Evolutionary dynamics and impacts of chromosome regions carrying R-gene clusters in rice.

To elucidate R-gene evolution, we compared the genomic compositions and structures of chromosome regions carrying R-gene clusters among cultivated and wild rice species. Map-based sequencing and gene annotation of orthologous genomic regions (1.2 to 1.9 Mb) close to the terminal end of the long arm of rice chromosome 11 revealed R-gene clusters within six cultivated and ancestral wild rice accessions. NBS-LRR R-genes were much more abundant in Asian cultivated rice (O. sativa L.) than in its ancestors, indicating that homologs of functional genes involved in the same pathway likely increase in number because of tandem duplication of chromosomal segments and were selected during cultivation. Phylogenetic analysis using amino acid sequences indicated that homologs of paired Pikm1-Pikm2 (NBS-LRR) genes conferring rice-blast resistance were likely conserved among all cultivated and wild rice species we examined, and the homolog of Xa3/Xa26 (LRR-RLK) conferring bacterial blight resistance was lacking only in Kasalath.

Correction: EAT1 transcription factor, a non-cell-autonomous regulator of pollen production, activates meiotic small RNA biogenesis in rice anther tapetum.

[This corrects the article DOI: 10.1371/journal.pgen.1007238.].

EAT1 transcription factor, a non-cell-autonomous regulator of pollen production, activates meiotic small RNA biogenesis in rice anther tapetum.

The 24-nucleotides (nt) phased secondary small interfering RNA (phasiRNA) is a unique class of plant small RNAs abundantly expressed in monocot anthers at early meiosis. Previously, 44 intergenic regions were identified as the loci for longer precursor RNAs of 24-nt phasiRNAs (24-PHASs) in the rice genome. However, the regulatory mechanism that determines spatiotemporal expression of these RNAs has remained elusive. ETERNAL TAPETUM1 (EAT1) is a basic-helix-loop-helix (bHLH) transcription factor indispensable for induction of programmed cell death (PCD) in postmeiotic anther tapetum, the somatic nursery for pollen production. In this study, EAT1-dependent non-cell-autonomous regulation of male meiosis was evidenced from microscopic observation of the eat1 mutant, in which meiosis with aberrantly decondensed chromosomes was retarded but accomplished somehow, eventually resulting in abortive microspores due to an aberrant tapetal PCD. EAT1 protein accumulated in tapetal-cell nuclei at early meiosis and postmeiotic microspore stages. Meiotic EAT1 promoted transcription of 24-PHAS RNAs at 101 loci, and importantly, also activated DICER-LIKE5 (DCL5, previous DCL3b in rice) mRNA transcription that is required for processing of double-stranded 24-PHASs into 24-nt lengths. From the results of the chromatin-immunoprecipitation and transient expression analyses, another tapetum-expressing bHLH protein, TDR INTERACTING PROTEIN2 (TIP2), was suggested to be involved in meiotic small-RNA biogenesis. The transient assay also demonstrated that UNDEVELOPED TAPETUM1 (UDT1)/bHLH164 is a potential interacting partner of both EAT1 and TIP2 during early meiosis. This study indicates that EAT1 is one of key regulators triggering meiotic phasiRNA biogenesis in anther tapetum, and that other bHLH proteins, TIP2 and UDT1, also play some important roles in this process. Spatiotemporal expression control of these bHLH proteins is a clue to orchestrate precise meiosis progression and subsequent pollen production non-cell-autonomously.

Detection of novel QTLs qDTH4.5 and qDTH6.3, which confer late heading under short-day conditions, by SSR marker-based and QTL-seq analysis.

Heading date is one of the most important traits in rice breeding. It is governed by multiple genes, including known quantitative trait loci (QTLs). In general, almost all japonica cultivars, including Nipponbare, head early under short-day (SD) conditions, but some indica cultivars, including Kasalath, head late. To explain this difference, we identified QTLs controlling heading date under SD conditions. We used NILs, CSSLs, and BILs from a cross between Nipponbare and Kasalath, and evaluated days to heading (DTH) under SD conditions. No NILs or CSSLs showed late heading, but two BILs (BIL-55 and BIL-78) had almost the same DTH as Kasalath. We developed an F2 population from a cross between BIL-55 and Nipponbare and performed QTL analysis using SSR markers. The late-heading phenotype was controlled by two known genes and at least two novel QTLs on chromosomes 4 and 6, named qDTH4.5 and qDTH6.3. These QTLs were confirmed by QTL-seq. The QTLs and polymorphisms detected here will provide useful information for further genetic studies and breeding under SD conditions at lower latitudes.

