Antagonistic regulation of the gibberellic acid response during stem growth in rice.

The size of plants is largely determined by growth of the stem. Stem elongation is stimulated by gibberellic acid1-3. Here we show that internode stem elongation in rice is regulated antagonistically by an 'accelerator' and a 'decelerator' in concert with gibberellic acid. Expression of a gene we name ACCELERATOR OF INTERNODE ELONGATION 1 (ACE1), which encodes a protein of unknown function, confers cells of the intercalary meristematic region with the competence for cell division, leading to internode elongation in the presence of gibberellic acid. By contrast, upregulation of DECELERATOR OF INTERNODE ELONGATION 1 (DEC1), which encodes a zinc-finger transcription factor, suppresses internode elongation, whereas downregulation of DEC1 allows internode elongation. We also show that the mechanism of internode elongation that is mediated by ACE1 and DEC1 is conserved in the Gramineae family. Furthermore, an analysis of genetic diversity suggests that mutations in ACE1 and DEC1 have historically contributed to the selection of shorter plants in domesticated populations of rice to increase their resistance to lodging, and of taller plants in wild species of rice for adaptation to growth in deep water. Our identification of these antagonistic regulatory factors enhances our understanding of the gibberellic acid response as an additional mechanism that regulates internode elongation and environmental fitness, beyond biosynthesis and gibberellic acid signal transduction.

Regulator of Awn Elongation 3, an E3 ubiquitin ligase, is responsible for loss of awns during African rice domestication.

Two species of rice have been independently domesticated from different ancestral wild species in Asia and Africa. Comparison of mutations that underlie phenotypic and physiological alterations associated with domestication traits in these species gives insights into the domestication history of rice in both regions. Asian cultivated rice, Oryza sativa, and African cultivated rice, Oryza glaberrima, have been modified and improved for common traits beneficial for humans, including erect plant architecture, nonshattering seeds, nonpigmented pericarp, and lack of awns. Independent mutations in orthologous genes associated with these traits have been documented in the two cultivated species. Contrary to this prevailing model, selection for awnlessness targeted different genes in O. sativa and O. glaberrima. We identify Regulator of Awn Elongation 3 (RAE3) a gene that encodes an E3 ubiquitin ligase and is responsible for the awnless phenotype only in O. glaberrima. A 48-bp deletion may disrupt the substrate recognition domain in RAE3 and diminish awn elongation. Sequencing analysis demonstrated low nucleotide diversity in a ~600-kb region around the derived rae3 allele on chromosome 6 in O. glaberrima compared with its wild progenitor. Identification of RAE3 sheds light on the molecular mechanism underlying awn development and provides an example of how selection on different genes can confer the same domestication phenotype in Asian and African rice.

Effectiveness of Create ML in microscopy image classifications: a simple and inexpensive deep learning pipeline for non-data scientists.

Observing chromosomes is a time-consuming and labor-intensive process, and chromosomes have been analyzed manually for many years. In the last decade, automated acquisition systems for microscopic images have advanced dramatically due to advances in their controlling computer systems, and nowadays, it is possible to automatically acquire sets of tiling-images consisting of large number, more than 1000, of images from large areas of specimens. However, there has been no simple and inexpensive system to efficiently select images containing mitotic cells among these images. In this paper, a classification system of chromosomal images by deep learning artificial intelligence (AI) that can be easily handled by non-data scientists was applied. With this system, models suitable for our own samples could be easily built on a Macintosh computer with Create ML. As examples, models constructed by learning using chromosome images derived from various plant species were able to classify images containing mitotic cells among samples from plant species not used for learning in addition to samples from the species used. The system also worked for cells in tissue sections and tetrads. Since this system is inexpensive and can be easily trained via deep learning using scientists' own samples, it can be used not only for chromosomal image analysis but also for analysis of other biology-related images.

Loss of function at RAE2, a previously unidentified EPFL, is required for awnlessness in cultivated Asian rice.

