[Show abstract][Hide abstract] ABSTRACT: Gene targeting (GT) is a technique used to modify endogenous genes in target genomes precisely via homologous recombination (HR). Although GT plants are produced using genetic transformation techniques, if the difference between the endogenous and the modified gene is limited to point mutations, GT crops can be considered equivalent to non-genetically-modified mutant crops generated by conventional mutagenesis techniques. However, it is difficult to guarantee the non-incorporation of DNA fragments from Agrobacterium in GT plants created by Agrobacterium-mediated GT despite screening with conventional Southern blot and/or PCR techniques. Here, we report a comprehensive analysis of herbicide-tolerant rice plants generated by inducing point mutations in the rice ALS gene via Agrobacterium-mediated GT. We performed genome comparative genomic hybridization (CGH) array analysis and whole genome sequencing to evaluate the molecular composition of GT rice plants. Thus far, no integration of Agrobacterium-derived DNA fragments has been detected in GT rice plants. However, more than 1,000 single nucleotide polymorphisms (SNPs) and insertion/deletion (InDels) were found in GT plants. Among these mutations, 20-100 variants might have some effect on expression levels and/or protein function. Information about additive mutations should be useful in clearing out uninvited mutations by backcrossing.
Plant and Cell Physiology 11/2014; · 4.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Anhydrobiosis represents an extreme example of tolerance adaptation to water loss, where an organism can survive in an ametabolic state until water returns. Here we report the first comparative analysis examining the genomic background of extreme desiccation tolerance, which is exclusively found in larvae of the only anhydrobiotic insect, Polypedilum vanderplanki. We compare the genomes of P. vanderplanki and a congeneric desiccation-sensitive midge P. nubifer. We determine that the genome of the anhydrobiotic species specifically contains clusters of multi-copy genes with products that act as molecular shields. In addition, the genome possesses several groups of genes with high similarity to known protective proteins. However, these genes are located in distinct paralogous clusters in the genome apart from the classical orthologues of the corresponding genes shared by both chironomids and other insects. The transcripts of these clustered paralogues contribute to a large majority of the mRNA pool in the desiccating larvae and most likely define successful anhydrobiosis. Comparison of expression patterns of orthologues between two chironomid species provides evidence for the existence of desiccation-specific gene expression systems in P. vanderplanki.
[Show abstract][Hide abstract] ABSTRACT: Analyzing barley gene expression profiles in response to abiotic stress is critical to understanding how barley manages stress, and provides vital information to improve environmental stress tolerance for stable crop production. We developed an Agilent 60-mer oligo DNA microarray with 42,491 probe sets based on the sequences of 36,632 barley (Hordeum vulgare L.) full-length cDNA clones and conducted global expression profiling on barley seedlings subjected to desiccation, salt, cold and abscisic acid (ABA). We identified 281 genes that were differentially expressed in response to desiccation, salt, and cold stresses and ABA treatment. Among them, a class C heat shock transcription factor (HvHsfC1) and a homeodomain leucine zipper (HD-Zip) family I transcription factor (HvHox22) showed more than tenfold and fourfold higher expression, respectively, in response to the stimuli. Heat shock and HD-Zip transcription factors function as important regulators in stress responses in rice (Oryza sativa) and Arabidopsis thaliana; our results suggest these two transcription factors also play important roles in abiotic stress responses in barley. We mapped HvHox22 to the long arm of chromosome 2H and HvHsfC1 to the long arm of 4H, where drought resistance quantitative trait loci were previously detected. Our microarray data and identification of these stress response genes provide key information for dissection of the mechanism of abiotic stress tolerance in barley.
[Show abstract][Hide abstract] ABSTRACT: Soybeans exhibit a nitrogen-fixing symbiosis with soil bacteria of the genera Bradyrhizobium and Ensifer/Sinorhizobium in a unique organ, the root nodule. It has been well known that nodulation of soybean is controlled by several host genes referred to as Rj (rj) genes. Among these genes, a dominant allele, Rj4, restricts nodulation with specific bacterial strains such as B. elkanii USDA61 and B. japonicum Is-34. These incompatible strains fail to invade the host epidermal cells as revealed by observations using DsRed-labelled bacteria. Here, we describe the molecular identification of the Rj4 gene by using map-based cloning with several mapping populations. The Rj4 gene encoded a thaumatin-like protein (TLP) that belongs to pathogenesis-related (PR) protein family 5. In rj4/rj4-genotype soybeans and wild-soybeans, we found six missense mutations and two consecutive amino acid deletions in the rj4 gene as compared with the Rj4 allele. We also found that the rj4/rj4-genotype soybeans were fully complemented by the expression of the Rj4 gene by using hairy root transformation. Whereas the expression of many TLPs and other PR proteins is induced by biotic/abiotic stress, the Rj4 gene expression appears to be constitutive in roots including root nodules.
