ABSTRACT
Wheat blast, a devastating disease having spread recently from South America to Asia and Africa, is caused by Pyricularia oryzae (synonym of Magnaporthe oryzae) pathotype Triticum, which first emerged in Brazil in 1985. Rmg8 and Rmg7, genes for resistance to wheat blast found in common wheat and tetraploid wheat, respectively, recognize the same avirulence gene, AVR-Rmg8. Here we show that an ancestral resistance gene, which had obtained an ability to recognize AVR-Rmg8 before the differentiation of Triticum and Aegilops, has expanded its target pathogens. Molecular cloning revealed that Rmg7 was an allele of Pm4, a gene for resistance to wheat powdery mildew on 2AL, whereas Rmg8 was its homoeologue on 2BL ineffective against wheat powdery mildew. Rmg8 variants with the ability to recognize AVR-Rmg8 were distributed not only in Triticum spp. but also in Aegilops speltoides, Aegilops umbellulata and Aegilops comosa. This result suggests that the origin of resistance gene(s) recognizing AVR-Rmg8 dates back to the time before differentiation of A, B, S, U and M genomes, that is, ~5 Myr before the emergence of its current target, the wheat blast fungus. Phylogenetic analyses suggested that, in the evolutionary process thereafter, some of their variants gained the ability to recognize the wheat powdery mildew fungus and evolved into genes controlling dual resistance to wheat powdery mildew and wheat blast.
Subject(s)
Ascomycota , Disease Resistance , Plant Diseases , Triticum , Triticum/microbiology , Triticum/genetics , Triticum/immunology , Plant Diseases/microbiology , Plant Diseases/immunology , Plant Diseases/genetics , Disease Resistance/genetics , Ascomycota/physiology , Genes, Plant , Evolution, Molecular , Aegilops/genetics , Aegilops/microbiology , Plant Proteins/genetics , Plant Proteins/metabolism , PhylogenyABSTRACT
In bread wheat (Triticum aestivum L.), fine-tuning the heading time is essential to maximize grain yield. Photoperiod-1 (Ppd-1) and VERNALIZATION 1 (Vrn-1) are major genes affecting photoperiod sensitivity and vernalization requirements, respectively. These genes have predominantly governed heading timing. However, Ppd-1 and Vrn-1 significantly impact heading dates, necessitating another gene that can slightly modify heading dates for fine-tuning. In this study, we developed an early heading mutant from the ethyl methanesulfonate-mutagenized population of the Japanese winter wheat cultivar "Kitahonami." MutMap analysis identified a nonsense mutation in the clock component gene Wheat PHYTOCLOCK 1/LUX ARRHYTHMO (WPCL-D1) as the probable SNP responsible for the early heading mutant on chromosome 3D. Segregation analysis using F2 and F3 populations confirmed that plants carrying the wpcl-D1 allele headed significantly earlier than those with the functional WPCL-D1. The early heading mutant exhibited increased expression levels of Ppd-1 and circadian clock genes, such as WPCL1 and LATE ELONGATED HYPOCOTYL (LHY). Notably, the transcript accumulation levels of Ppd-A1 and Ppd-D1 were influenced by the copy number of the functional WPCL1 gene. These results suggest that a loss-of-function mutation in WPCL-D1 is the causal mutation for the early heading phenotype. Adjusting the functional copy number of WPCL1 will be beneficial in fine-tuning of heading dates. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01478-5.