Domestication of crops based on artificial selection has contributed numerous beneficial traits for agriculture. Wild characteristics such as red pericarp and seed shattering were lost in both Asian (Oryza sativa) and African (Oryza glaberrima) cultivated rice species as a result of human selection on common genes. Awnedness, in contrast, is a trait that has been lost in both cultivated species due to selection on different sets of genes. In a previous report, we revealed that at least three loci regulate awn development in rice; however, the molecular mechanism underlying awnlessness remains unknown. Here we isolate and characterize a previously unidentified EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) family member named REGULATOR OF AWN ELONGATION 2 (RAE2) and identify one of its requisite processing enzymes, SUBTILISIN-LIKE PROTEASE 1 (SLP1). The RAE2 precursor is specifically cleaved by SLP1 in the rice spikelet, where the mature RAE2 peptide subsequently induces awn elongation. Analysis of RAE2 sequence diversity identified a highly variable GC-rich region harboring multiple independent mutations underlying protein-length variation that disrupt the function of the RAE2 protein and condition the awnless phenotype in Asian rice. Cultivated African rice, on the other hand, retained the functional RAE2 allele despite its awnless phenotype. Our findings illuminate the molecular function of RAE2 in awn development and shed light on the independent domestication histories of Asian and African cultivated rice.

Rare allele of a previously unidentified histone H4 acetyltransferase enhances grain weight, yield, and plant biomass in rice.

Grain weight is an important crop yield component; however, its underlying regulatory mechanisms are largely unknown. Here, we identify a grain-weight quantitative trait locus (QTL) encoding a new-type GNAT-like protein that harbors intrinsic histone acetyltransferase activity (OsglHAT1). Our genetic and molecular evidences pinpointed the QTL-OsglHAT1's allelic variations to a 1.2-kb region upstream of the gene body, which is consistent with its function as a positive regulator of the traits. Elevated OsglHAT1 expression enhances grain weight and yield by enlarging spikelet hulls via increasing cell number and accelerating grain filling, and increases global acetylation levels of histone H4. OsglHAT1 localizes to the nucleus, where it likely functions through the regulation of transcription. Despite its positive agronomical effects on grain weight, yield, and plant biomass, the rare allele elevating OsglHAT1 expression has so far escaped human selection. Our findings reveal the first example, to our knowledge, of a QTL for a yield component trait being due to a chromatin modifier that has the potential to improve crop high-yield breeding.

Construction of rice chromosome segment substitution lines harboring Oryza barthii genome and evaluation of yield-related traits.

Rice (Oryza sativa L.) is one of the most important staple food in the world. To meet the increasing demand for food, a strategy for improving rice yield is needed. Alleles of wild relatives are useful because they confer adaptation to plants under diverse harsh environments and have the potential to improve rice. O. barthii is a wild rice species endemic to Africa and the known progenitor of the African cultivated rice, O. glaberrima. To explore the genetic potential of the O. barthii as a genetic resource, 40 chromosome segment substitution lines (CSSL) of O. barthii in the background of the elite japonica cultivar Koshihikari were developed and evaluated to identify QTLs associated with 10 traits related to flag leaf morphology, grain yield and other agronomic traits. More than 90% of the entire genome of the donor parent was represented in contiguous or overlapping chromosome segments in the CSSLs. Evaluation of the CSSLs for several agriculturally important traits identified candidate chromosome segments that harbors QTLs associated with yield and yield-related traits. These results suggest that alleles from O. barthii might be used as a novel genetic resource for improving the yield-related traits in cultivars of O. sativa.

A Small Molecule That Represses Translation of G-Quadruplex-Containing mRNA.

The G-quadruplexes form highly stable nucleic acid structures, which are implicated in various biological processes in both DNA and RNA. Although DNA G-quadruplexes have been studied in great detail, biological roles of RNA G-quadruplexes have received less attention. Here, a screening of a chemical library permitted identification of a small-molecule tool that binds selectively to RNA G-quadruplex structures. The polyaromatic molecule, RGB-1, stabilizes RNA G-quadruplex, but not DNA versions or other RNA structures. RGB-1 intensified the G-quadruplex-mediated inhibition of RNA translation in mammalian cells, decreased expression of the NRAS proto-oncogene in breast cancer cells, and permitted identification of a novel sequence that forms G-quadruplex in NRAS mRNA. RGB-1 may serve as a unique tool for understanding cellular roles of RNA G-quadruplex structures.

Development of chromosome segment substitution lines harboring Oryza nivara genomic segments in Koshihikari and evaluation of yield-related traits.

