Low mutation rate of spontaneous mutants enables detection of causative genes by comparing whole genome sequences.

In the early 1900s, mutation breeding to select varieties with desirable traits using spontaneous mutation was actively conducted around the world, including Japan. In rice, the number of fixed mutations per generation was estimated to be 1.38-2.25. Although this low mutation rate was a major problem for breeding in those days, in the modern era with the development of next-generation sequencing (NGS) technology, it was conversely considered to be an advantage for efficient gene identification. In this paper, we proposed an in silico approach using NGS to compare the whole genome sequence of a spontaneous mutant with that of a closely related strain with a nearly identical genome, to find polymorphisms that differ between them, and to identify the causal gene by predicting the functional variation of the gene caused by the polymorphism. Using this approach, we found four causal genes for the dwarf mutation, the round shape grain mutation and the awnless mutation. Three of these genes were the same as those previously reported, but one was a novel gene involved in awn formation. The novel gene was isolated from Bozu-Aikoku, a mutant of Aikoku with the awnless trait, in which nine polymorphisms were predicted to alter gene function by their whole-genome comparison. Based on the information on gene function and tissue-specific expression patterns of these candidate genes, Os03g0115700/LOC_Os03g02460, annotated as a short-chain dehydrogenase/reductase SDR family protein, is most likely to be involved in the awnless mutation. Indeed, complementation tests by transformation showed that it is involved in awn formation. Thus, this method is an effective way to accelerate genome breeding of various crop species by enabling the identification of useful genes that can be used for crop breeding with minimal effort for NGS analysis.

Integrated genome-wide differentiation and association analyses identify causal genes underlying breeding-selected grain quality traits in japonica rice.

Improving grain quality is a primary objective in contemporary rice breeding. Japanese modern rice breeding has developed two different types of rice, eating and sake-brewing rice, with different grain characteristics, indicating the selection of variant gene alleles during the breeding process. Given the critical importance of promptly and efficiently identifying genes selected in past breeding for future molecular breeding, we conducted genome scans for divergence, genome-wide association studies, and map-based cloning. Consequently, we successfully identified two genes, OsMnS and OsWOX9D, both contributing to rice grain traits. OsMnS encodes a mannan synthase that increases the white core frequency in the endosperm, a desirable trait for sake brewing but decreases the grain appearance quality. OsWOX9D encodes a grass-specific homeobox-containing transcription factor, which enhances grain width for better sake brewing. Furthermore, haplotype analysis revealed that their defective alleles were selected in East Asia, but not Europe, during modern improvement. In addition, our analyses indicate that a reduction in grain mannan content during African rice domestication may also be caused a defective OsMnS allele due to breeding selection. This study not only reveals the delicate balance between grain appearance quality and nutrition in rice but also provides a new strategy for isolating causal genes underlying complex traits, based on the concept of "breeding-assisted genomics" in plants.

Genome-wide association study identifies a gene responsible for temperature-dependent rice germination.

Environment is an important determinant of agricultural productivity; therefore, crops have been bred with traits adapted to their environment. It is assumed that the physiology of seed germination is optimised for various climatic conditions. Here, to understand the genetic basis underlying seed germination, we conduct a genome-wide association study considering genotype-by-environment interactions on the germination rate of Japanese rice cultivars under different temperature conditions. We find that a 4 bp InDel in one of the 14-3-3 family genes, GF14h, preferentially changes the germination rate of rice under optimum temperature conditions. The GF14h protein constitutes a transcriptional regulatory module with a bZIP-type transcription factor, OREB1, and a florigen-like protein, MOTHER OF FT AND TFL 2, to control the germination rate by regulating abscisic acid (ABA)-responsive genes. The GF14h loss-of-function allele enhances ABA signalling and reduces the germination rate. This allele is found in rice varieties grown in the northern area and in modern cultivars of Japan and China, suggesting that it contributes to the geographical adaptation of rice. This study demonstrates the complicated molecular system involved in the regulation of seed germination in response to temperature, which has allowed rice to be grown in various geographical locations.

