[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: Mother of FT and TFL1 (MFT) participates in the regulation of seed dormancy in wheat. A single-nucleotide polymorphism (SNP) in the promoter of MFT on chromosome 3A (MFT-3A) can explain the difference in the levels of MFT-3A expression, and the difference in seed dormancy, between Chinese Spring (CS) and the CS line with a substituted chromosome 3A from Zenkoujikomugi (Zen) (CS(Zen3A)). Using a cleaved amplified polymorphic sequence (CAPS) marker for this SNP, we analyzed the distribution of the SNP in 694 cultivars, including 117 Japanese cultivars and 551 cultivars from a wheat core collection. The Zen-type SNP predominantly occurs in cultivars used in Japan, where the rainy season overlaps with the wheat harvest season. We also sequenced the ~800 bp genomic fragment amplified by the CAPS marker and categorized the sequences into 16 groups, G1–G16. Only G11 has the Zen-type SNP; all the others have the CS-type SNP. This indicates that the CS-type allele (T) is ancestral, and during cultivation, nucleotide substitution occurred to give the Zen-type allele (C), which has been adopted to improve pre-harvest sprouting tolerance. Phylogenetic analysis indicates that G11 is derived from G3, which occurs worldwide. Our results suggest that the CAPS marker provides a valuable tool to select the Zen-type MFT-3A allele in wheat breeding programs for improving pre-harvest sprouting tolerance.
[Show abstract][Hide abstract] ABSTRACT: We elucidated the genome sequence of Glycine max cv. Enrei to provide a reference for characterization of Japanese domestic soybean cultivars. The whole genome sequence obtained using a next-generation sequencer was used for reference mapping into the current genome assembly of G. max cv. Williams 82 obtained by the Soybean Genome Sequencing Consortium in the USA. After sequencing and assembling the whole genome shotgun reads, we obtained a data set with about 928 Mbs total bases and 60,838 gene models. Phylogenetic analysis provided glimpses into the ancestral relationships of both cultivars and their divergence from the complex that include the wild relatives of soybean. The gene models were analyzed in relation to traits associated with anthocyanin and flavonoid biosynthesis and an overall profile of the proteome. The sequence data are made available in DAIZUbase in order to provide a comprehensive informatics resource for comparative genomics of a wide range of soybean cultivars in Japan and a reference tool for improvement of soybean cultivars worldwide.
International Journal of Genomics 07/2015; 2015:358127. DOI:10.1155/2015/358127 · 0.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Bunching onion (Allium fistulosum L.) is one of the most important vegetables in Japan. Although expressed sequence tag (EST)-derived markers for bulb onion (A. cepa L.) have been developed from medium-scale sequencing, comparable EST sequences in bunching onion are lacking. In this study, we obtained 54,903 bunching onion unigenes using transcriptome shotgun assembly (TSA) and two next-generation sequencing technologies, GS-FLX and HiSeq 2000. When bunching onion and bulb onion unigenes were compared, 10,688 were estimated as reciprocal best-hit relationships. In the bunching onion TSA sequences, we discovered 2,396 di- to pentanucleotide simple sequence repeat (SSR) motifs and 5,505 exon-intron boundary sites. Moreover, we detected 9,002 single nucleotide polymorphisms and 4,335 insertion-deletion (InDel) by comparing sequence reads obtained from two inbred lines, “F” and “A.” TSA-derived SSR, cleaved amplified polymorphic sequences, InDels and intron-spanning markers were used to develop a linkage map. The genetic map, designated the FA map, contained 17 linkage groups with 364 markers (190 bunching onion TSAs, 96 bunching onion genomic SSRs, 39 bulb onion ESTs and 4 other markers) and covered a distance of 1,150 cM.
[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, >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 unwanted mutations by backcrossing.
[Show abstract][Hide abstract] ABSTRACT: Background
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.
Sorghum populations derived from Nakei-MS3B and M36001 accumulated apigeninidin, or both apigeninidin and luteolinidin, in different proportions in lesions caused by B. sorghicola infection, suggesting that the relative proportions of the two 3-deoxyanthocyanidins determine color variation. QTL analysis and genomic sequencing indicated that two closely linked loci on chromosome 4, containing the flavonoid 3′-hydroxylase (F3′H) and Tannin1 (Tan1) genes, were responsible for the lesion color variation. The F3′H locus in Nakei-MS3B had a genomic deletion resulting in the fusion of two tandemly arrayed F3′H genes. The recessive allele at the Tan1 locus derived from M36001 had a genomic insertion and encoded a non-functional WD40 repeat transcription factor. Whole-mRNA sequencing revealed that expression of the fused F3′H gene was conspicuously induced in purple sorghum lines. The levels of expression of F3′H matched the relative proportions of apigeninidin and luteolinidin.
Expression of F3′H is responsible for the synthesis of luteolinidin; the expression level of this gene is therefore critical in determining color variation in sorghum leaves infected with B. sorghicola.
Electronic supplementary material
The online version of this article (doi:10.1186/1756-0500-7-761) contains supplementary material, which is available to authorized users.
BMC Research Notes 10/2014; 7(1):761. DOI:10.1186/1756-0500-7-761
[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 is 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-labeled
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, using hairy root transformation, that the rj4/rj4 genotype soybeans were fully complemented by the expression of the Rj4 gene. Whereas the expression of many TLPs and other PR proteins is induced by biotic/abiotic stress, Rj4 gene expression appears to be constitutive in roots including root nodules.
[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: 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. DOI:10.1371/journal.pone.0096946 · 3.23 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.
[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.
DNA Research 02/2014; 21(4). DOI:10.1093/dnares/dsu006 · 5.48 Impact Factor
[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: 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.