Background: Accurate detection of polymorphisms with a next generation sequencer data is an impor... more Background: Accurate detection of polymorphisms with a next generation sequencer data is an important element of current genetic analysis. However, there is still no detection pipeline that is completely reliable. Result: We demonstrate two new detection methods of polymorphisms focusing on the Polymorphic Edge (PED). In the matching between two homologous sequences, the first mismatched base to appear is the SNP, or the edge of the structural variation. The first method is based on k-mers from short reads and detects polymorphic edges with k-mers for which there is no match between target and control, making it possible to detect SNPs by direct comparison of short-reads in two datasets (target and control) without a reference genome sequence. The second method is based on bidirectional alignment to detect polymorphic edges, not only SNPs but also insertions, deletions, inversions and translocations. Using these two methods, we succeed in making a high-quality comparison map between rice cultivars showing good match to the theoretical value of introgression, and in detecting specific large deletions across cultivars. Conclusions: Using Polymorphic Edge Detection (PED), the k-mer method is able to detect SNPs by direct comparison of short-reads in two datasets without genomic alignment step, and the bidirectional alignment method is able to detect SNPs and structural variations from even single-end short-reads. The PED is an efficient tool to obtain accurate data for both SNPs and structural variations.
Rice is a major crop feeding the majority of the global population, and increasing its sink stren... more Rice is a major crop feeding the majority of the global population, and increasing its sink strength is one of the modes to alleviate the declining availability of food for the rapidly growing world population. We demonstrate a role for an important rice vacuolar invertase isoform, OsINV3, in sink strength determination. OsINV3 mutants showed shorter panicles with lighter and smaller grains, owing to a smaller cell size on the outer and inner surfaces of the palea and lemma as observed by scanning electron microscopy. Further, strong promoter::GUS expression was observed in the palea, lemma and the rachis branches in the young elongating panicles, which supported the role of OsINV3 in cell expansion and thus, in spikelet size and panicle length determination. Size of the spikelet was found to directly influence the grain weight, which was confirmed by the lack of differences in weights of hulled grain for differently segregated alleles in the heterozygous lines. Assessment of field ...
Acta Crystallographica Section B Structural Crystallography and Crystal Chemistry
The structure of 8-thioxoadenosine monohydrate, C10HtaNsOaS. H20 , has been determined. The cryst... more The structure of 8-thioxoadenosine monohydrate, C10HtaNsOaS. H20 , has been determined. The crystals are monoclinic, space group P2~, with a = 8.758 (1), b = 6.717(1), c = 11.462 (2) A, /] = 95.22(1) °, Z = 2. The structure was refined to R = 0.048. The conformation about C (4')-C (5') is gauche-trans, and the puckering of the ribose is C(2')-endo-C(3')-exo. The molecule exhibits a syn conformation about the glycosyl bond in spite of the absence of an intramolecular O(5')-H... N(3) hydrogen bond. The bases are stacked into columns, overlapping mainly at the S substituents of adjacent molecules. All the donor atoms are involved in hydrogen bonds, while the S atom acts as a hydrogen-bond acceptor.
Lateral roots play an important role in the acquisition of nutrients and anchorage of the whole p... more Lateral roots play an important role in the acquisition of nutrients and anchorage of the whole plant. To better understand the mechanisms underlying lateral root development, we isolated a new lateral-rootless mutant lrt2 in screening for 2,4-dichlorophenoxyacetic acid (2,4-D) resistance in M 2 lines of rice (Oryza sativa L. cv. Nipponbare) generated by tissue culture. lrt2 failed to form lateral roots and exhibited altered root response to gravity. Analysis for auxin resistance showed that lrt2 was less sensitive to various auxins including 2,4-D, indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA) compared with wild type, but was similarly sensitive to auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). This suggests that the reduced sensitivity to auxin in lrt2 might be caused by a disruption in auxin response rather than in auxin transport. Genetic analysis indicated that the lateral-rootless phenotype of lrt2 is due to a recessive mutation. To map the lrt2 gene, we tested molecular markers by bulk segregant analysis. The lrt2 gene was localized to a 10.8 cM interval on the short arm of chromosome 2, flanked by two sequence-tagged site (STS) markers Lrt2P1 and Lrt2P2.
