Hiroyuki Kanamori

The genome sequence and structure of rice chromosome 1

The rice species Oryza sativa is considered to be a model plant because of its small genome size, extensive genetic map, relative ease of transformation and synteny with other cereal crops. Here we report the essentially complete sequence of chromosome 1, the longest chromosome in the rice genome. We summarize characteristics of the chromosome structure and the biological insight gained from the sequence. The analysis of 43.3 megabases (Mb) of non-overlapping sequence reveals 6,756 protein coding genes, of which 3,161 show homology to proteins of Arabidopsis thaliana, another model plant. About 30% (2,073) of the genes have been functionally categorized. Rice chromosome 1 is (G + C)-rich, especially in its coding regions, and is characterized by several gene families that are dispersed or arranged in tandem repeats. Comparison with a draft sequence indicates the importance of a high-quality finished sequence.

Improvement of the Oryza sativa Nipponbare reference genome using next generation sequence and optical map data

BackgroundRice research has been enabled by access to the high quality reference genome sequence generated in 2005 by the International Rice Genome Sequencing Project (IRGSP). To further facilitate genomic-enabled research, we have updated and validated the genome assembly and sequence for the Nipponbare cultivar of Oryza sativa (japonica group).ResultsThe Nipponbare genome assembly was updated by revising and validating the minimal tiling path of clones with the optical map for rice. Sequencing errors in the revised genome assembly were identified by re-sequencing the genome of two different Nipponbare individuals using the Illumina Genome Analyzer II/IIx platform. A total of 4,886 sequencing errors were identified in 321 Mb of the assembled genome indicating an error rate in the original IRGSP assembly of only 0.15 per 10,000 nucleotides. A small number (five) of insertions/deletions were identified using longer reads generated using the Roche 454 pyrosequencing platform. As the re-sequencing data were generated from two different individuals, we were able to identify a number of allelic differences between the original individual used in the IRGSP effort and the two individuals used in the re-sequencing effort. The revised assembly, termed Os-Nipponbare-Reference-IRGSP-1.0, is now being used in updated releases of the Rice Annotation Project and the Michigan State University Rice Genome Annotation Project, thereby providing a unified set of pseudomolecules for the rice community.ConclusionsA revised, error-corrected, and validated assembly of the Nipponbare cultivar of rice was generated using optical map data, re-sequencing data, and manual curation that will facilitate on-going and future research in rice. Detection of polymorphisms between three different Nipponbare individuals highlights that allelic differences between individuals should be considered in diversity studies.

Six-rowed barley originated from a mutation in a homeodomain-leucine zipper I-class homeobox gene

Increased seed production has been a common goal during the domestication of cereal crops, and early cultivators of barley (Hordeum vulgare ssp. vulgare) selected a phenotype with a six-rowed spike that stably produced three times the usual grain number. This improved yield established barley as a founder crop for the Near Eastern Neolithic civilization. The barley spike has one central and two lateral spikelets at each rachis node. The wild-type progenitor (H. vulgare ssp. spontaneum) has a two-rowed phenotype, with additional, strictly rudimentary, lateral rows; this natural adaptation is advantageous for seed dispersal after shattering. Until recently, the origin of the six-rowed phenotype remained unknown. In the present study, we isolated vrs1 (six-rowed spike 1), the gene responsible for the six-rowed spike in barley, by means of positional cloning. The wild-type Vrs1 allele (for two-rowed barley) encodes a transcription factor that includes a homeodomain with a closely linked leucine zipper motif. Expression of Vrs1 was strictly localized in the lateral-spikelet primordia of immature spikes, suggesting that the VRS1 protein suppresses development of the lateral rows. Loss of function of Vrs1 resulted in complete conversion of the rudimentary lateral spikelets in two-rowed barley into fully developed fertile spikelets in the six-rowed phenotype. Phylogenetic analysis demonstrated that the six-rowed phenotype originated repeatedly, at different times and in different regions, through independent mutations of Vrs1.