Plant and Cell Physiology 07/2014; · 4.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The allohexaploid bread wheat genome consists of three closely related subgenomes
(A, B, and D), but a clear understanding of their phylogenetic history has been lacking.
We used genome assemblies of bread wheat and five diploid relatives to analyze
genome-wide samples of gene trees, as well as to estimate evolutionary relatedness
and divergence times.We show that the A and B genomes diverged from a common
ancestor ~7 million years ago and that these genomes gave rise to the D genome through
homoploid hybrid speciation 1 to 2 million years later. Our findings imply that the
present-day bread wheat genome is a product of multiple rounds of hybrid speciation
(homoploid and polyploid) and lay the foundation for a new framework for understanding
the wheat genome as a multilevel phylogenetic mosaic.
[Show abstract][Hide abstract] ABSTRACT: Plant growth is severely affected by toxic concentrations of the non-essential heavy metal cadmium (Cd). Comprehensive transcriptome analysis by RNA-Seq following cadmium exposure is required to further understand plant responses to Cd and facilitate future systems-based analyses of the underlying regulatory networks. In this study, rice plants were hydroponically treated with 50 µM Cd for 24 hours and ∼60,000 expressed transcripts, including transcripts that could not be characterized by microarray-based approaches, were evaluated. Upregulation of various ROS-scavenging enzymes, chelators and metal transporters demonstrated the appropriate expression profiles to Cd exposure. Gene Ontology enrichment analysis of the responsive transcripts indicated the upregulation of many drought stress-related genes under Cd exposure. Further investigation into the expression of drought stress marker genes such as DREB suggested that expression of genes in several drought stress signal pathways was activated under Cd exposure. Furthermore, qRT-PCR analyses of randomly selected Cd-responsive metal transporter transcripts under various metal ion stresses suggested that the expression of Cd-responsive transcripts might be easily affected by other ions. Our transcriptome analysis demonstrated a new transcriptional network linking Cd and drought stresses in rice. Considering our data and that Cd is a non-essential metal, the network underlying Cd stress responses and tolerance, which plants have developed to adapt to other stresses, could help to acclimate to Cd exposure. Our examination of this transcriptional network provides useful information for further studies of the molecular mechanisms of plant adaptation to Cd exposure and the improvement of tolerance in crop species.
PLoS ONE 05/2014; 9(5):e96946. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The development of single nucleotide polymorphism (SNP) markers in Japanese pear (Pyrus pyrifolia Nakai) offers the opportunity to use DNA markers for marker-assisted selection in breeding programs because of their high abundance, codominant inheritance, and potential for automated high-throughput analysis. We developed a 1,536-SNP bead array without a reference genome sequence from more than 44,000 base changes on the basis of a large-scale expressed sequence tag (EST) analysis combined with 454 genome sequencing data of Japanese pear ‘Housui’. Among the 1,536 SNPs on the array, 756 SNPs were genotyped, and 609 SNP loci were mapped to linkage groups on a genetic linkage map of ‘Housui’, based on progeny of an interspecific cross between European pear (Pyrus communis L.) ‘Bartlett’ and ‘Housui’. The newly constructed genetic linkage map consists of 951 loci, comprising 609 new SNPs, 110 pear genomic simple sequence repeats (SSRs), 25 pear EST–SSRs, 127 apple SSRs, 61 pear SNPs identified by the “potential intron polymorphism” method, and 19 other loci. The map covers 22 linkage groups spanning 1341.9 cM with an average distance of 1.41 cM between markers and is anchored to reference genetic linkage maps of European pears and apples. A total of 514 contigs containing mapped SNP loci showed significant similarity to known proteins by functional annotation analysis.
