Comparative analysis of metabolite profiling and free radical scavenging activity in phenotypic variants of OsCOP1 colored rice mutant seed.

Interest in colored rice has been increasing due to its health benefits. This study examined the metabolite profiling of CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) mutated rice seed (yel-mutant). The wild-type (WT) and the yel-mutant having yellow (y)- and purple (p)-pericarp variants from Chucheong (cc) and Samkwang (sk) cultivars were investigated for differences in bioactive metabolite profiles and free radical scavenging activity. The total fatty acid content decreased by >50% in the yel-mutant against the WT, while no significant difference was observed between yellow- and purple-pericarp variants (p < 0.05). The yel-mutant of both cultivars showed significantly higher flavone contents than their WT (non-detected). Most of the metabolites examined were highly produced in the yel-cc-p and the yel-sk-y than in the other phenotypic variants studied. This study provides further useful information for colored rice breeding by revealing the detailed biofunctional metabolic profile under COP1 mutation in colored rice.

Reinterpretation of anthocyanins biosynthesis in developing black rice seeds through gene expression analysis.

The biosynthesis of anthocyanins is still questionable in regulating the quantities of anthocyanins biosynthesized in rice seeds and the expression levels of transcription factors and the structural genes involved in the biosynthetic pathway of anthocyanins. We herein investigated the relationship between the accumulated anthocyanin contents and the expression levels of genes related to the biosynthesis of anthocyanins in rice seeds. Liquid chromatography/mass spectrometry-mass spectrometry analysis of cyanidin 3-glucoside (C3G) in rice seeds showed no accumulation of C3G in white and red rice cultivars, and the differential accumulation of C3G among black rice cultivars. RNA-seq analysis in rice seeds, including white, red, and black rice cultivars, at twenty days after heading (DAH) further exhibited that the genes involved in the biosynthesis of anthocyanins were differentially upregulated in developing seeds of black rice. We further verified these RNA-seq results through gene expression analysis by a quantitative real-time polymerase chain reaction in developing seeds of white, red, and black rice cultivars at 20 DAH. Of these genes related to the biosynthesis of anthocyanins, bHLHs, MYBs, and WD40, which are regulators, and the structural genes, including chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), flavonoid 3´-hydroxylase (F3´H), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS), were differentially upregulated in black rice seeds. The correlation analysis revealed that the quantities of C3G biosynthesized in black rice seeds were positively correlated to the expression levels of bHLHs, MYBs and WD40, CHS, F3H, F3´H, DFR, and ANS. In addition, we present bHLH2 (LOC_Os04g47040) and MYBs (LOC_Os01g49160, LOC_Os01g74410, and LOC_Os03g29614) as new putative transcription factor genes for the biosynthesis of anthocyanins in black rice seeds. It is expected that this study will help to improve the understanding of the molecular levels involved in the biosynthesis of anthocyanins in black rice seeds.

Identification and characterization of a novel gene controlling floral organ number in rice (Oryza sativa L.).

Floral organ number is crucial for successful seed setting and mature grain development. Although some genes and signaling pathways controlling floral organ number have been studied, the underlying mechanism is complicated and requires further investigation. In this study, a floral organ number mutant was generated by the ethyl methanesulfonate treatment of the Korean japonica rice cultivar Ilpum. In the floral organ number mutant, 37% of the spikelets showed an increase in the number of floral organs, especially stamens and pistils. Histological analysis revealed that the number of ovaries was determined by the number of stigmas; spikelets with two or three stigmas contained only one ovary, whereas spikelets with four stigmas possessed two ovaries. The floral organ number mutant showed pleiotropic phenotypes including multiple grains, early flowering, short plant height, and reduced tiller number compared with the wild-type. Genetic and MutMap analyses revealed that floral organ number is controlled by a single recessive gene located between the 8.0 and 20.0 Mb region on chromosome 8. Calculation of SNP-index confirmed Os08g0299000 as the candidate gene regulating floral organ number, which was designated as FLORAL ORGAN NUMBER7 (FON7). A single nucleotide polymorphism (G to A) was discovered at the intron splicing donor site of FON7, which caused the skipping of the entire sixth exon in the mutant, resulting in the deletion of 144 bp. Furthermore, the T-DNA-tagged line displayed the same floral organ number phenotype as the fon7 mutant. These results provide valuable insight into the mechanism of floral organ differentiation and formation in rice.