ABSTRACT
Closed fertilization in flowers, or cleistogamy, reduces the risk of fungal infection in Triticeae crops. In barley (Hordeum vulgare), cleistogamy is determined by a single recessive gene, cly1, which results from a single nucleotide polymorphism within the microRNA172 target site of the Apetala2 (AP2) transcription factor gene. The recessive cly1 allele negatively regulates the development of lodicules, keeping florets closed at anthesis. However, cleistogamy is not evident in hexaploid wheat (Triticum aestivum) cultivars. This study aimed at identifying mutations in wheat AP2 orthologs by ethyl methane sulfonate-induced mutagenesis and high-resolution melt analysis. Although flowers of AP2 mutants induced in the A and D genomes opened at anthesis, their lodicule size was significantly smaller, especially in the direction of depth, than that of wild-type plants. One of the mutants that carried a nucleotide replacement in AP2 from the D genome produced a compact spike caused by a substantial decrease in rachis internode length, analogous to the barley dense spike. Cleistogamous hexaploid wheat might be generated by combining effective mutant alleles of AP2-homoeologous genes.
ABSTRACT
BACKGROUND: Gamma-irradiated mutants of Triticum aestivum L., hexaploid wheat, provide novel and agriculturally important traits and are used as breeding materials. However, the identification of causative genomic regions of mutant phenotypes is challenging because of the large and complicated genome of hexaploid wheat. Recently, the combined use of high-quality reference genome sequences of common wheat and cost-effective resequencing technologies has made it possible to evaluate genome-wide polymorphisms, even in complex genomes. RESULTS: To investigate whether the genome sequencing approach can effectively detect structural variations, such as deletions, frequently caused by gamma irradiation, we selected a grain-hardness mutant from the gamma-irradiated population of Japanese elite wheat cultivar "Kitahonami." The Hardness (Ha) locus, including the puroindoline protein-encoding genes Pina-D1 and Pinb-D1 on the short arm of chromosome 5D, primarily regulates the grain hardness variation in common wheat. We performed short-read genome sequencing of wild-type and grain-hardness mutant plants, and subsequently aligned their short reads to the reference genome of the wheat cultivar "Chinese Spring." Genome-wide comparisons of depth-of-coverage between wild-type and mutant strains detected ~ 130 Mbp deletion on the short arm of chromosome 5D in the mutant genome. Molecular markers for this deletion were applied to the progeny populations generated by a cross between the wild-type and the mutant. A large deletion in the region including the Ha locus was associated with the mutant phenotype, indicating that the genome sequencing is a powerful and efficient approach for detecting a deletion marker of a gamma-irradiated mutant phenotype. In addition, we investigated a pre-harvest sprouting tolerance mutant and identified a 67.8 Mbp deletion on chromosome 3B where Viviparous-B1 and GRAS family transcription factors are located. Co-dominant markers designed to detect the deletion-polymorphism confirmed the association with low germination rate, leading to pre-harvest sprouting tolerance. CONCLUSIONS: Short read-based genome sequencing of gamma-irradiated mutants facilitates the identification of large deletions linked to mutant phenotypes when combined with segregation analyses in progeny populations. This method allows effective application of mutants with agriculturally important traits in breeding using marker-assisted selection.
Subject(s)
Plant Proteins , Triticum , Chromosome Mapping , Phenotype , Plant Breeding , Plant Proteins/genetics , Triticum/geneticsABSTRACT
The seed protein α-gliadin is a major component of wheat flour and causes gluten-related diseases. However, due to the complexity of this multigene family with a genome structure composed of dozens of copies derived from tandem and genome duplications, little was known about the variation between accessions, and thus little effort has been made to explicitly target α-gliadin for bread wheat breeding. Here, we analyzed genomic variation in α-gliadins across 11 recently published chromosome-scale assemblies of hexaploid wheat, with validation using long-read data. We unexpectedly found that the Gli-B2 locus is not a single contiguous locus but is composed of two subloci, suggesting the possibility of recombination between the two during breeding. We confirmed that the number of immunogenic epitopes among 11 accessions varied. The D subgenome of a European spelt line also contained epitopes, in agreement with its hybridization history. Evolutionary analysis identified amino acid sites under diversifying selection, suggesting their functional importance. The analysis opens the way for improved grain quality and safety through wheat breeding.
