phenotype in the ehd2 mutants suggests that Ehd2 is pivotal for the floral transition in rice. Map-based cloning revealed that Ehd2 encodes a putative transcription factor with zinc finger motifs orthologous to the INDETERMINATE1 (ID1) gene, which promotes flowering in maize (Zea mays). Ehd2 mRNA in rice tissues accumulated most abundantly in developing leaves, but was present at very low levels around the shoot apex and in roots, patterns that are similar to those of ID1. To assign the position of Ehd2 within the flowering pathway of rice, we compared transcript levels of previously isolated flowering-time genes, such as Ehd1, a member of the unique pathway, Hd3a, and Rice FT-like1 (RFT1; rice florigens), between the wild-type plants and the ehd2 mutants. Severely reduced expression of these genes in ehd2 under both short- and long-day conditions suggests that Ehd2 acts as a flowering promoter mainly by up-regulating Ehd1 and by up-regulating the downstream Hd3a and RFT1 genes in the unique genetic network of photoperiodic flowering in rice.

Collection and comparative analysis of 1888 full-length cDNAs from wild rice Oryza rufipogon Griff. W1943.

A huge amount of cDNA and EST resources have been developed for cultivated rice species Oryza sativa; however, only few cDNA resources are available for wild rice species. In this study, we isolated and completely sequenced 1888 putative full-length cDNA (FLcDNA) clones from wild rice Oryza rufipogon Griff. W1943 for comparative analysis between wild and cultivated rice species. Two cDNA libraries were constructed from 3-week-old leaf samples under either normal or cold-treated conditions. Homology searching of these cDNA sequences revealed that >96.8% of the wild rice cDNAs were matched to the cultivated rice O. sativa ssp. japonica cv. Nipponbare genome sequence. However, <22% of them were fully matched to the cv. Nipponbare genome sequence. The comparative analysis showed that O. rufipogon W1943 had greater similarity to O. sativa ssp. japonica than to ssp. indica cultivars. In addition, 17 novel rice cDNAs were identified, and 41 putative tissue-specific expression genes were defined through searching the rice massively parallel signature-sequencing database. In conclusion, these FLcDNA clones are a resource for further function verification and could be broadly utilized in rice biological studies.

Isolation of a regulatory gene of anthocyanin biosynthesis in tuberous roots of purple-fleshed sweet potato.

Many transcriptional factors harboring the R2R3-MYB domain, basic helix-loop-helix domain, or WD40 repeats have been identified in various plant species as regulators of flavonoid biosynthesis in flowers, seeds, and fruits. However, the regulatory elements of flavonoid biosynthesis in underground organs have not yet been elucidated. We isolated the novel MYB genes IbMYB1 and IbMYB2s from purple-fleshed sweet potato (Ipomoea batatas L. Lam. cv Ayamurasaki). IbMYB1 was predominantly expressed in the purple flesh of tuberous roots but was not detected (or only scarcely) in other anthocyanin-containing tissues such as nontuberous roots, stems, leaves, or flowers. IbMYB1 was also expressed in the tuberous roots of other purple-fleshed cultivars but not in those of orange-, yellow-, or white-fleshed cultivars. Although the orange- or yellow-fleshed cultivars contained anthocyanins in the skins of their tuberous roots, we could not detect IbMYB1 transcripts in these tissues. These results suggest that IbMYB1 controls anthocyanin biosynthesis specifically in the flesh of tuberous roots. The results of transient and stable transformation experiments indicated that expression of IbMYB1 alone was sufficient for induction of all structural anthocyanin genes and anthocyanin accumulation in the flesh of tuberous roots, as well as in heterologous tissues or heterologous plant species.

Genome-wide searching of single-nucleotide polymorphisms among eight distantly and closely related rice cultivars (Oryza sativa L.) and a wild accession (Oryza rufipogon Griff.).

We searched the genomes of eight rice cultivars (Oryza sativa L. ssp. japonica and ssp. indica) and a wild rice accession (Oryza rufipogon Griffith) for nucleotide polymorphisms, and identified 7805 polymorphic loci, including single-nucleotide polymorphisms (SNPs) and insertions/deletions (InDels), in predicted intergenic regions. Polymorphisms are useful as DNA markers for genetic analysis or positional cloning with segregating populations of crosses. Pairwise comparison between cultivars and a neighbor-joining tree calculated from SNPs agreed very well with relationships between rice strains predicted from pedigree data or calculated with other DNA markers such as p-SINE1 and simple sequence repeats (SSRs), suggesting that whole-genome SNP information can be used for analysis of evolutionary relationships. Using multiple SNPs to identify alleles, we drew a map to illustrate the alleles shared among the eight cultivars and the accession. The map revealed that most of the genome is mono- or di-allelic among japonica cultivars, whereas alleles well conserved among modern japonica paddy rice cultivars were often shared with indica cultivars or wild rice, suggesting that the genome structure of modern cultivars is composed of chromosomal segments from various genetic backgrounds. Use of allele-sharing analysis and association analysis were also tested and are discussed.

Search for and analysis of single nucleotide polymorphisms (SNPs) in rice (Oryza sativa, Oryza rufipogon) and establishment of SNP markers.

We searched for SNPs in 417 regions distributed throughout the genome of three Oryza sativa ssp. japonica cultivars, two indica cultivars, and a wild rice (O. rufipogon). We found 2800 SNPs in approximately 250,000 aligned bases for an average of one SNP every 89 bp, or one SNP every 232 bp between two randomly selected strains. Graphic representation of the frequency of SNPs along each chromosome showed uneven distribution of polymorphism-rich and -poor regions, but little obvious association with the centromere or telomere. The 94 SNPs that we found between the closely related cultivars 'Nipponbare' and 'Koshihikari' can be converted into molecular markers. Our establishment of 213 co-dominant SNP markers distributed throughout the genome illustrates the immense potential of SNPs as molecular markers not only for genome research, but also for molecular breeding of rice.

Positional cloning of rice semidwarfing gene, sd-1: rice "green revolution gene" encodes a mutant enzyme involved in gibberellin synthesis.

A rice semidwarfing gene, sd-1, known as the "green revolution gene," was isolated by positional cloning and revealed to encode gibberellin 20-oxidase, the key enzyme in the gibberellin biosynthesis pathway. Analysis of 3477 segregants using several PCR-based marker technologies, including cleaved amplified polymorphic sequence, derived-CAPS, and single nucleotide polymorphisms revealed 1 ORF in a 6-kb candidate interval. Normal-type rice cultivars have an identical sequence in this region, consisting of 3 exons (558, 318, and 291 bp) and 2 introns (105 and 1471 bp). Dee-Geo-Woo-Gen-type sd-1 mutants have a 383-bp deletion from the genome (278-bp deletion from the expressed sequence), from the middle of exon 1 to upstream of exon 2, including a 105-bp intron, resulting in a frame-shift that produces a termination codon after the deletion site. The radiation-induced sd-1 mutant Calrose 76 has a 1-bp substitution in exon 2, causing an amino acid substitution (Leu [CTC] to Phe [TTC]). Expression analysis suggests the existence of at least one more locus of gibberellin 20-oxidase which may prevent severe dwarfism from developing in sd-1 mutants.