GWAS with principal component analysis identifies a gene comprehensively controlling rice architecture.

Elucidation of the genetic control of rice architecture is crucial due to the global demand for high crop yields. Rice architecture is a complex trait affected by plant height, tillering, and panicle morphology. In this study, principal component analysis (PCA) on 8 typical traits related to plant architecture revealed that the first principal component (PC), PC1, provided the most information on traits that determine rice architecture. A genome-wide association study (GWAS) using PC1 as a dependent variable was used to isolate a gene encoding rice, SPINDLY (OsSPY), that activates the gibberellin (GA) signal suppression protein SLR1. The effect of GA signaling on the regulation of rice architecture was confirmed in 9 types of isogenic plant having different levels of GA responsiveness. Further population genetics analysis demonstrated that the functional allele of OsSPY associated with semidwarfism and small panicles was selected in the process of rice breeding. In summary, the use of PCA in GWAS will aid in uncovering genes involved in traits with complex characteristics.

Evolution and diversification of the plant gibberellin receptor GID1.

The plant gibberellin (GA) receptor GID1 shows sequence similarity to carboxylesterase (CXE). Here, we report the molecular evolution of GID1 from establishment to functionally diverse forms in eudicots. By introducing 18 mutagenized rice GID1s into a rice gid1 null mutant, we identified the amino acids crucial for GID1 activity in planta. We focused on two amino acids facing the C2/C3 positions of ent-gibberellane, not shared by lycophytes and euphyllophytes, and found that adjustment of these residues resulted in increased GID1 affinity toward GA4, new acceptance of GA1 and GA3 carrying C13-OH as bioactive ligands, and elimination of inactive GAs. These residues rendered the GA perception system more sophisticated. We conducted phylogenetic analysis of 169 GID1s from 66 plant species and found that, unlike other taxa, nearly all eudicots contain two types of GID1, named A- and B-type. Certain B-type GID1s showed a unique evolutionary characteristic of significantly higher nonsynonymous-to-synonymous divergence in the region determining GA4 affinity. Furthermore, these B-type GID1s were preferentially expressed in the roots of Arabidopsis, soybean, and lettuce and might be involved in root elongation without shoot elongation for adaptive growth under low-temperature stress. Based on these observations, we discuss the establishment and adaption of GID1s during plant evolution.

Loss of red opsin genes relaxes sexual isolation between skin-colour variants of medaka.

Colour vision is often essential for animals. Fine discrimination of colours enhances the ability of animals to find food, predators, or mating partners. Using two colour variants of medaka (Oryzias latipes), which mate assortatively depending on visual cues (pale grey versus dark orange), we recently established red colour-blind strains by knocking out the red opsin (long-wavelength-sensitive) genes and elucidated that the fish were indeed insensitive to red light. In the present study, we investigated the mate choice of these red-blind fish. The colour variants with normal colour vision strongly preferred to mate with their own strain. The red-blind ones also preferred their own strain; i.e. they still mated assortatively. However, their preference was significantly weaker than that of fish with normal colour vision. In other words, the red-blind fish showed increased sexual interest in the other colour variant. These results indicated that reduced sensitivity to red light also reduced their ability to discriminate colours. This empirical evidence directly demonstrates that a change in cone-opsin repertoire changes mating decision behaviours, which would affect gene flow and speciation processes between conspecific colour variants in nature, as suggested in other studies.

Sorghum DW1 positively regulates brassinosteroid signaling by inhibiting the nuclear localization of BRASSINOSTEROID INSENSITIVE 2.