ABSTRACT
MAIN CONCLUSION: The distribution of early flowering alleles of VRN-A3 was found to be biased to low latitudes, and these alleles may contribute to environmental adaptability to low latitudes in cultivated emmer wheat. In wheat (Triticum spp.), the flowering time is an important trait for successful seed production and yield by adapting to the regional environment. An early flowering allele of VRN-A3 with 7- and 25-bp insertions in the promoter region (Vrn-A3a-h1) has recently been reported from the analysis of an emmer wheat (Triticum turgidum L. ssp. dicoccum) accession, TN26. This early flowering allele of VRN-A3 might be associated with the regional adaptation of wheat. In this study, we elucidated its geographic distribution to assess the importance of the early flowering allele of VRN-A3 in worldwide wheat collection. From sequence analysis, we identified six VRN-A3 alleles with the 7- and 25-bp insertions, namely, Vrn-A3a-h2, Vrn-A3a-h3, Vrn-A3a-h4, Vrn-A3a-h5, Vrn-A3a-h6, and Vrn-A3c-h2 from wild emmer wheat, while we identified two VRN-A3 alleles with these insertions, Vrn-A3a-h2 and Vrn-A3c-h1 from cultivated tetraploid and hexaploid wheat species in addition to Vrn-A3a-h1. Among VRN-A3 alleles distributed in cultivated wheat, we found that Vrn-A3a-h2 promoted early heading, whereas Vrn-A3c-h1 did not affect heading time. Our analysis showed that the distribution of early flowering alleles of VRN-A3 dominated in cultivated emmer wheat in Ethiopia and India, which actually showed an early flowering phenotype. This implied that the early flowering alleles of VRN-A3 contribute to adaptability to a low-latitude environment in cultivated emmer wheat. We could not find durum (T. turgidum L. ssp. durum) and bread wheat (T. aestivum L. ssp. aestivum) accessions with these early flowering alleles. Our findings indicated that Vrn-A3a-h1 and Vrn-A3a-h2 were useful for breeding of early flowering cultivars in durum and bread wheat varieties.
Subject(s)
Plant Breeding , Triticum , Alleles , Ethiopia , Polyploidy , Triticum/geneticsABSTRACT
The cloning of agriculturally important genes is often complicated by haplotype variation across crop cultivars. Access to pan-genome information greatly facilitates the assessment of structural variations and rapid candidate gene identification. Here, we identified the red glume 1 (Rg-B1) gene using association genetics and haplotype analyses in ten reference grade wheat genomes. Glume color is an important trait to characterize wheat cultivars. Red glumes are frequent among Central European spelt, a dominant wheat subspecies in Europe before the 20th century. We used genotyping-by-sequencing to characterize a global diversity panel of 267 spelt accessions, which provided evidence for two independent introductions of spelt into Europe. A single region at the Rg-B1 locus on chromosome 1BS was associated with glume color in the diversity panel. Haplotype comparisons across ten high-quality wheat genomes revealed a MYB transcription factor as candidate gene. We found extensive haplotype variation across the ten cultivars, with a particular group of MYB alleles that was conserved in red glume wheat cultivars. Genetic mapping and transient infiltration experiments allowed us to validate this particular MYB transcription factor variants. Our study demonstrates the value of multiple high-quality genomes to rapidly resolve copy number and haplotype variations in regions controlling agriculturally important traits.