The Nipponbare genome and the next-generation of rice genomics research in Japan.

The map-based genome sequence of the japonica rice cultivar Nipponbare remains to date as the only monocot genome that has been sequenced to a high-quality level. It has become the reference sequence for understanding the diversity among thousands of rice cultivars and its wild relatives as well as the major cereal crops that comprised the food source for the entire human race. This review focuses on the accomplishments in rice genomics in Japan encompassing the last 10 years which have led into deeper understanding of the genome, characterization of many agronomic traits, comprehensive analysis of the transcriptome, and the map-based cloning of many genes associated with agronomic traits.

The radish genome and comprehensive gene expression profile of tuberous root formation and development.

Understanding the processes that regulate plant sink formation and development at the molecular level will contribute to the areas of crop breeding, food production and plant evolutionary studies. We report the annotation and analysis of the draft genome sequence of the radish Raphanus sativus var. hortensis (long and thick root radish) and transcriptome analysis during root development. Based on the hybrid assembly approach of next-generation sequencing, a total of 383 Mb (N50 scaffold: 138.17 kb) of sequences of the radish genome was constructed containing 54,357 genes. Syntenic and phylogenetic analyses indicated that divergence between Raphanus and Brassica coincide with the time of whole genome triplication (WGT), suggesting that WGT triggered diversification of Brassiceae crop plants. Further transcriptome analysis showed that the gene functions and pathways related to carbohydrate metabolism were prominently activated in thickening roots, particularly in cell proliferating tissues. Notably, the expression levels of sucrose synthase 1 (SUS1) were correlated with root thickening rates. We also identified the genes involved in pungency synthesis and their transcription factors.

A Chromosome Segment Substitution Library of Weedy Rice for Genetic Dissection of Complex Agronomic and Domestication Traits.

Chromosome segment substitution lines (CSSLs) are a powerful alternative for locating quantitative trait loci (QTL), analyzing gene interactions, and providing starting materials for map-based cloning projects. We report the development and characterization of a CSSL library of a U.S. weedy rice accession 'PSRR-1' with genome-wide coverage in an adapted rice cultivar 'Bengal' background. The majority of the CSSLs carried a single defined weedy rice segment with an average introgression segment of 2.8 % of the donor genome. QTL mapping results for several agronomic and domestication traits from the CSSL population were compared with those obtained from two recombinant inbred line (RIL) populations involving the same weedy rice accession. There was congruence of major effect QTLs between both types of populations, but new and additional QTLs were detected in the CSSL population. Although, three major effect QTLs for plant height were detected on chromosomes 1, 4, and 8 in the CSSL population, the latter two escaped detection in both RIL populations. Since this was observed for many traits, epistasis may play a major role for the phenotypic variation observed in weedy rice. High levels of shattering and seed dormancy in weedy rice might result from an accumulation of many small effect QTLs. Several CSSLs with desirable agronomic traits (e.g. longer panicles, longer grains, and higher seed weight) identified in this study could be useful for rice breeding. Since weedy rice is a reservoir of genes for many weedy and agronomic attributes, the CSSL library will serve as a valuable resource to discover latent genetic diversity for improving crop productivity and understanding the plant domestication process through cloning and characterization of the underlying genes.

Mapping of seed shattering loci provides insights into origin of weedy rice and rice domestication.

Seed shattering is an important trait that distinguishes crop cultivars from the wild and weedy species. The genetics of seed shattering was investigated in this study to provide insights into rice domestication and the evolution of weedy rice. Quantitative trait locus (QTL) analysis, conducted in 2 recombinant inbred populations involving 2 rice cultivars and a weedy rice accession of the southern United States, revealed 3-5 QTLs that controlled seed shattering with 38-45% of the total phenotypic variation. Two QTLs on chromosomes 4 and 10 were consistent in both populations. Both cultivar and weedy rice contributed alleles for increased seed shattering. Genetic backgrounds affected both QTL number and the magnitude of QTL effects. The major QTL qSH4 and a minor QTL qSH3 were validated in near-isogenic lines, with the former conferring a significantly higher degree of seed shattering than the latter. Although the major QTL qSH4 overlapped with the sh4, the presence of the nonshattering single nucleotide polymorphism allele in the weedy rice accession suggested involvement of a linked locus or an alternative molecular genetic mechanism. Overlapping of several QTLs with those from earlier studies indicated that weedy rice may have been derived from the wild species Oryza rufipogon. Natural hybridization of rice cultivars with the highly variable O. rufipogon present in different geographic regions might be responsible for the evolution of a wide range of phenotypic and genotypic variabilities seen in weedy rice populations worldwide.

