A Koshihikari X Oryza rufipogon Introgression Line with a High Capacity to Take up Nitrogen to Maintain Growth and Panicle Development under Low Nitrogen Conditions.

Nitrogen (N) is an important macronutrient for plant growth and development. Currently, N fertilizers are required for the efficient production of modern crops such as rice due to their limited capacity to take up N when present at low concentrations. Wild rice represents a useful genetic resource for improving crop responses to low nutrient stress. Here, we describe the isolation and characterization of an introgression line, KRIL37, that carries a small region of the Oryza rufipogon genome in the Oryza sativa L. cv Koshihikari (KH) background. This line was found to grow better under low N conditions and have similar or lower C/N ratios in aerial portions compared to those in the parental KH cultivar, suggesting that KRIL37 has a higher capacity to take up and assimilate N when present at low concentrations. KRIL37 performance in the field was also better than that of KH cultivated without N and fertilizer (-F). Transcriptome analyses of 3-week-old seedlings based on RNA-sequencing revealed that KH induced a wider suite of genes than the tolerant line KRIL37 in response to low N conditions. Some ammonium transporters and N assimilation genes were found to be induced under low N in KRIL37, but not in KH. Our findings suggest that the superior growth performance of KRIL37 under limited N conditions could be due to the expression of wild alleles influencing N uptake and assimilation. Our study demonstrates the potential to use wild rice genomes to improve modern crops for low nutrient tolerance.

Genome-wide marker development for the wheat D genome based on single nucleotide polymorphisms identified from transcripts in the wild wheat progenitor Aegilops tauschii.

KEY MESSAGE: 13,347 high-confidence SNPs were discovered through transcriptome sequencing of Aegilops tauschii, which are useful for genomic analysis and molecular breeding of hexaploid wheat. In organisms with large and complex genomes, such as wheat, RNA-seq analysis is cost-effective for discovery of genome-wide single nucleotide polymorphisms (SNPs). In this study, deep sequencing of the spike transcriptome from two Aegilops tauschii accessions representing two major lineages led to the discovery of 13,347 high-confidence (HC) SNPs in 4,872 contigs. After removing redundant SNPs detected in the leaf transcriptome from the same accessions in an earlier study, 10,589 new SNPs were discovered. In total, 5,642 out of 5,808 contigs with HC SNPs were assigned to the Ae. tauschii draft genome sequence. On average, 732 HC polymorphic contigs were mapped in silico to each Ae. tauschii chromosome. Based on the polymorphic data, we developed markers to target the short arm of chromosome 2D and validated the polymorphisms using 20 Ae. tauschii accessions. Of the 29 polymorphic markers, 28 were successfully mapped to 2DS in the diploid F2 population of Ae. tauschii. Among ten hexaploid wheat lines, which included wheat synthetics and common wheat cultivars, 25 of the 43 markers were polymorphic. In the hexaploid F2 population between a common wheat cultivar and a synthetic wheat line, 23 of the 25 polymorphic markers between the parents were available for genotyping of the F2 plants and 22 markers mapped to chromosome 2DS. These results indicate that molecular markers that developed from polymorphisms between two distinct lineages of Ae. tauschii might be useful for analysis not only of the diploid, but also of the hexaploid wheat genome.

Discovery of high-confidence single nucleotide polymorphisms from large-scale de novo analysis of leaf transcripts of Aegilops tauschii, a wild wheat progenitor.

Construction of high-resolution genetic maps is important for genetic and genomic research, as well as for molecular breeding. Single nucleotide polymorphisms (SNPs) are the predominant class of genetic variation and can be used as molecular markers. Aegilops tauschii, the D-genome donor of common wheat, is considered a valuable genetic resource for wheat improvement. Our previous study implied that Ae. tauschii accessions can be genealogically divided into two major lineages. In this study, the transcriptome of two Ae. tauschii accessions from each lineage, lineage 1 (L1) and 2 (L2), was sequenced, yielding 9435 SNPs and 739 insertion/deletion polymorphisms (indels) after de novo assembly of the reads. Based on 36 contig sequences, 31 SNPs and six indels were validated on 20 diverse Ae. tauschii accessions. Because almost all of the SNP markers were polymorphic between L1 and L2, and the D-genome donor of common wheat is presumed to belong to L2, these markers are available for D-genome typing in crosses between common wheat varieties and L1-derived synthetic wheat. Due to the conserved synteny between wheat and barley chromosomes, the high-density expressed sequence tag barley map and the hypothetical gene order in barley can be applied to develop markers on target chromosomal regions in wheat.