Subject(s)
Color , Genetic Variation , Genome, Plant , Haplotypes , Metagenomics , Plant Proteins/genetics , Transcription Factors/genetics , Triticum/genetics , Chromosomes, Plant , DNA Copy Number Variations , Gene Dosage , Gene Expression Regulation, Plant , Genome-Wide Association Study , High-Throughput Nucleotide Sequencing , Phenotype , Plant Proteins/metabolism , Polymorphism, Single Nucleotide , Transcription Factors/metabolism , Triticum/metabolismABSTRACT
Bread wheat is a major crop that has long been the focus of basic and breeding research. Assembly of its genome has been difficult because of its large size and allohexaploid nature (AABBDD genome). Following the first reported assembly of the genome of the experimental strain Chinese Spring (CS), the 10+ Wheat Genomes Project was launched to produce multiple assemblies of worldwide modern cultivars. The only Asian cultivar in the project is Norin 61, a representative Japanese cultivar adapted to grow across a broad latitudinal range, mostly characterized by a wet climate and a short growing season. Here, we characterize the key aspects of its chromosome-scale genome assembly spanning 15 Gb with a raw scaffold N50 of 22 Mb. Analysis of the repetitive elements identified chromosomal regions unique to Norin 61 that encompass a tandem array of the pathogenesis-related 13 family. We report novel copy-number variations in the B homeolog of the florigen gene FT1/VRN3, pseudogenization of its D homeolog and the association of its A homeologous alleles with the spring/winter growth habit. Furthermore, the Norin 61 genome carries typical East Asian functional variants different from CS, ranging from a single nucleotide to multi-Mb scale. Examples of such variation are the Fhb1 locus, which confers Fusarium head-blight resistance, Ppd-D1a, which confers early flowering, Glu-D1f for Asian noodle quality and Rht-D1b, which introduced semi-dwarfism during the green revolution. The adoption of Norin 61 as a reference assembly for functional and evolutionary studies will enable comprehensive characterization of the underexploited Asian bread wheat diversity.
Subject(s)
Disease Resistance/genetics , Flowers/growth & development , Genes, Plant/genetics , Genome, Plant/genetics , Triticum/genetics , Chromosome Mapping , Chromosomes, Plant/genetics , Cytogenetics , Asia, Eastern , Flowers/genetics , Fusarium , Genes, Plant/physiology , Genetic Association Studies , Genetic Variation/genetics , Genetic Variation/physiology , Genome, Plant/physiology , Genotype , Phylogeny , Sequence Alignment , Sequence Analysis, DNA , Triticum/growth & development , Triticum/immunology , Triticum/physiologyABSTRACT
Cadmium (Cd) is as an extremely toxic metal that can contaminate agricultural soils. To reduce the risk of Cd intake in food cereals, the development of cultivars with low grain Cd concentration (GCC) is an effective countermeasure. We analyzed quantitative trait loci (QTLs) for GCC in a doubled haploid (DH) common wheat (Triticum aestivum L.) population derived from 'Chugoku 165' (low GCC) × 'Chukei 10-22' (high GCC). We found novel loci for low GCC on the short arm of chromosome 4B and on the long arm of chromosome 6B. These QTLs accounted for 9.4%-25.4% (4B) and 9.0%-17.8% (6B) of the phenotypic variance in the DH population. An association analysis with 43 cultivars identified 3 loci at these QTLs: QCdc.4B-kita, QCdc.6B-kita1, and QCdc.6B-kita2. In contrast to durum wheat and barley, no QTL was detected on the chromosomes of homeologous group 5 for heavy metal P1B-type ATPase 3. These results will contribute to marker-assisted selection for low GCC in breeding of common wheat.
ABSTRACT
The reference genome sequence of wheat 'Chinese Spring' (CS) is now available (IWGSC RefSeq v1.0), but the core sequences defining the nucleolar organizer regions (NORs) have not been characterized. We estimated that the total copy number of the rDNA units in the wheat genome is 11 160, of which 30.5%, 60.9% and 8.6% are located on Nor-B1 (1B), Nor-B2 (6B) and other NORs, respectively. The total length of the NORs is estimated to be 100 Mb, corresponding to approximately 10% of the unassembled portion of the genome not represented in RefSeq v1.0. Four subtypes (S1-S4) of the rDNA units were identified based on differences within the 3' external transcribed spacer regions in Nor-B1 and Nor-B2, and quantitative PCR indicated locus-specific variation in rDNA subtype contents. Expression analyses of rDNA subtypes revealed that S1 was predominantly expressed and S2 weakly expressed, in contrast to the relative abundance of rDNA subtypes in the wheat genome. These results suggest a regulation mechanism of differential rDNA expression based on sequence differences. S3 expression increased in the ditelosomic lines Dt1BL and Dt6BL, suggesting that S3 is subjected to chromosome-mediated silencing. Structural differences were detected in the regions surrounding the NOR among homoeologous chromosomes of groups 1 and 6. The adjacent regions distal to the major NORs were expanded compared with their homoeologous counterparts, and the gene density of these expanded regions was relatively low. We provide evidence that these regions are likely to be important for autoregulation of the associated major NORs as well as silencing of minor NORs.