Tree Genetics & Genomes 04/2014; 10(4). · 2.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Having a deep genetic structure evolved during its domestication and adaptation, the Asian cultivated rice (Oryza sativa) displays considerable physiological and morphological variations. Here, we describe deep whole-genome sequencing of the aus rice cultivar Kasalath by using the advanced next-generation sequencing (NGS) technologies to gain a better understanding of the sequence and structural changes among highly differentiated cultivars. The de novo assembled Kasalath sequences represented 91.1% (330.55 Mb) of the genome and contained 35 139 expressed loci annotated by RNA-Seq analysis. We detected 2 787 250 single-nucleotide polymorphisms (SNPs) and 7393 large insertion/deletion (indel) sites (>100 bp) between Kasalath and Nipponbare, and 2 216 251 SNPs and 3780 large indels between Kasalath and 93-11. Extensive comparison of the gene contents among these cultivars revealed similar rates of gene gain and loss. We detected at least 7.39 Mb of inserted sequences and 40.75 Mb of unmapped sequences in the Kasalath genome in comparison with the Nipponbare reference genome. Mapping of the publicly available NGS short reads from 50 rice accessions proved the necessity and the value of using the Kasalath whole-genome sequence as an additional reference to capture the sequence polymorphisms that cannot be discovered by using the Nipponbare sequence alone.
[Show abstract][Hide abstract] ABSTRACT: QTL cloning of genes controlling rice agronomic traits has elucidated the contributions of such genes to phenotypes. However, we face difficulties in the isolation of genes with relatively small effects on phenotypes. Next-generation sequencing technology allows us to explore the allelic differences among diverse populations by rapid full-genome sequencing. The concomitant use of information from QTL mapping and genome sequencing could facilitate the allele mining of natural genetic variants affecting agronomic traits in rice. To this end, we have been developing chromosomal segment substitution lines (CSSLs) by using diverse rice accessions and high-depth short-read re-sequencing of diverse donor cultivars. Phenotyping of CSSLs provides us with accurate information on the genetic effects of a single chromosomal segment and the position of the candidate genes. Sequence comparisons of relevant small regions between multiple donor cultivars and a common recurrent parent subsequently reduce the number of candidate genes. Additional information about the candidate genes, such as functional annotations and expression profiles (for example, expression levels at different stages), also accelerates allele mining. The combination of QTL mapping for heading date (Hd) by using CSSLs and sequence information can serve as an example of the efficient allele mining of genes associated with Hd. Additional information has helped us to reduce the number of candidates. To verify the phenotypic effects of sequence changes in a few genes, the development of overexpressing or RNAi lines and the selection of gene disruption lines for candidate genes is underway. The effective strategies for allele mining will be discussed.
International Plant and Animal Genome Conference XXII 2014; 01/2014
[Show abstract][Hide abstract] ABSTRACT: Sorghum (Sorghum bicolor L. Moench) accumulates 3-deoxyanthocyanidins and exhibits orange to purple coloration on parts of the leaf in response to infection with the fungus Bipolaris sorghicola. We aimed to identify the key genes determining this color variation.
[Show abstract][Hide abstract] ABSTRACT: An ordered draft sequence of the 17-gigabase hexaploid bread wheat (Triticum aestivum) genome has been produced by sequencing isolated chromosome arms. We have annotated 124,201 gene loci distributed nearly evenly across the homeologous chromosomes and subgenomes. Comparative gene analysis of wheat subgenomes and extant diploid and tetraploid wheat relatives showed that high sequence similarity and structural conservation are retained, with limited gene loss, after polyploidization. However, across the genomes there was evidence of dynamic gene gain, loss, and duplication since the divergence of the wheat lineages. A high degree of transcriptional autonomy and no global dominance was found for the subgenomes. These insights into the genome biology of a polyploid crop provide a springboard for faster gene isolation, rapid genetic marker development, and precise breeding to meet the needs of increasing food demand worldwide.