Chromosome segment substitution lines (CSSLs) are rich genetic resources that can be mined for novel, agriculturally useful loci or that can be used directly as materials for breeding. To date, a number of rice CSSLs have been developed by crossing rice cultivars with its wild relatives as a means to tap into the potential of wild alleles in rice improvement. Oryza nivara is a wild relative of rice that is thought to be a progenitor of O. sativa spp. indica. In the present study, 26 CSSLs that covers the entire genome of O. nivara as contiguous, overlapping segments in the genomic background of a japonica cultivar, O. sativa cv. Koshihikari were developed. Evaluation of the CSSLs for several agriculturally important traits identified candidate chromosome segments that harbors QTLs associated with yield and yield-related traits. The results of the study revealed the potential of O. nivara as a source of novel alleles that can be used to improve the existing japonica cultivar.

Development and evaluation of chromosome segment substitution lines (CSSLs) carrying chromosome segments derived from Oryza rufipogon in the genetic background of Oryza sativa L.

The wild relatives of rice (Oryza sativa L.) are useful sources of alleles that have evolved to adapt in diverse environments around the world. Oryza rufipogon, the known progenitor of the cultivated rice, harbors genes that have been lost in cultivated varieties through domestication or evolution. This makes O. rufipogon an ideal source of value-added traits that can be utilized to improve the existing rice cultivars. To explore the potential of the rice progenitor as a genetic resource for improving O. sativa, 33 chromosome segment substitution lines (CSSLs) of O. rufipogon (W0106) in the background of the elite japonica cultivar Koshihikari were developed and evaluated for several agronomic traits. Over 90% of the entire genome was introgressed from the donor parent into the CSSLs. A total of 99 putative QTLs were detected, of which 15 were identified as major effective QTLs that have significantly large effects on the traits examined. Among the 15 major effective QTLs, a QTL on chromosome 10 showed a remarkable positive effect on the number of grains per panicle. Comparison of the putative QTLs identified in this study and previous studies indicated a wide genetic diversity between O. rufipogon accessions.

MCPtaggR: R package for accurate genotype calling in reduced representation sequencing data by eliminating error-prone markers based on genome comparison.

Reduced representation sequencing (RRS) offers cost-effective, high-throughput genotyping platforms such as genotyping-by-sequencing (GBS). RRS reads are typically mapped onto a reference genome. However, mapping reads harbouring mismatches against the reference can potentially result in mismapping and biased mapping, leading to the detection of error-prone markers that provide incorrect genotype information. We established a genotype-calling pipeline named mappable collinear polymorphic tag genotyping (MCPtagg) to achieve accurate genotyping by eliminating error-prone markers. MCPtagg was designed for the RRS-based genotyping of a population derived from a biparental cross. The MCPtagg pipeline filters out error-prone markers prior to genotype calling based on marker collinearity information obtained by comparing the genome sequences of the parents of a population to be genotyped. A performance evaluation on real GBS data from a rice F2 population confirmed its effectiveness. Furthermore, our performance test using a genome assembly that was obtained by genome sequence polishing on an available genome assembly suggests that our pipeline performs well with converted genomes, rather than necessitating de novo assembly. This demonstrates its flexibility and scalability. The R package, MCPtaggR, was developed to provide functions for the pipeline and is available at https://github.com/tomoyukif/MCPtaggR.

Genetic background influences mineral accumulation in rice straw and grains under different soil pH conditions.

Mineral element accumulation in plants is influenced by soil conditions and varietal factors. We investigated the dynamic accumulation of 12 elements in straw at the flowering stage and in grains at the mature stage in eight rice varieties with different genetic backgrounds (Japonica, Indica, and admixture) and flowering times (early, middle, and late) grown in soil with various pH levels. In straw, Cd, As, Mn, Zn, Ca, Mg, and Cu accumulation was influenced by both soil pH and varietal factors, whereas P, Mo, and K accumulation was influenced by pH, and Fe and Ni accumulation was affected by varietal factors. In grains, Cd, As, Mn, Cu, Ni, Mo, Ca, and Mg accumulation was influenced by both pH and varietal factors, whereas Zn, Fe, and P accumulation was affected by varietal factors, and K accumulation was not altered. Only As, Mn, Ca and Mg showed similar trends in the straw and grains, whereas the pH responses of Zn, P, K, and Ni differed between them. pH and flowering time had synergistic effects on Cd, Zn, and Mn in straw and on Cd, Ni, Mo, and Mn in grains. Soil pH is a major factor influencing mineral uptake in rice straw and grains, and genetic factors, flowering stage factors, and their interaction with soil pH contribute in a combined manner.