Subject(s)
Gene Expression Regulation, Plant/genetics , Genes, Plant/genetics , Nucleolus Organizer Region/genetics , RNA, Plant/genetics , RNA, Ribosomal/genetics , Triticum/genetics , Chromosomes, Plant/genetics , DNA Copy Number Variations/genetics , Genetic Loci/genetics , Genome, Plant/genetics , In Situ Hybridization, Fluorescence , Nucleolus Organizer Region/metabolism , Polymerase Chain Reaction , RNA, Plant/metabolism , RNA, Ribosomal/metabolism , Triticum/metabolismABSTRACT
A core collection of Japanese wheat varieties (JWC) consisting of 96 accessions was established based on their passport data and breeding pedigrees. To clarify the molecular basis of the JWC collection, genome-wide single-nucleotide polymorphism (SNP) genotyping was performed using the genotyping-by-sequencing (GBS) approach. Phylogenetic tree and population structure analyses using these SNP data revealed the genetic diversity and relationships among the JWC accessions, classifying them into four groups; "varieties in the Hokkaido area", "modern varieties in the northeast part of Japan", "modern varieties in the southwest part of Japan" and "classical varieties including landraces". This clustering closely reflected the history of wheat breeding in Japan. Furthermore, to demonstrate the utility of the JWC collection, we performed a genome-wide association study (GWAS) for three traits, namely, "days to heading in autumn sowing", "days to heading in spring sowing" and "culm length". We found significantly associated SNP markers with each trait, and some of these were closely linked to known major genes for heading date or culm length on the genetic map. Our study indicates that this JWC collection is a useful set of germplasm for basic and applied research aimed at understanding and utilizing the genetic diversity among Japanese wheat varieties.
ABSTRACT
Rice growth is severely affected by toxic concentrations of the nonessential heavy metal cadmium (Cd). To elucidate the molecular basis of the response to Cd stress, we performed mRNA sequencing of rice following our previous study on exposure to high concentrations of Cd (Oono et al., 2014). In this study, rice plants were hydroponically treated with low concentrations of Cd and approximately 211 million sequence reads were mapped onto the IRGSP-1.0 reference rice genome sequence. Many genes, including some identified under high Cd concentration exposure in our previous study, were found to be responsive to low Cd exposure, with an average of about 11,000 transcripts from each condition. However, genes expressed constitutively across the developmental course responded only slightly to low Cd concentrations, in contrast to their clear response to high Cd concentration, which causes fatal damage to rice seedlings according to phenotypic changes. The expression of metal ion transporter genes tended to correlate with Cd concentration, suggesting the potential of the RNA-Seq strategy to reveal novel Cd-responsive transporters by analyzing gene expression under different Cd concentrations. This study could help to develop novel strategies for improving tolerance to Cd exposure in rice and other cereal crops.