[Show abstract][Hide abstract] ABSTRACT: Background and AimsThe timing of flowering has a direct impact on successful seed production in plants. Flowering of soybean (Glycine max) is controlled by several E loci, and previous studies identified the genes responsible for the flowering loci E1, E2, E3 and E4. However, natural variation in these genes has not been fully elucidated. The aims of this study were the identification of new alleles, establishment of allele diagnoses, examination of allelic combinations for adaptability, and analysis of the integrated effect of these loci on flowering.Methods
The sequences of these genes and their flanking regions were determined for 39 accessions by primer walking. Systematic discrimination among alleles was performed using DNA markers. Genotypes at the E1-E4 loci were determined for 63 accessions covering several ecological types using DNA markers and sequencing, and flowering times of these accessions at three sowing times were recorded.Key ResultsA new allele with an insertion of a long interspersed nuclear element (LINE) at the promoter of the E1 locus (e1-re) was identified. Insertion and deletion of 36 bases in the eighth intron (E2-in and E2-dl) were observed at the E2 locus. Systematic discrimination among the alleles at the E1-E3 loci was achieved using PCR-based markers. Allelic combinations at the E1-E4 loci were found to be associated with ecological types, and about 62-66 % of variation of flowering time could be attributed to these loci.Conclusions
The study advances understanding of the combined roles of the E1-E4 loci in flowering and geographic adaptation, and suggests the existence of unidentified genes for flowering in soybean.
[Show abstract][Hide abstract] ABSTRACT: Common wheat (Triticum aestivum L.) is one of the most important cereals in the world. To improve wheat quality and productivity, the genomic sequence of wheat must be determined. The large genome size (∼17 Gb/1 C) and the hexaploid status of wheat have hampered the genome sequencing of wheat. However, flow sorting of individual chromosomes has allowed us to purify and separately shotgun-sequence a pair of telocentric chromosomes. Here, we describe a result from the survey sequencing of wheat chromosome 6B (914 Mb/1 C) using massively parallel 454 pyrosequencing. From the 4.94 and 5.51 Gb shotgun sequence data from the two chromosome arms of 6BS and 6BL, 235 and 273 Mb sequences were assembled to cover ∼55.6 and 54.9% of the total genomic regions, respectively. Repetitive sequences composed 77 and 86% of the assembled sequences on 6BS and 6BL, respectively. Within the assembled sequences, we predicted a total of 4798 non-repetitive gene loci with the evidence of expression from the wheat transcriptome data. The numbers and chromosomal distribution patterns of the genes for tRNAs and microRNAs in wheat 6B were investigated, and the results suggested a significant involvement of DNA transposon diffusion in the evolution of these non-protein-coding RNA genes. A comparative analysis of the genomic sequences of wheat 6B and monocot plants clearly indicated the evolutionary conservation of gene contents.
[Show abstract][Hide abstract] ABSTRACT: 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 the 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 our further understanding of the molecular mechanisms underlying the evolution and functions of the rice genome. This article is protected by copyright. All rights reserved.
The Plant Journal 08/2013; 76(4):699-708. · 6.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rice has developed several morphological and physiological strategies to adapt to phosphate starvation in the soil. In order to elucidate the molecular basis of response to phosphate starvation, we performed mRNA sequencing of 4 rice cultivars with variation in growth response to Pi starvation as indicated by the shoot/root dry weight ratio. Approximately 254 million sequence reads were mapped onto the IRGSP-1.0 reference rice genome sequence and an average of about 5,000 transcripts from each cultivar were found to be responsive under phosphate starvation. Comparative analysis of the RNA-Seq profiles of the 4 cultivars revealed similarities as well as distinct differences in expression of these responsive transcripts. We elucidated a set of core responsive transcripts including annotated and unannotated transcripts commonly expressed in the 4 cultivars but with different levels of expression. De novo assembly of unmapped reads to the Nipponbare genome generated a set of sequence contigs representing potential new transcripts that may be involved in tolerance to phosphate starvation. This study can be used for identification of genes and gene networks associated with environmental stress and the development of novel strategies for improving tolerance to phosphate starvation in rice and other cereal crops.