GBScleanR: robust genotyping error correction using a hidden Markov model with error pattern recognition.

Reduced-representation sequencing (RRS) provides cost-effective and time-saving genotyping platforms. Despite the outstanding advantage of RRS in throughput, the obtained genotype data usually contain a large number of errors. Several error correction methods employing the hidden Markov model (HMM) have been developed to overcome these issues. These methods assume that markers have a uniform error rate with no bias in the allele read ratio. However, bias does occur because of uneven amplification of genomic fragments and read mismapping. In this paper, we introduce an error correction tool, GBScleanR, which enables robust and precise error correction for noisy RRS-based genotype data by incorporating marker-specific error rates into the HMM. The results indicate that GBScleanR improves the accuracy by more than 25 percentage points at maximum compared to the existing tools in simulation data sets and achieves the most reliable genotype estimation in real data even with error-prone markers.

Initial Result of Stroke Care at the Stroke Center in a New Hospital Opened by the Merger of Three Facilities with Different Management Bases: Effect of Stroke Center on Mechanical Thrombectomy.

Objective: The most important function required for the stroke center is prompt treatment for acute stroke. We report the initial results of stroke care under the new medical care system of stroke center in a new hospital that merges three hospitals with different management bases to verify the effect of stroke center on mechanical thrombectomy. Methods: We investigated changes in the number of inpatients and surgical treatments compared with the past 3 years (Stages I, II, and III) with stage IV one year after the new hospital was opened, and examined the effect of establishing a stroke center on mechanical thrombectomy for acute main cerebral artery occlusion. Results: From stage I to stage IV, the number of hospitalized patients increased from 396, 485, 482 to 630, respectively, and the proportion of patients with cerebrovascular disease increased from 57.6%, 55.7%, 60.4% to 68.3%, respectively. Total surgical treatment increased from 137, 195, 224 to 297, respectively, especially endovascular therapy increased markedly from 22, 36, 68 to 118, respectively. The main treatment contents of endovascular treatment in stage IV were ruptured cerebral aneurysm embolization 22 cases, unruptured cerebral aneurysm embolization 13 cases, carotid artery stenting 23 cases, other intracranial or extracranial artery angioplasty/stenting 9 cases, and mechanical thrombectomy 34 cases. In particular, mechanical thrombectomy was significantly increased to 34 in stage IV, compared to 4 in stage I, 4 in stage II, and 17 in stage III (degree of contribution [DC] 25.0%, contribution ratio [CR] 34.0%). Conclusion: With the establishment of the stroke center, the number of cases of acute cerebral infarction within the adaptation time who received mechanical thrombectomy remarkably increased. It is considered that the effect and validity of function aggregation by establishing stroke center are shown.

Cervical spinal cord infarction in a patient with cervical spondylosis triggered by straining during bowel movement.

An 82-year-old man experienced sudden-onset left shoulder pain and hemiparesis, which subsided on the third day. MRI revealed an ischaemic lesion at the C3-4 level together with disk herniation and cord compression from C3/4 to C6/7. On the fourth day, however, similar symptoms recurred when straining during defecation, and then progressed in a fluctuating manner and finally resulted in quadriparesis and respiratory impairment. A follow-up MRI showed fresh infarction of the anterior spinal artery (ASA) territory extending from C2 to C5. Straining during defecation may have induced the second exacerbation by elevating the venous pressure in the craniospinal axis and intervertebral disc pressure and causing minor damage and intraluminal thrombus in the ASA.

Nutrient-Based Chemical Library as a Source of Energy Metabolism Modulators.

Covalent conjugates of multiple nutrients often exhibit greater biological activities than each individual nutrient and more predictable safety profiles than completely unnatural chemical entities. Here, we report the construction and application of a focused chemical library of 308 covalent conjugates of a variety of small-molecule nutrients. Screening of the library with a reporter gene of sterol regulatory element-binding protein (SREBP), a master regulator of mammalian lipogenesis, led to the discovery of a conjugate of docosahexaenoic acid (DHA), glucosamine, and amino acids as an inhibitor of SREBP (molecule 1, DHG). Mechanistic analyses indicate that molecule 1 impairs the SREBP activity by inhibiting glucose transporters and thereby activating AMP-activated protein kinase (AMPK). Oral administration of molecule 1 suppressed the intestinal absorption of glucose in mice. These results suggest that such synthetic libraries of nutrient conjugates serve as a source of novel chemical tools and pharmaceutical seeds that modulate energy metabolism.