Subject(s)
Cadmium/toxicity , Gene Expression Profiling , Oryza/genetics , Transcriptome/genetics , Gene Expression Regulation, Plant/drug effects , Oryza/drug effects , Oryza/growth & development , Plant Proteins/biosynthesis , Plant Proteins/genetics , Plant Roots/genetics , Plant Roots/growth & development , RNA, Messenger/genetics , Seedlings/genetics , Seedlings/growth & developmentABSTRACT
BACKGROUND: A complete genome sequence is an essential tool for the genetic improvement of wheat. Because the wheat genome is large, highly repetitive and complex due to its allohexaploid nature, the International Wheat Genome Sequencing Consortium (IWGSC) chose a strategy that involves constructing bacterial artificial chromosome (BAC)-based physical maps of individual chromosomes and performing BAC-by-BAC sequencing. Here, we report the construction of a physical map of chromosome 6B with the goal of revealing the structural features of the third largest chromosome in wheat. RESULTS: We assembled 689 informative BAC contigs (hereafter reffered to as contigs) representing 91% of the entire physical length of wheat chromosome 6B. The contigs were integrated into a radiation hybrid (RH) map of chromosome 6B, with one linkage group consisting of 448 loci with 653 markers. The order and direction of 480 contigs, corresponding to 87% of the total length of 6B, were determined. We also characterized the contigs that contained a part of the nucleolus organizer region or centromere based on their positions on the RH map and the assembled BAC clone sequences. Analysis of the virtual gene order along 6B using the information collected for the integrated map revealed the presence of several chromosomal rearrangements, indicating evolutionary events that occurred on chromosome 6B. CONCLUSIONS: We constructed a reliable physical map of chromosome 6B, enabling us to analyze its genomic structure and evolutionary progression. More importantly, the physical map should provide a high-quality and map-based reference sequence that will serve as a resource for wheat chromosome 6B.
Subject(s)
Chromosomes, Artificial, Bacterial/genetics , Physical Chromosome Mapping/methods , Triticum/genetics , Chromosomes, Plant , Evolution, Molecular , Gene Order , Gene Rearrangement , Genetic Markers , Nucleolus Organizer RegionABSTRACT
KEY MESSAGE: High-resolution genetic linkage mapping and BAC physical mapping narrowed the fertility restorer locus Rfm1 in barley to a sub-centimorgan genetic interval and a 208-kb physical interval. Rfm1 restores the fertility of msm1 and msm2 male-sterile cytoplasms in barley. The fertility restoration gene is located on the short arm of chromosome 6H (6HS), and we pursued a positional cloning of this gene. Starting from a previous result that has delimited Rfm1 within a 10.8 cM region on 6HS, we developed novel CAPS and SSR markers tightly linked to the gene in barley using the sequence information from the syntenic region of rice and barley genome assemblies. Next, we performed fine mapping of the Rfm1 locus. To isolate recombinants, we surveyed 3,638 F2 plants derived from a cross between the CMS strain and the Rf strain with adjacent markers (NAS2090 and NAS1080). This analysis identified 175 recombinant plants from the F2 population to build a high-resolution map with nine markers tightly linked to the Rfm1 locus. Rfm1 was located within the 0.14 cM region delimited by two markers (NAS9113 and NAS9200). Using these flanking markers as well as marker cosegregating with Rfm1 (NAS9133), we screened the BAC libraries of the cultivar Morex, an rfm1 carrier. We isolated 11 BAC clones and constructed a BAC physical map using their fingerprints. Finally, we delimited the Rfm1 locus encompassing the rfm1 allele on a 208-kb contig composed of three minimally overlapping BAC clones. This precise localization of the Rfm1 locus in the barley genome is expected to greatly accelerate the future map-based cloning of the Rfm1 gene by sequence analysis and its genetic transformation for the complementation of cytoplasmic male-sterile plants.
Subject(s)
Genetic Linkage , Hordeum/genetics , Physical Chromosome Mapping , Plant Infertility/genetics , Chromosomes, Artificial, Bacterial , Chromosomes, Plant , Comparative Genomic Hybridization , DNA, Plant/genetics , Flowers/anatomy & histology , Genes, Plant , Genetic Markers , GenotypeABSTRACT
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 â¼60,000 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.