Two Adjacent Nucleotide-Binding Site–Leucine-Rich Repeat Class Genes Are Required to Confer Pikm-Specific Rice Blast Resistance

The rice blast resistance gene Pikm was cloned by a map-based cloning strategy. High-resolution genetic mapping and sequencing of the gene region in the Pikm-containing cultivar Tsuyuake narrowed down the candidate region to a 131-kb genomic interval. Sequence analysis predicted two adjacently arranged resistance-like genes, Pikm1-TS and Pikm2-TS, within this candidate region. These genes encoded proteins with a nucleotide-binding site (NBS) and leucine-rich repeats (LRRs) and were considered the most probable candidates for Pikm. However, genetic complementation analysis of transgenic lines individually carrying these two genes negated the possibility that either Pikm1-TS or Pikm2-TS alone was Pikm. Instead, it was revealed that transgenic lines carrying both of these genes expressed blast resistance. The results of the complementation analysis and an evaluation of the resistance specificity of the transgenic lines to blast isolates demonstrated that Pikm-specific resistance is conferred by cooperation of Pikm1-TS and Pikm2-TS. Although these two genes are not homologous with each other, they both contain all the conserved motifs necessary for an NBS–LRR class gene to function independently as a resistance gene.

Evolutionary history of GS3, a gene conferring grain length in rice

Unlike maize and wheat, where artificial selection is associated with an almost uniform increase in seed or grain size, domesticated rice exhibits dramatic phenotypic diversity for grain size and shape. Here we clone and characterize GS3, an evolutionarily important gene controlling grain size in rice. We show that GS3 is highly expressed in young panicles in both short- and long-grained varieties but is not expressed in leaves or panicles after flowering, and we use genetic transformation to demonstrate that the dominant allele for short grain complements the long-grain phenotype. An association study revealed that a C to A mutation in the second exon of GS3 (A allele) was associated with enhanced grain length in Oryza sativa but was absent from other Oryza species. Linkage disequilibrium (LD) was elevated and there was a 95.7% reduction in nucleotide diversity (θπ) across the gene in accessions carrying the A allele, suggesting positive selection for long grain. Haplotype analysis traced the origin of the long-grain allele to a Japonica-like ancestor and demonstrated introgression into the Indica gene pool. This study indicates a critical role for GS3 in defining the seed morphologies of modern subpopulations of O. sativa and enhances the potential for genetic manipulation of grain size in rice.

Barley grain with adhering hulls is controlled by an ERF family transcription factor gene regulating a lipid biosynthesis pathway

In contrast to other cereals, typical barley cultivars have caryopses with adhering hulls at maturity, known as covered (hulled) barley. However, a few barley cultivars are a free-threshing variant called naked (hulless) barley. The covered/naked caryopsis is controlled by a single locus (nud) on chromosome arm 7HL. On the basis of positional cloning, we concluded that an ethylene response factor (ERF) family transcription factor gene controls the covered/naked caryopsis phenotype. This conclusion was validated by (i) fixation of the 17-kb deletion harboring the ERF gene among all 100 naked cultivars studied; (ii) two x-ray-induced nud alleles with a DNA lesion at a different site, each affecting the putative functional motif; and (iii) gene expression strictly localized to the testa. Available results indicate the monophyletic origin of naked barley. The Nud gene has homology to the Arabidopsis WIN1/SHN1 transcription factor gene, whose deduced function is control of a lipid biosynthesis pathway. Staining with a lipophilic dye (Sudan black B) detected a lipid layer on the pericarp epidermis only in covered barley. We infer that, in covered barley, the contact of the caryopsis surface, overlaid with lipids to the inner side of the hull, generates organ adhesion.

Comprehensive Sequence Analysis of 24,783 Barley Full-Length cDNAs Derived from 12 Clone Libraries

Full-length cDNA (FLcDNA) libraries consisting of 172,000 clones were constructed from a two-row malting barley cultivar (Hordeum vulgare ‘Haruna Nijo’) under normal and stressed conditions. After sequencing the clones from both ends and clustering the sequences, a total of 24,783 complete sequences were produced. By removing duplicates between these and publicly available sequences, 22,651 representative sequences were obtained: 17,773 were novel barley FLcDNAs, and 1,699 were barley specific. Highly conserved genes were found in the barley FLcDNA sequences for 721 of 881 rice (Oryza sativa) trait genes with 50% or greater identity. These FLcDNA resources from our Haruna Nijo cDNA libraries and the full-length sequences of representative clones will improve our understanding of the biological functions of genes in barley, which is the cereal crop with the fourth highest production in the world, and will provide a powerful tool for annotating the barley genome sequences that will become available in the near future.