The shoot is important for high-affinity nitrate uptake in Egeria densa, a submerged vascular plant.

To understand the mechanisms of nitrate uptake by submerged vascular plants, a cDNA for a high-affinity nitrate transporter, NRT2, was isolated from Egeria densa, a submerged monocot. The deduced EdNRT2 protein was similar to the proteins of a conserved NRT2 group in higher plants. Real-time reverse transcription-PCR analysis revealed that after feeding whole plants with 0.2 mM nitrate, the EdNRT2 transcripts were induced in both shoots and roots within 0.5 h, reached the maximum by 1-3 h and then decreased. The EdNRT2 transcript levels in shoots were comparable to those in roots. When nitrate was applied separately to shoots and roots, the EdNRT2 transcripts were induced only in nitrate-treated organs and reached the maximum levels comparable to those in organs when nitrate was applied to whole plants. (15)N-nitrate feeding experiments demonstrated that both shoots and roots are responsible for nitrate uptake and that biomass and (15)N content in shoots was even higher than that in roots. We concluded that EdNRT2 is involved in high-affinity nitrate uptake by shoots and roots of E. densa, that nitrate is taken up independently by shoots and roots and that shoots play an important role in nitrate uptake from aquatic ecosystem.

Suppression of a NAC-like transcription factor gene improves boron-toxicity tolerance in rice.

We identified a gene responsible for tolerance to boron (B) toxicity in rice (Oryza sativa), named BORON EXCESS TOLERANT1. Using recombinant inbred lines derived from the B-toxicity-sensitive indica-ecotype cultivar IR36 and the tolerant japonica-ecotype cultivar Nekken 1, the region responsible for tolerance to B toxicity was narrowed to 49 kb on chromosome 4. Eight genes are annotated in this region. The DNA sequence in this region was compared between the B-toxicity-sensitive japonica cultivar Wataribune and the B-toxicity-tolerant japonica cultivar Nipponbare by eco-TILLING analysis and revealed a one-base insertion mutation in the open reading frame sequence of the gene Os04g0477300. The gene encodes a NAC (NAM, ATAF, and CUC)-like transcription factor and the function of the transcript is abolished in B-toxicity-tolerant cultivars. Transgenic plants in which the expression of Os04g0477300 is abolished by RNA interference gain tolerance to B toxicity.

OryzaExpress: an integrated database of gene expression networks and omics annotations in rice.

Similarity of gene expression profiles provides important clues for understanding the biological functions of genes, biological processes and metabolic pathways related to genes. A gene expression network (GEN) is an ideal choice to grasp such expression profile similarities among genes simultaneously. For GEN construction, the Pearson correlation coefficient (PCC) has been widely used as an index to evaluate the similarities of expression profiles for gene pairs. However, calculation of PCCs for all gene pairs requires large amounts of both time and computer resources. Based on correspondence analysis, we developed a new method for GEN construction, which takes minimal time even for large-scale expression data with general computational circumstances. Moreover, our method requires no prior parameters to remove sample redundancies in the data set. Using the new method, we constructed rice GENs from large-scale microarray data stored in a public database. We then collected and integrated various principal rice omics annotations in public and distinct databases. The integrated information contains annotations of genome, transcriptome and metabolic pathways. We thus developed the integrated database OryzaExpress for browsing GENs with an interactive and graphical viewer and principal omics annotations (http://riceball.lab.nig.ac.jp/oryzaexpress/). With integration of Arabidopsis GEN data from ATTED-II, OryzaExpress also allows us to compare GENs between rice and Arabidopsis. Thus, OryzaExpress is a comprehensive rice database that exploits powerful omics approaches from all perspectives in plant science and leads to systems biology.

Changes in the gene expression of the white rot fungus Phanerochaete chrysosporium due to the addition of atropine.