A BAC physical map of aus rice cultivar 'Kasalath', and the map-based genomic sequence of 'Kasalath' chromosome 1.

Comparative analysis using available genomic resources within closely related species is an effective way to investigate genomic sequence and structural diversity. Rice (Oryza sativa L.) has undergone significant physiological and morphological changes during its domestication and local adaptation. We present a complete bacterial artificial chromosome (BAC) physical map for the aus rice cultivar 'Kasalath', which covers 90% of the sequence of temperate japonica rice cultivar 'Nipponbare'. Examination of physical distances between computational and experimental measurements of 'Kasalath' BAC insert size revealed the presence of more than 500 genomic regions that appear to have significant chromosome structural changes between the two cultivars. In particular, a genomic region on the long arm of 'Kasalath' chromosome 11 carrying a disease-resistance gene cluster was greatly expanded relative to the 'Nipponbare' genome. We also decoded 41.37 Mb of high-quality genomic sequence from 'Kasalath' chromosome 1. Extensive comparisons of chromosome 1 between 'Kasalath' and 'Nipponbare' led to the discovery of 317,843 single-nucleotide polymorphisms (SNPs) and 66,331 insertion/deletion (indel) sites. Nearly two-thirds of the expressed genes on rice chromosome 1 carried natural variations involving SNPs and/or indels that resulted in substitutions, insertions or deletions of amino acids in one cultivar relative to the other. We also observed gain and loss of genes caused by large indels. This study provides an important framework and an invaluable dataset for further understanding of the molecular mechanisms underlying the evolution and functions of the rice genome.

Rice Annotation Project Database (RAP-DB): an integrative and interactive database for rice genomics.

The Rice Annotation Project Database (RAP-DB, http://rapdb.dna.affrc.go.jp/) has been providing a comprehensive set of gene annotations for the genome sequence of rice, Oryza sativa (japonica group) cv. Nipponbare. Since the first release in 2005, RAP-DB has been updated several times along with the genome assembly updates. Here, we present our newest RAP-DB based on the latest genome assembly, Os-Nipponbare-Reference-IRGSP-1.0 (IRGSP-1.0), which was released in 2011. We detected 37,869 loci by mapping transcript and protein sequences of 150 monocot species. To provide plant researchers with highly reliable and up to date rice gene annotations, we have been incorporating literature-based manually curated data, and 1,626 loci currently incorporate literature-based annotation data, including commonly used gene names or gene symbols. Transcriptional activities are shown at the nucleotide level by mapping RNA-Seq reads derived from 27 samples. We also mapped the Illumina reads of a Japanese leading japonica cultivar, Koshihikari, and a Chinese indica cultivar, Guangluai-4, to the genome and show alignments together with the single nucleotide polymorphisms (SNPs) and gene functional annotations through a newly developed browser, Short-Read Assembly Browser (S-RAB). We have developed two satellite databases, Plant Gene Family Database (PGFD) and Integrative Database of Cereal Gene Phylogeny (IDCGP), which display gene family and homologous gene relationships among diverse plant species. RAP-DB and the satellite databases offer simple and user-friendly web interfaces, enabling plant and genome researchers to access the data easily and facilitating a broad range of plant research topics.

DaizuBase, an integrated soybean genome database including BAC-based physical maps.