Composition and Structure of the Centromeric Region of Rice Chromosome 8

Understanding the organization of eukaryotic centromeres has both fundamental and applied importance because of their roles in chromosome segregation, karyotypic stability, and artificial chromosome-based cloning and expression vectors. Using clone-by-clone sequencing methodology, we obtained the complete genomic sequence of the centromeric region of rice (Oryza sativa) chromosome 8. Analysis of 1.97 Mb of contiguous nucleotide sequence revealed three large clusters of CentO satellite repeats (68.5 kb of 155-bp repeats) and >220 transposable element (TE)–related sequences; together, these account for ∼60% of this centromeric region. The 155-bp repeats were tandemly arrayed head to tail within the clusters, which had different orientations and were interrupted by TE-related sequences. The individual 155-bp CentO satellite repeats showed frequent transitions and transversions at eight nucleotide positions. The 40 TE elements with highly conserved sequences were mostly gypsy-type retrotransposons. Furthermore, 48 genes, showing high BLAST homology to known proteins or to rice full-length cDNAs, were predicted within the region; some were close to the CentO clusters. We then performed a genome-wide survey of the sequences and organization of CentO and RIRE7 families. Our study provides the complete sequence of a centromeric region from either plants or animals and likely will provide insight into the evolutionary and functional analysis of plant centromeres.

Cleistogamous flowering in barley arises from the suppression of microRNA-guided HvAP2 mRNA cleavage

The cleistogamous flower sheds its pollen before opening, forcing plants with this habit to be almost entirely autogamous. Cleistogamy also provides a means of escape from cereal head blight infection and minimizes pollen-mediated gene flow. The lodicule in cleistogamous barley is atrophied. We have isolated cleistogamy 1 (Cly1) by positional cloning and show that it encodes a transcription factor containing two AP2 domains and a putative microRNA miR172 targeting site, which is an ortholog of Arabidopsis thaliana AP2. The expression of Cly1 was concentrated within the lodicule primordia. We established a perfect association between a synonymous nucleotide substitution at the miR172 targeting site and cleistogamy. Cleavage of mRNA directed by miR172 was detectable only in a noncleistogamous background. We conclude that the miR172-derived down-regulation of Cly1 promotes the development of the lodicules, thereby ensuring noncleistogamy, although the single nucleotide change at the miR172 targeting site results in the failure of the lodicules to develop properly, producing the cleistogamous phenotype.

Insights into the Musa genome: Syntenic relationships to rice and between Musa species

BackgroundMusa species (Zingiberaceae, Zingiberales) including bananas and plantains are collectively the fourth most important crop in developing countries. Knowledge concerning Musa genome structure and the origin of distinct cultivars has greatly increased over the last few years. Until now, however, no large-scale analyses of Musa genomic sequence have been conducted. This study compares genomic sequence in two Musa species with orthologous regions in the rice genome.ResultsWe produced 1.4 Mb of Musa sequence from 13 BAC clones, annotated and analyzed them along with 4 previously sequenced BACs. The 443 predicted genes revealed that Zingiberales genes share GC content and distribution characteristics with eudicot and Poaceae genomes. Comparison with rice revealed microsynteny regions that have persisted since the divergence of the Commelinid orders Poales and Zingiberales at least 117 Mya. The previously hypothesized large-scale duplication event in the common ancestor of major cereal lineages within the Poaceae was verified. The divergence time distributions for Musa-Zingiber (Zingiberaceae, Zingiberales) orthologs and paralogs provide strong evidence for a large-scale duplication event in the Musa lineage after its divergence from the Zingiberaceae approximately 61 Mya. Comparisons of genomic regions from M. acuminata and M. balbisiana revealed highly conserved genome structure, and indicated that these genomes diverged circa 4.6 Mya.ConclusionThese results point to the utility of comparative analyses between distantly-related monocot species such as rice and Musa for improving our understanding of monocot genome evolution. Sequencing the genome of M. acuminata would provide a strong foundation for comparative genomics in the monocots. In addition a genome sequence would aid genomic and genetic analyses of cultivated Musa polyploid genotypes in research aimed at localizing and cloning genes controlling important agronomic traits for breeding purposes.