We evaluated a large collection of Tos17 mutant panel lines for their reaction to three different... more We evaluated a large collection of Tos17 mutant panel lines for their reaction to three different races of Magnaporthe oryzae and identified a lesion mimic mutant, NF4050-8, that showed lesions similar to naturally occurring spl5 mutant and enhanced resistance to all the three blast races tested. Nested modified-AFLP using Tos17-specific primers and southern hybridization experiments of segregating individuals indicated that the lesion mimic phenotype in NF4050-8 is most likely due to a nucleotide change acquired during the culturing process and not due to Tos17 insertion per se. Inheritance and genetic analyses in two japonica 9 indica populations identified an overlapping genomic region of 13 cM on short arm of chromosome 7 that was linked with the lesion mimic phenotype. High-resolution genetic mapping using 950 F 3 and 3,821 F 4 plants of NF4050-8 9 CO39 delimited a 35 kb region flanked by NBARC1 (5.262 Mb) and RM8262 (5.297 Mb), which contained 6 ORFs; 3 of them were 'resistance gene related' with typical NBS-LRR signatures. One of them harbored a NB-ARC domain, which had been previously demonstrated to be associated with cell death in animals. Microarray analysis of NF4050-8 revealed significant up-regulation of numerous defense/ pathogenesis-related genes and down-regulation of heme peroxidase genes. Real-time PCR analysis of WRKY45 and PR1b genes suggested possible constitutive activation of a defense signaling pathway downstream of salicylic acid but independent of NH1 in these mutant lines of rice. Communicated by J.-B. Veyrieras.
ABSTRACT We applied the full-length cDNA overexpressor (FOX) gene-hunting system for systematic a... more ABSTRACT We applied the full-length cDNA overexpressor (FOX) gene-hunting system for systematic and genome-wide functional analysis of rice genes. In this study, we constructed a novel binary vector carrying the Gateway site-specific recombination cassette and then constructed rice FOX libraries containing a maximum of 13,823 independent, full-length cDNAs (fl-cDNAs) that correspond to approximately half the total number of rice fl-cDNA clones. By introducing the FOX libraries via Agrobacterium, we generated 2,586 FOX-rice lines exhibiting various visible alterations (e.g., plant height, tillers, leaves, and heading dates). The introduced fl-cDNAs, integrated into individual transgenic rice genomes, were amplified by genomic PCR and identified using sequencing analysis. The fl-cDNAs were PCR-amplified in 2,251 (94.2%) of the 2,389 FOX-rice lines that were examined, identifying 1,920 independent fl-cDNAs in the FOX lines. In addition to the previously generated FOX-rice plants, our new collection of FOX-rice lines produced through the Gateway system should be a useful tool for the efficient identification of gene functions in rice. Moreover, this Gateway-based technology should be applicable to other species in which a collection of fl-cDNA clones is available.
Background: Accurate detection of polymorphisms with a next generation sequencer data is an impor... more Background: Accurate detection of polymorphisms with a next generation sequencer data is an important element of current genetic analysis. However, there is still no detection pipeline that is completely reliable. Result: We demonstrate two new detection methods of polymorphisms focusing on the Polymorphic Edge (PED). In the matching between two homologous sequences, the first mismatched base to appear is the SNP, or the edge of the structural variation. The first method is based on k-mers from short reads and detects polymorphic edges with k-mers for which there is no match between target and control, making it possible to detect SNPs by direct comparison of short-reads in two datasets (target and control) without a reference genome sequence. The second method is based on bidirectional alignment to detect polymorphic edges, not only SNPs but also insertions, deletions, inversions and translocations. Using these two methods, we succeed in making a high-quality comparison map between rice cultivars showing good match to the theoretical value of introgression, and in detecting specific large deletions across cultivars. Conclusions: Using Polymorphic Edge Detection (PED), the k-mer method is able to detect SNPs by direct comparison of short-reads in two datasets without genomic alignment step, and the bidirectional alignment method is able to detect SNPs and structural variations from even single-end short-reads. The PED is an efficient tool to obtain accurate data for both SNPs and structural variations.
Rice is a major crop feeding the majority of the global population, and increasing its sink stren... more Rice is a major crop feeding the majority of the global population, and increasing its sink strength is one of the modes to alleviate the declining availability of food for the rapidly growing world population. We demonstrate a role for an important rice vacuolar invertase isoform, OsINV3, in sink strength determination. OsINV3 mutants showed shorter panicles with lighter and smaller grains, owing to a smaller cell size on the outer and inner surfaces of the palea and lemma as observed by scanning electron microscopy. Further, strong promoter::GUS expression was observed in the palea, lemma and the rachis branches in the young elongating panicles, which supported the role of OsINV3 in cell expansion and thus, in spikelet size and panicle length determination. Size of the spikelet was found to directly influence the grain weight, which was confirmed by the lack of differences in weights of hulled grain for differently segregated alleles in the heterozygous lines. Assessment of field ...