Soybean [Glycine max (L) Merrill] is one of the most important leguminous crops and ranks fourth after to rice, wheat and maize in terms of world crop production. Soybean contains abundant protein and oil, which makes it a major source of nutritious food, livestock feed and industrial products. In Japan, soybean is also an important source of traditional staples such as tofu, natto, miso and soy sauce. The soybean genome was determined in 2010. With its enormous size, physical mapping and genome sequencing are the most effective approaches towards understanding the structure and function of the soybean genome. We constructed bacterial artificial chromosome (BAC) libraries from the Japanese soybean cultivar, Enrei. The end-sequences of approximately 100,000 BAC clones were analyzed and used for construction of a BAC-based physical map of the genome. BLAST analysis between Enrei BAC-end sequences and the Williams82 genome was carried out to increase the saturation of the map. This physical map will be used to characterize the genome structure of Japanese soybean cultivars, to develop methods for the isolation of agronomically important genes and to facilitate comparative soybean genome research. The current status of physical mapping of the soybean genome and construction of database are presented.

DDM1 (decrease in DNA methylation) genes in rice (Oryza sativa).

Regulation of cytosine methylation in the plant genome is of pivotal in determining the epigenetic states of chromosome regions. Relative tolerance of plant to deficiency in cytosine methylation provides unparalleled opportunities to study the mechanism for regulation of cytosine methylation. The Decrease in DNA Methylation 1 (DDM1) of Arabidopsis thaliana is one of the best characterized plant epigenetic regulators that are necessary for maintenance of cytosine methylation in genomic DNA. Although cytosine methylation could affect various aspects of plant growth and development including those related to agricultural importance, orthologs of DDM1 in plants other than Arabidopsis has not been studied in detail. In this study, we identified two rice genes with similarity to Arabidopsis DDM1 and designated them OsDDM1a and OsDDM1b. Both of the rice DDM1 homologs are transcribed during development and their amino acid sequences are 93 % identical to each other. Transgenic rice lines expressing the OsDDM1a cDNA in the antisense orientation exhibited genomic DNA hypomethylation. In those lines, repeated sequences were more severely affected than a single copy sequence as is the case in Arabidopsis ddm1 mutants. Transcripts derived from endogenous transposon-related loci were up-regulated in the antisense OsDDM1 lines, opening a possibility to identify and utilize potentially active transposons for rice functional genomics.

Distinct evolutionary patterns of Oryza glaberrima deciphered by genome sequencing and comparative analysis.

Here we present the genomic sequence of the African cultivated rice, Oryza glaberrima, and compare these data with the genome sequence of Asian cultivated rice, Oryza sativa. We obtained gene-enriched sequences of O. glaberrima that correspond to about 25% of the gene regions of the O. sativa (japonica) genome by methylation filtration and subtractive hybridization of repetitive sequences. While patterns of amino acid changes did not differ between the two species in terms of the biochemical properties, genes of O. glaberrima generally showed a larger synonymous-nonsynonymous substitution ratio, suggesting that O. glaberrima has undergone a genome-wide relaxation of purifying selection. We further investigated nucleotide substitutions around splice sites and found that eight genes of O. sativa experienced changes at splice sites after the divergence from O. glaberrima. These changes produced novel introns that partially truncated functional domains, suggesting that these newly emerged introns affect gene function. We also identified 2451 simple sequence repeats (SSRs) from the genomes of O. glaberrima and O. sativa. Although tri-nucleotide repeats were most common among the SSRs and were overrepresented in the protein-coding sequences, we found that selection against indels of tri-nucleotide repeats was relatively weak in both African and Asian rice. Our genome-wide sequencing of O. glaberrima and in-depth analyses provide rice researchers not only with useful genomic resources for future breeding but also with new insights into the genomic evolution of the African and Asian rice species.

Asymmetric distribution of gene expression in the centromeric region of rice chromosome 5.

There is controversy as to whether gene expression is silenced in the functional centromere. The complete genomic sequences of the centromeric regions in higher eukaryotes have not been fully elucidated, because the presence of highly repetitive sequences complicates many aspects of genomic sequencing. We performed resequencing, assembly, and sequence finishing of two P1-derived artificial chromosome clones in the centromeric region of rice (Oryza sativa L.) chromosome 5 (Cen5). The pericentromeric region, where meiotic recombination is silenced, is located at the center of chromosome 5 and is 2.14 Mb long; a total of six restriction-fragment-length polymorphism markers (R448, C1388, S20487S, E3103S, C53260S, and R2059) genetically mapped at 54.6 cM were located in this region. In the pericentromeric region, 28 genes were annotated on the short arm and 45 genes on the long arm. To quantify all transcripts in this region, we performed massive parallel sequencing of mRNA. Transcriptional density (total length of transcribed region/length of the genomic region) and expression level (number of uniquely mapped reads/length of transcribed region) were calculated on the basis of the mapped reads on the rice genome. Transcriptional density and expression level were significantly lower in Cen5 than in the average of the other chromosomal regions. Moreover, transcriptional density in Cen5 was significantly lower on the short arm than on the long arm; the distribution of transcriptional density was asymmetric. The genomic sequence of Cen5 has been integrated into the most updated reference rice genome sequence constructed by the International Rice Genome Sequencing Project.