Subject(s)
Cadmium/toxicity , Droughts , Gene Expression Profiling , Genomics , Oryza/drug effects , Signal Transduction/genetics , Stress, Physiological/genetics , Cadmium/metabolism , Cation Transport Proteins/genetics , Cation Transport Proteins/metabolism , Gene Expression Regulation, Plant/drug effects , Multigene Family/genetics , Oryza/cytology , Oryza/genetics , Oryza/physiology , Sequence Analysis, RNA , Signal Transduction/drug effects , Stress, Physiological/drug effects , Up-Regulation/drug effectsABSTRACT
BACKGROUND: The endoplasmic reticulum (ER) stress response is widely known to function in eukaryotes to maintain the homeostasis of the ER when unfolded or misfolded proteins are overloaded in the ER. To understand the molecular mechanisms of the ER stress response in rice (Oryza sativa L.), we previously analyzed the expression profile of stably transformed rice in which an ER stress sensor/transducer OsIRE1 was knocked-down, using the combination of preliminary microarray and quantitative RT-PCR. In this study, to obtain more detailed expression profiles of genes involved in the initial stages of the ER stress response in rice, we performed RNA sequencing of wild-type and transgenic rice plants produced by homologous recombination in which endogenous genomic OsIRE1 was replaced by missense alleles defective in ribonuclease activity. RESULTS: At least 38,076 transcripts were investigated by RNA sequencing, 380 of which responded to ER stress at a statistically significant level (195 were upregulated and 185 were downregulated). Furthermore, we successfully identified 17 genes from the set of 380 ER stress-responsive genes that were not included in the probe set of the currently available microarray chip in rice. Notably, three of these 17 genes were non-annotated genes, even in the latest version of the Rice Annotation Project Data Base (RAP-DB, version IRGSP-1.0). CONCLUSIONS: Therefore, RNA sequencing-mediated expression profiling provided valuable information about the ER stress response in rice plants and led to the discovery of new genes related to ER stress.
Subject(s)
Endoplasmic Reticulum Stress/genetics , Gene Expression Profiling/methods , Oryza/genetics , Sequence Analysis, RNA , Transcriptome/genetics , Base Sequence , Databases, Genetic , Down-Regulation/genetics , Gene Expression Regulation, Plant , Gene Ontology , Genes, Plant , Genetic Association Studies , Molecular Sequence Annotation , Plant Roots/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Real-Time Polymerase Chain Reaction , Seedlings/genetics , Sequence Homology, Nucleic AcidABSTRACT
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 (â¼17 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 â¼55.6 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.
Subject(s)
Chromosomes, Plant/genetics , Triticum/genetics , Chromosome Mapping , High-Throughput Nucleotide Sequencing , RNA, Untranslated/genetics , TranscriptomeABSTRACT
The spring-type near isogenic line (NIL) of the winter-type barley (Hordeum vulgare ssp. vulgare) var. Hayakiso 2 (HK2) was developed by introducing VERNALIZATION-H1 (Vrn-H1) for spring growth habit from the spring-type var. Indo Omugi. Contrary to expectations, the spring-type NIL flowered later than winter-type HK2. This phenotypic difference was controlled by a single gene, which cosegregated only with phytochrome C (HvPhyC) among three candidates around the Vrn-H1 region (Vrn-H1, HvPhyC, and CASEIN KINASE IIα), indicating that HvPhyC was the most likely candidate gene. Compared with the late-flowering allele HvPhyC-l from the NIL, the early-flowering allele HvPhyC-e from HK2 had a single nucleotide polymorphism T1139C in exon 1, which caused a nonsynonymous amino acid substitution of phenylalanine at position 380 by serine in the functionally essential GAF (3', 5'-cyclic-GMP phosphodiesterase, adenylate cyclase, formate hydrogen lyase activator protein) domain. Functional assay using a rice (Oryza sativa) phyA phyC double mutant line showed that both of the HvPhyC alleles are functional, but HvPhyC-e may have a hyperfunction. Expression analysis using NILs carrying HvPhyC-e and HvPhyC-l (NIL [HvPhyC-e] and NIL [HvPhyC-l], respectively) showed that HvPhyC-e up-regulated only the flowering promoter FLOWERING LOCUS T1 by bypassing the circadian clock genes and flowering integrator CONSTANS1 under a long photoperiod. Consistent with the up-regulation, NIL (HvPhyC-e) flowered earlier than NIL (HvPhyC-l) under long photoperiods. These results implied that HvPhyC is a key factor to control long-day flowering directly.