We constructed a LongSAGE (Long Serial Analysis of Gene Expression) library from a 3-d culture of Phanerochaete chrysosporium supplemented with atropine, which inhibits the production of lignin-degrading enzymes. The library (the atropine library) contains 13,108 LongSAGE tags and 6,783 unique tags. The gene expression profile represented by the tags was compared with those of two previously constructed libraries, one of which was constructed using 2-d cultures in which the fungus had not yet produced ligninolytic enzymes (the 2-d library) and the other was constructed using 3-d cultures in which the fungus had just started to produce the enzymes (the 3-d library). We found a total of 595 genes that were at least twice more highly or at least twice less highly expressed in the 3-d library than in the 2-d library or the atropine library, and the fluctuations were statistically significant. The relationships among these 595 genes were considered using cluster analysis. Of the 595 genes, 164 showed expression patterns similar to those of four ligninolytic enzyme genes, which were more expressed on day 3 than under any other conditions. Many of these 164 genes comprised genes possibly involved in lignin degradation, lipid metabolism, xenobiotic degradation, stress response, or signal transduction pathways.

Genetic analysis of root elongation induced by phosphorus deficiency in rice (Oryza sativa L.): fine QTL mapping and multivariate analysis of related traits.

Root elongation induced by phosphorus deficiency has been reported as one of the adaptive mechanisms in plants. Genetic differences were found in rice for the root elongation under phosphorus deficiency (REP), for which a distinct quantitative trait locus (QTL) was detected on the long arm of chromosome 6. Subsequently, the effect and position of the QTL, designated as qREP-6, were confirmed using chromosome segment substitution lines (CSSLs), in which the background of a japonica cultivar, 'Nipponbare' with non-REP, was partially substituted by chromosomal segments from an indica cultivar, 'Kasalath' with remarkable REP. Out of 54 CSSLs, two lines (CSSL28 and CSSL29) that retain a common 'Kasalath'-derived segment on the long arm of chromosome 6 showed a significantly high REP. The high REP lines also showed high adaptabilities such as enhanced tillering ability and shoot phosphorus content. Accordingly, conditional dependencies between the related traits were assessed using a graphical Gaussian model (GGM). Direct interactions between REP and root length, and between root length and tiller number were detected under P deficiency in CSSLs. Furthermore, qREP-6 for REP and qTNP-6 for tiller number under P deficiency were fine-mapped with an F(2) population of a cross between Nipponbare and CSSL29. A region containing qREP-6 accounted for more than half of the phenotypic variance, the most plausible interval of which contained 37 candidate genes. The result provides a foundation for cloning of the qREP-6 gene which will be applicable to study P deficiency-dependent response and to improve rice's adaptability to P deficiency stress.

A new method for gene discovery in large-scale microarray data.

Microarrays are an effective tool for monitoring genome-wide gene expression levels. In current microarray analyses, the majority of genes on arrays are frequently eliminated for further analysis because the changes in their expression levels (ratios) are considered to be not significant. This strategy risks failure to discover whole sets of genes related to a quantitative trait of interest, which is generally controlled by several loci that make various contributions. Here, we describe a high-throughput gene discovery method based on correspondence analysis with a new index for expression ratios [arctan (1/ratio)] and three artificial marker genes. This method allows us to quickly analyze the whole microarray dataset and discover up-/down-regulated genes related to a trait of interest. We employed an example dataset to show the theoretical advantage of this method. We then used the method to identify 88 cancer-related genes from a published microarray data from patients with breast cancer. This method also allows us to predict the phenotype of a given sample from the gene expression profile. This method can be easily performed and the result is also visible in 3D viewing software that we have developed.

Phosphorus deficiency-induced root elongation and its QTL in rice (Oryza sativa L.).

A significant level of root elongation was induced in rice (Oryza sativa) grown under phosphorus-deficient conditions. The root elongation clearly varied among a total of 62 varieties screened under two different phosphorus levels. Two contrasting varieties, 'Gimbozu', with a low elongating response and 'Kasalath', with a high elongating response, were chosen and crossed to produce a hybrid population for QTL analyses. QTLs for the phosphorus deficiency-induced root elongation were detected by two linkage maps, i.e., one with 82 F3 families constructed by 97 simple sequence repeat (SSR) and sequence-tag site markers and another with 97 F8 lines by 790 amplified fragment length polymorphism and SSR markers. A single QTL for the elongation response was detected on chromosome 6, with a LOD score of 4.5 in both maps and explained about 20% of total phenotypic variance. In addition, this QTL itself, or a region tightly linked with it, partly explained an ability to reduce accumulation of excess iron in the shoots. The identified QTL will be useful to improve rice varieties against a complex nutritional disorder caused by phosphorus deficiency and iron toxicity.