Acta Crystallographica Section B Structural Crystallography and Crystal Chemistry
The structure of 8-thioxoadenosine monohydrate, C10HtaNsOaS. H20 , has been determined. The cryst... more The structure of 8-thioxoadenosine monohydrate, C10HtaNsOaS. H20 , has been determined. The crystals are monoclinic, space group P2~, with a = 8.758 (1), b = 6.717(1), c = 11.462 (2) A, /] = 95.22(1) °, Z = 2. The structure was refined to R = 0.048. The conformation about C (4')-C (5') is gauche-trans, and the puckering of the ribose is C(2')-endo-C(3')-exo. The molecule exhibits a syn conformation about the glycosyl bond in spite of the absence of an intramolecular O(5')-H... N(3) hydrogen bond. The bases are stacked into columns, overlapping mainly at the S substituents of adjacent molecules. All the donor atoms are involved in hydrogen bonds, while the S atom acts as a hydrogen-bond acceptor.
Lateral roots play an important role in the acquisition of nutrients and anchorage of the whole p... more Lateral roots play an important role in the acquisition of nutrients and anchorage of the whole plant. To better understand the mechanisms underlying lateral root development, we isolated a new lateral-rootless mutant lrt2 in screening for 2,4-dichlorophenoxyacetic acid (2,4-D) resistance in M 2 lines of rice (Oryza sativa L. cv. Nipponbare) generated by tissue culture. lrt2 failed to form lateral roots and exhibited altered root response to gravity. Analysis for auxin resistance showed that lrt2 was less sensitive to various auxins including 2,4-D, indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA) compared with wild type, but was similarly sensitive to auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). This suggests that the reduced sensitivity to auxin in lrt2 might be caused by a disruption in auxin response rather than in auxin transport. Genetic analysis indicated that the lateral-rootless phenotype of lrt2 is due to a recessive mutation. To map the lrt2 gene, we tested molecular markers by bulk segregant analysis. The lrt2 gene was localized to a 10.8 cM interval on the short arm of chromosome 2, flanked by two sequence-tagged site (STS) markers Lrt2P1 and Lrt2P2.
We evaluated a large collection of Tos17 mutant panel lines for their reaction to three different... more We evaluated a large collection of Tos17 mutant panel lines for their reaction to three different races of Magnaporthe oryzae and identified a lesion mimic mutant, NF4050-8, that showed lesions similar to naturally occurring spl5 mutant and enhanced resistance to all the three blast races tested. Nested modified-AFLP using Tos17-specific primers and southern hybridization experiments of segregating individuals indicated that the lesion mimic phenotype in NF4050-8 is most likely due to a nucleotide change acquired during the culturing process and not due to Tos17 insertion per se. Inheritance and genetic analyses in two japonica 9 indica populations identified an overlapping genomic region of 13 cM on short arm of chromosome 7 that was linked with the lesion mimic phenotype. High-resolution genetic mapping using 950 F 3 and 3,821 F 4 plants of NF4050-8 9 CO39 delimited a 35 kb region flanked by NBARC1 (5.262 Mb) and RM8262 (5.297 Mb), which contained 6 ORFs; 3 of them were 'resistance gene related' with typical NBS-LRR signatures. One of them harbored a NB-ARC domain, which had been previously demonstrated to be associated with cell death in animals. Microarray analysis of NF4050-8 revealed significant up-regulation of numerous defense/ pathogenesis-related genes and down-regulation of heme peroxidase genes. Real-time PCR analysis of WRKY45 and PR1b genes suggested possible constitutive activation of a defense signaling pathway downstream of salicylic acid but independent of NH1 in these mutant lines of rice. Communicated by J.-B. Veyrieras.
ABSTRACT We applied the full-length cDNA overexpressor (FOX) gene-hunting system for systematic a... more ABSTRACT We applied the full-length cDNA overexpressor (FOX) gene-hunting system for systematic and genome-wide functional analysis of rice genes. In this study, we constructed a novel binary vector carrying the Gateway site-specific recombination cassette and then constructed rice FOX libraries containing a maximum of 13,823 independent, full-length cDNAs (fl-cDNAs) that correspond to approximately half the total number of rice fl-cDNA clones. By introducing the FOX libraries via Agrobacterium, we generated 2,586 FOX-rice lines exhibiting various visible alterations (e.g., plant height, tillers, leaves, and heading dates). The introduced fl-cDNAs, integrated into individual transgenic rice genomes, were amplified by genomic PCR and identified using sequencing analysis. The fl-cDNAs were PCR-amplified in 2,251 (94.2%) of the 2,389 FOX-rice lines that were examined, identifying 1,920 independent fl-cDNAs in the FOX lines. In addition to the previously generated FOX-rice plants, our new collection of FOX-rice lines produced through the Gateway system should be a useful tool for the efficient identification of gene functions in rice. Moreover, this Gateway-based technology should be applicable to other species in which a collection of fl-cDNA clones is available.
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Papers by Akio Miyao