Subject(s)
Flowers/physiology , Hordeum/physiology , Photoperiod , Phytochrome/metabolism , Amino Acid Sequence , Crosses, Genetic , Epistasis, Genetic , Flowers/genetics , Gene Expression Regulation, Plant , Genes, Plant/genetics , Genetic Linkage , Haplotypes/genetics , Hordeum/genetics , Inbreeding , Molecular Sequence Data , Oryza/genetics , Phytochrome/chemistry , Phytochrome/genetics , Plants, Genetically Modified , Transformation, GeneticABSTRACT
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.
Subject(s)
Oryza/physiology , Phosphates/deficiency , RNA, Plant/genetics , Transcriptome/genetics , Gene Expression Profiling/methods , Gene Expression Regulation, Plant/genetics , Gene Expression Regulation, Plant/physiology , Genotype , Oryza/genetics , Oryza/growth & development , Plant Roots/metabolism , Plant Shoots/metabolism , Polymerase Chain Reaction/methods , Stress, Physiological/genetics , Stress, Physiological/physiologyABSTRACT
BACKGROUND: Phosphorus (P) is an essential macronutrient for plant growth and development. To modulate their P homeostasis, plants must balance P uptake, mobilisation, and partitioning to various organs. Despite the worldwide importance of wheat as a cultivated food crop, molecular mechanisms associated with phosphate (Pi) starvation in wheat remain unclear. To elucidate these mechanisms, we used RNA-Seq methods to generate transcriptome profiles of the wheat variety 'Chinese Spring' responding to 10 days of Pi starvation. RESULTS: We carried out de novo assembly on 73.8 million high-quality reads generated from RNA-Seq libraries. We then constructed a transcript dataset containing 29,617 non-redundant wheat transcripts, comprising 15,047 contigs and 14,570 non-redundant full-length cDNAs from the TriFLDB database. When compared with barley full-length cDNAs, 10,656 of the 15,047 contigs were unalignable, suggesting that many might be distinct from barley transcripts. The average expression level of the contigs was lower than that of the known cDNAs, implying that these contigs included transcripts that were rarely represented in the full-length cDNA library. Within the non-redundant transcript set, we identified 892-2,833 responsive transcripts in roots and shoots, corresponding on average to 23.4% of the contigs not covered by cDNAs in TriFLDB under Pi starvation. The relative expression level of the wheat IPS1 (Induced by Phosphate Starvation 1) homologue, TaIPS1, was 341-fold higher in roots and 13-fold higher in shoots; this finding was further confirmed by qRT-PCR analysis. A comparative analysis of the wheat- and rice-responsive transcripts for orthologous genes under Pi-starvation revealed commonly upregulated transcripts, most of which appeared to be involved in a general response to Pi starvation, namely, an IPS1-mediated signalling cascade and its downstream functions such as Pi remobilisation, Pi uptake, and changes in Pi metabolism. CONCLUSIONS: Our transcriptome profiles demonstrated the impact of Pi starvation on global gene expression in wheat. This study revealed that enhancement of the Pi-mediated signalling cascade using IPS1 is a potent adaptation mechanism to Pi starvation that is conserved in both wheat and rice and validated the effectiveness of using short-read next-generation sequencing data for wheat transcriptome analysis in the absence of reference genome information.