[Show abstract][Hide abstract] ABSTRACT: The diamondback moth (DBM), Plutella xylostella, is one of the most harmful insect pests for crucifer crops worldwide. DBM has rapidly evolved high resistance to most conventional insecticides such as pyrethroids, organophosphates, fipronil, spinosad, Bacillus thuringiensis, and diamides. Therefore, it is important to develop genomic and transcriptomic DBM resources for analysis of genes related to insecticide resistance, both to clarify the mechanism of resistance of DBM and to facilitate the development of insecticides with a novel mode of action for more effective and environmentally less harmful insecticide rotation. To contribute to this goal, we developed KONAGAbase, a genomic and transcriptomic database for DBM (KONAGA is the Japanese word for DBM).Description: KONAGAbase provides (1) transcriptomic sequences of 37,340 ESTs/mRNAs and 147,370 RNA-seq contigs which were clustered and assembled into 84,570 unigenes (30,695 contigs, 50,548 pseudo singletons, and 3,327 singletons); and (2) genomic sequences of 88,530 WGS contigs with 246,244 degenerate contigs and 106,455 singletons from which 6,310 de novo identified repeat sequences and 34,890 predicted gene-coding sequences were extracted. The unigenes and predicted gene-coding sequences were clustered and 32,800 representative sequences were extracted as a comprehensive putative gene set. These sequences were annotated with BLAST descriptions, Gene Ontology (GO) terms, and Pfam descriptions, respectively. KONAGAbase contains rich graphical user interface (GUI)-based web interfaces for easy and efficient searching, browsing, and downloading sequences and annotation data. Five useful search interfaces consisting of BLAST search, keyword search, BLAST result-based search, GO tree-based search, and genome browser are provided. KONAGAbase is publicly available from our website (http://dbm.dna.affrc.go.jp/px/) through standard web browsers.
KONAGAbase provides DBM comprehensive transcriptomic and draft genomic sequences with useful annotation information with easy-to-use web interfaces, which helps researchers to efficiently search for target sequences such as insect resistance-related genes. KONAGAbase will be continuously updated and additional genomic/transcriptomic resources and analysis tools will be provided for further efficient analysis of the mechanism of insecticide resistance and the development of effective insecticides with a novel mode of action for DBM.
[Show abstract][Hide abstract] ABSTRACT: Establishment of a complete genomic sequence of silkworm, the model species of Lepidoptera, laid a foundation for its functional genomics. A more complete annotation of the genome will benefit functional and comparative studies and accelerate extensive industrial applications for this insect. To realize these goals, we embarked upon a large-scale full-length cDNA collection from 21 full-length cDNA libraries derived from 14 tissues of the domesticated silkworm and performed full-sequencing by primer walking for 11,104 full-length cDNAs. The large average intron size was 1,904 bp, resulting from a high accumulation of transposons. Using gene models predicted by GLEAN and published mRNAs, we identified 16,823 gene loci on the silkworm genome assembly. Orthology analysis of 153 species including 11 insects revealed that among three Lepidoptera including Monarch and Heliconius butterflies, the 403 largest silkworm-specific genes were mainly composed of protective immunity, hormone-related, and characteristic structural proteins. Analysis of testis-/ovary-specific genes revealed distinctive features of sexual dimorphism including depletion of ovary-specific genes on the Z chromosome in contrast to an enrichment of testis-specific genes. Over 40% of genes expressed in specific tissues mapped in tissue-specific chromosomal clusters. The newly obtained FL-cDNA sequences enabled us to annotate the genome of this lepidopteran model insect more accurately, enhancing genomic and functional studies of Lepidoptera and comparative analyses with other insect orders, and yielding new insights into the evolution and organization of lepidopteran-specific genes.
[Show abstract][Hide abstract] ABSTRACT: Epicuticular wax (bloom) plays important roles in protecting the tissues of sorghum (Sorghum bicolor (L.) Moench) plants from abiotic stresses. However, reducing wax content provides resistance to greenbug and sheath blight-a useful trait in agricultural crops. We generated a sorghum bloomless (bm) mutant by gamma irradiation. One bm population segregated for individuals with and without epicuticular wax at a frequency of 72:22, suggesting that the bm mutation was under the control of a single recessive nuclear gene. Genes differentially expressed in the wild-type and the bm mutant were identified by RNA-seq technology. Of the 31 downregulated genes, Sb06g023280 was the most differentially expressed and was similar to WBC11, which encodes an ABC transporter responsible for wax secretion in Arabidopsis. An inversion of about 1.4 Mb was present in the region upstream of the Sb06g023280 gene in the bm mutant; it is likely that this inversion changed the promoter sequence of the Sb06g023280 gene. Using genomic PCR, we confirmed that six independent F2 bm mutant-phenotype plants carried the same inversion. Therefore, we concluded that the inversion involving the Sb06g023280 gene inhibited wax secretion in the bloomless sorghum.
Theoretical and Applied Genetics 03/2013; · 3.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
Rice 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).
The 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.
A 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.