Semi-dwarf traits have been widely introgressed into cereal crops to improve lodging resistance. In sorghum (Sorghum bicolor L. Moench), four major unlinked dwarfing genes, Dw1-Dw4, have been introduced to reduce plant height, and among them, Dw3 and Dw1 have been cloned. Dw3 encodes a gene involved in auxin transport, whereas, Dw1 was recently isolated and identified as a gene encoding a protein of unknown function. In this study, we show that DW1 is a novel component of brassinosteroid (BR) signaling. Sorghum possessing the mutated allele of Dw1 (dw1), showed similar phenotypes to rice BR-deficient mutants, such as reduced lamina joint bending, attenuated skotomorphogenesis, and insensitivity against feedback regulation of BR-related genes. Furthermore, DW1 interacted with a negative regulator of BR signaling, BRASSINOSTEROID INSENSITIVE 2 (BIN2), and inhibited its nuclear localization, indicating that DW1 positively regulates BR signaling by inhibiting the function of BIN2. In contrast to rice and wheat breeding which used gibberellin (GA) deficiency to reduce plant height, sorghum breeding modified auxin and BR signaling. This difference may result from GA deficiency in rice and wheat does not cause deleterious side effects on plant morphology, whereas in sorghum it leads to abnormal culm bending.

Screening of rice mutants with improved saccharification efficiency results in the identification of CONSTITUTIVE PHOTOMORPHOGENIC 1 and GOLD HULL AND INTERNODE 1.

MAIN CONCLUSION: The screening of rice mutants with improved cellulose to glucose saccharification efficiency (SE) identifies reduced xylan and/or ferulic acid, and a qualitative change of lignin to impact SE. To ensure the availability of sustainable energy, considerable effort is underway to utilize lignocellulosic plant biomass as feedstock for the production of biofuels. However, the high cost of degrading plant cell wall components to fermentable sugars (saccharification) has been problematic. One way to overcome this barrier is to develop plants possessing cell walls that are amenable to saccharification. In this study, we aimed to identify new molecular factors that influence saccharification efficiency (SE) in rice. By screening 22 rice mutants, we identified two lines, 122 and 108, with improved SE. Reduced xylan and ferulic acid within the cell wall of line 122 were probable reasons of improved SE. Line 108 showed reduced levels of thioglycolic-released lignin; however, the amount of Klason lignin was comparable to the wild-type, indicating that structural changes had occurred in the 108 lignin polymer which resulted in improved SE. Positional cloning revealed that the genes responsible for improved SE in 122 and 108 were rice CONSTITUTIVE PHOTOMORPHOGENIC 1 (OsCOP1) and GOLD HULL AND INTERNODE 1 (GH1), respectively, which have not been previously reported to influence SE. The screening of mutants for improved SE is an efficient approach to identify novel genes that affect SE, which is relevant in the development of crops as biofuel sources.

SMALL ORGAN SIZE 1 and SMALL ORGAN SIZE 2/DWARF AND LOW-TILLERING Form a Complex to Integrate Auxin and Brassinosteroid Signaling in Rice.

Although auxin and brassinosteroid (BR) synergistically control various plant responses, the molecular mechanism underlying the auxin-BR crosstalk is not well understood. We previously identified SMOS1, an auxin-regulated APETALA2-type transcription factor, as the causal gene of the small organ size 1 (smos1) mutant that is characterized by a decreased final size of various organs in rice. In this study, we identified another smos mutant, smos2, which shows the phenotype indistinguishable from smos1. SMOS2 was identical to the previously reported DWARF AND LOW-TILLERING (DLT), which encodes a GRAS protein involved in BR signaling. SMOS1 and SMOS2/DLT physically interact to cooperatively enhance transcriptional transactivation activity in yeast and in rice nuclei. Consistently, the expression of OsPHI-1, a direct target of SMOS1, is upregulated only when SMOS1 and SMOS2/DLT proteins are both present in rice cells. Taken together, our results suggest that SMOS1 and SMOS2/DLT form a keystone complex on auxin-BR signaling crosstalk in rice.

DELLA protein functions as a transcriptional activator through the DNA binding of the indeterminate domain family proteins.