Assembling the genome of the African wild rice Oryza longistaminata by exploiting synteny in closely related Oryza species.

The African wild rice species Oryza longistaminata has several beneficial traits compared to cultivated rice species, such as resistance to biotic stresses, clonal propagation via rhizomes, and increased biomass production. To facilitate breeding efforts and functional genomics studies, we de-novo assembled a high-quality, haploid-phased genome. Here, we present our assembly, with a total length of 351 Mb, of which 92.2% was anchored onto 12 chromosomes. We detected 34,389 genes and 38.1% of the genome consisted of repetitive content. We validated our assembly by a comparative linkage analysis and by examining well-characterized gene families. This genome assembly will be a useful resource to exploit beneficial alleles found in O. longistaminata. Our results also show that it is possible to generate a high-quality, functionally complete rice genome assembly from moderate SMRT read coverage by exploiting synteny in a closely related Oryza species.

Ethylene-gibberellin signaling underlies adaptation of rice to periodic flooding.

Most plants do poorly when flooded. Certain rice varieties, known as deepwater rice, survive periodic flooding and consequent oxygen deficiency by activating internode growth of stems to keep above the water. Here, we identify the gibberellin biosynthesis gene, SD1 (SEMIDWARF1), whose loss-of-function allele catapulted the rice Green Revolution, as being responsible for submergence-induced internode elongation. When submerged, plants carrying the deepwater rice-specific SD1 haplotype amplify a signaling relay in which the SD1 gene is transcriptionally activated by an ethylene-responsive transcription factor, OsEIL1a. The SD1 protein directs increased synthesis of gibberellins, largely GA4, which promote internode elongation. Evolutionary analysis shows that the deepwater rice-specific haplotype was derived from standing variation in wild rice and selected for deepwater rice cultivation in Bangladesh.

Adapting Genotyping-by-Sequencing for Rice F2 Populations.

Rapid and cost-effective genotyping of large mapping populations can be achieved by sequencing a reduced representation of the genome of every individual in a given population, and using that information to generate genetic markers. A customized genotyping-by-sequencing (GBS) pipeline was developed to genotype a rice F2 population from a cross of Oryza sativa ssp. japonica cv. Nipponbare and the African wild rice species O. longistaminata While most GBS pipelines aim to analyze mainly homozygous populations, we attempted to genotype a highly heterozygous F2 population. We show how species- and population-specific improvements of established protocols can drastically increase sample throughput and genotype quality. Using as few as 50,000 reads for some individuals (134,000 reads on average), we were able to generate up to 8154 informative SNP markers in 1081 F2 individuals. Additionally, the effects of enzyme choice, read coverage, and data postprocessing are evaluated. Using GBS-derived markers, we were able to assemble a genetic map of 1536 cM. To demonstrate the usefulness of our GBS pipeline, we determined quantitative trait loci (QTL) for the number of tillers. We were able to map four QTL to chromosomes 1, 3, 4, and 8, and partially confirm their effects using introgression lines. We provide an example of how to successfully use GBS with heterozygous F2 populations. By using the comparatively low-cost MiSeq platform, we show that the GBS method is flexible and cost-effective, even for smaller laboratories.

Construction of a versatile SNP array for pyramiding useful genes of rice.

DNA marker-assisted selection (MAS) has become an indispensable component of breeding. Single nucleotide polymorphisms (SNP) are the most frequent polymorphism in the rice genome. However, SNP markers are not readily employed in MAS because of limitations in genotyping platforms. Here the authors report a Golden Gate SNP array that targets specific genes controlling yield-related traits and biotic stress resistance in rice. As a first step, the SNP genotypes were surveyed in 31 parental varieties using the Affymetrix Rice 44K SNP microarray. The haplotype information for 16 target genes was then converted to the Golden Gate platform with 143-plex markers. Haplotypes for the 14 useful allele are unique and can discriminate among all other varieties. The genotyping consistency between the Affymetrix microarray and the Golden Gate array was 92.8%, and the accuracy of the Golden Gate array was confirmed in 3 F2 segregating populations. The concept of the haplotype-based selection by using the constructed SNP array was proofed.