The role of casein kinase II in flowering time regulation has diversified during evolution.

Casein kinase II (CK2) is a protein kinase with an evolutionarily conserved function as a circadian clock component in several organisms, including the long-day plant Arabidopsis (Arabidopsis thaliana). The circadian clock component CIRCADIAN CLOCK ASSOCIATED1 (CCA1) is a CK2 target in Arabidopsis, where it influences photoperiodic flowering. In rice (Oryza sativa), a short-day plant, Heading date6 (Hd6) encodes a CK2alpha subunit that delays flowering time under long-day conditions. Here, we demonstrate that control of flowering time in rice by the Hd6 CK2alpha subunit requires a functional Hd1 gene (an Arabidopsis CONSTANS ortholog) and is independent of the circadian clock mechanism. Our findings from overexpressing the dominant-negative CK2 allele in rice support the independence of CK2 function from the circadian clock. This lack of control of the circadian clock by Hd6 CK2alpha might be due to the presence of glutamate in OsLHY (a CCA1 ortholog in rice) instead of the serine at the corresponding CK2 target site in CCA1. However, this glutamate is critical for the control of the OsPRR1 gene (a rice ortholog of the Arabidopsis TOC1/PRR1 gene) by OsLHY for regulation of the circadian clock. We also demonstrated that the other conserved CK2 target sites in OsLHY conferred robust rhythmic expression of OsLHY-LUC under diurnal conditions. These findings imply that the role of CK2 in flowering-time regulation in higher plants has diversified during evolution.

Comparative analysis of complete orthologous centromeres from two subspecies of rice reveals rapid variation of centromere organization and structure.

Centromeres are sites for assembly of the chromosomal structures that mediate faithful segregation at mitosis and meiosis. This function is conserved across species, but the DNA components that are involved in kinetochore formation differ greatly, even between closely related species. To shed light on the nature, evolutionary timing and evolutionary dynamics of rice centromeres, we decoded a 2.25-Mb DNA sequence covering the centromeric region of chromosome 8 of an indica rice variety, 'Kasalath' (Kas-Cen8). Analysis of repetitive sequences in Kas-Cen8 led to the identification of 222 long terminal repeat (LTR)-retrotransposon elements and 584 CentO satellite monomers, which account for 59.2% of the region. A comparison of the Kas-Cen8 sequence with that of japonica rice 'Nipponbare' (Nip-Cen8) revealed that about 66.8% of the Kas-Cen8 sequence was collinear with that of Nip-Cen8. Although the 27 putative genes are conserved between the two subspecies, only 55.4% of the total LTR-retrotransposon elements in 'Kasalath' had orthologs in 'Nipponbare', thus reflecting recent proliferation of a considerable number of LTR-retrotransposons since the divergence of two rice subspecies of indica and japonica within Oryza sativa. Comparative analysis of the subfamilies, time of insertion, and organization patterns of inserted LTR-retrotransposons between the two Cen8 regions revealed variations between 'Kasalath' and 'Nipponbare' in the preferential accumulation of CRR elements, and the expansion of CentO satellite repeats within the core domain of Cen8. Together, the results provide insights into the recent proliferation of LTR-retrotransposons, and the rapid expansion of CentO satellite repeats, underlying the dynamic variation and plasticity of plant centromeres.

Development in rice genome research based on accurate genome sequence.

Rice is one of the most important crops in the world. Although genetic improvement is a key technology for the acceleration of rice breeding, a lack of genome information had restricted efforts in molecular-based breeding until the completion of the high-quality rice genome sequence, which opened new opportunities for research in various areas of genomics. The syntenic relationship of the rice genome to other cereal genomes makes the rice genome invaluable for understanding how cereal genomes function. Producing an accurate genome sequence is not an easy task, and it is becoming more important as sequence deviations among, and even within, species highlight functional or evolutionary implications for comparative genomics.