DELLA protein is a key negative regulator of gibberellin (GA) signaling. Although how DELLA regulates downstream gene expression remains unclear, DELLA has been proposed to function as a transcriptional activator. However, because DELLA lacks a DNA-binding domain, intermediate protein(s) mediating the DELLA/DNA interaction are believed to be necessary for activating DELLA target genes. Here, using yeast hybrid screenings, we identified five members of indeterminate domain (IDD) protein family which bind physically to both DELLA and the promoter sequence of the GA-positive regulator SCARECROW-LIKE 3 (SCL3), which previously was characterized as a DELLA direct target gene. Transient assays using Arabidopsis protoplasts demonstrated that a luciferase reporter controlled by the SCL3 promoter was additively transactivated by REPRESSOR of ga1-3 (RGA) and IDDs. Phenotypic analysis of transgenic plants expressing AtIDD3 (one of the 16 IDDs in the Arabidopsis genome) fused with the plant-specific repression domain (SRDX) supported the possibility that AtIDD3 is positively involved in GA signaling. In addition, we found that SCL3 protein also interacts with IDDs, resulting in the suppression of its target gene expression. In this context, DELLA and SCL3 interact competitively with IDD proteins to regulate downstream gene expression. These results suggest that the coregulators DELLA and SCL3, using IDDs as transcriptional scaffolds for DNA binding, antagonistically regulate the expression of their downstream targets to control the GA signaling pathway.

A rice gid1 suppressor mutant reveals that gibberellin is not always required for interaction between its receptor, GID1, and DELLA proteins.

To investigate gibberellin (GA) signaling using the rice (Oryza sativa) GA receptor GIBBERELLIN-INSENSITIVE DWARF1 (GID1) mutant gid1-8, we isolated a suppressor mutant, Suppressor of gid1-1 (Sgd-1). Sgd-1 is an intragenic mutant containing the original gid1-8 mutation (L45F) and an additional amino acid substitution (P99S) in the loop region. GID1(P99S) interacts with the rice DELLA protein SLENDER RICE1 (SLR1), even in the absence of GA. Substitution of the 99th Pro with other amino acids revealed that substitution with Ala (P99A) caused the highest level of GA-independent interaction. Physicochemical analysis using surface plasmon resonance revealed that GID1(P99A) has smaller K(a) (association) and K(d) (dissociation) values for GA(4) than does wild-type GID1. This suggests that the GID1(P99A) lid is at least partially closed, resulting in both GA-independent and GA-hypersensitive interactions with SLR1. One of the three Arabidopsis thaliana GID1s, At GID1b, can also interact with DELLA proteins in the absence of GA, so we investigated whether GA-independent interaction of At GID1b depends on a mechanism similar to that of rice GID1(P99A). Substitution of the loop region or a few amino acids of At GID1b with those of At GID1a diminished its GA-independent interaction with GAI while maintaining the GA-dependent interaction. Soybean (Glycine max) and Brassica napus also have GID1s similar to At GID1b, indicating that these unique GID1s occur in various dicots and may have important functions in these plants.

Characterization of the molecular mechanism underlying gibberellin perception complex formation in rice.

The DELLA protein SLENDER RICE1 (SLR1) is a repressor of gibberellin (GA) signaling in rice (Oryza sativa), and most of the GA-associated responses are induced upon SLR1 degradation. It is assumed that interaction between GIBBERELLIN INSENSITIVE DWARF1 (GID1) and the N-terminal DELLA/TVHYNP motif of SLR1 triggers F-box protein GID2-mediated SLR1 degradation. We identified a semidominant dwarf mutant, Slr1-d4, which contains a mutation in the region encoding the C-terminal GRAS domain of SLR1 (SLR1(G576V)). The GA-dependent degradation of SLR1(G576V) was reduced in Slr1-d4, and compared with SLR1, SLR1(G576V) showed reduced interaction with GID1 and almost none with GID2 when tested in yeast cells. Surface plasmon resonance of GID1-SLR1 and GID1-SLR1(G576V) interactions revealed that the GRAS domain of SLR1 functions to stabilize the GID1-SLR1 interaction by reducing its dissociation rate and that the G576V substitution in SLR1 diminishes this stability. These results suggest that the stable interaction of GID1-SLR1 through the GRAS domain is essential for the recognition of SLR1 by GID2. We propose that when the DELLA/TVHYNP motif of SLR1 binds with GID1, it enables the GRAS domain of SLR1 to interact with GID1 and that the stable GID1-SLR1 complex is efficiently recognized by GID2.