Identification of volatile organic compounds related to the eating quality of cooked japonica rice.

Eating quality (EQ) of rice has a complex nature composed of physicochemical properties. Nevertheless, breeding programs evaluating EQ through sensory test or taste-evaluation instruments have been laborious, time-consuming and inefficient. EQ is affected by both taste and aroma. However, in actual breeding programs, aroma of cooked rice has been considered the least due to lack of information. Here we identified a total of 41 volatile compounds potentially affecting the EQ of non-aromatic, cooked japonica rice, identified by GC-MS, sensory panel test, and Toyo taste-meter analyses. Partial least squares discriminant analysis demonstrated an outstanding classification effect of the identified volatile compounds on eating-quality discrimination. Several volatile compounds related to lipid oxidation and fatty acid degradation were identified to affect the EQ in japonica rice. Of them, 1-octen-3-ol, 1-ethyl-3,5-dimethylbenzene, 2,6,11-trimethyldodecane, 3-ethyloctane, 2,7,10-trimethyldodecane, methyl salicylate, 2-octanone, and heptanal were selected as important compounds. The discriminant model for the classification of the quality of cultivars was robust and accurate, an r-squared value was 0.91, a q squared value was 0.85, and an accuracy was 1.0. Overall, the results of this study characterize EQ of rice cultivars based on volatile compounds, suggesting the application of metabolite profiling data for rice breeding of high eating quality.

Mutations in OsDET1, OsCOP10, and OsDDB1 confer embryonic lethality and alter flavonoid accumulation in Rice (Oryza sativa L.) seed.

Morphological and biochemical changes accompanying embryogenesis and seed development are crucial for plant survival and crop productivity. Here, we identified a novel yellowish-pericarp embryo lethal (yel) mutant of the japonica rice cultivar Sindongjin (Oryza sativa L.), namely, yel-sdj. Seeds of the yel-sdj mutant showed a yellowish pericarp and black embryo, and were embryonic lethal. Compared with wild-type seeds, the yel-sdj mutant seeds exhibited significantly reduced grain size, grain weight, and embryo weight, and a remarkably lower rate of embryo retention in kernels subjected to milling. However, the volume of air space between embryo and endosperm, density of embryo, and total phenolic content (TPC) and antioxidant activity of mature grains were significantly higher in the yel-sdj mutant than in the wild type. Genetic analysis and mapping revealed that the yel-sdj mutant was non-allelic to the oscop1 null mutants yel-hc, yel-cc, and yel-sk, and its phenotype was controlled by a single recessive gene, LOC_Os01g01484, an ortholog of Arabidopsis thaliana DE-ETIOLATED 1 (DET1). The yel-sdj mutant carried a 7 bp deletion in the second exon of OsDET1. Seeds of the osdet1 knockout mutant, generated via CRISPR/Cas9-based gene editing, displayed the yel mutant phenotype. Consistent with the fact that OsDET1 interacts with CONSTITUTIVE PHOTOMORPHOGENIC 10 (OsCOP10) and UV-DAMAGED DNA BINDING PROTEIN 1 (OsDDB1) to form the COP10-DET1-DDB1 (CDD), seeds of oscop10 and osddb1 knockout mutants also showed the yel phenotype. These findings will enhance our understanding of the functional roles of OsDET1 and the CDD complex in embryogenesis and flavonoid biosynthesis in rice seeds.

Histological characterization of anther structure in Tetep-cytoplasmic male sterility and fine mapping of restorer-of-fertility gene in rice.

Cytoplasmic male sterility (CMS) is a maternally inherited trait that inhibits plants from producing or releasing viable pollen. CMS is caused by mitochondrial-nuclear interaction, and can be rescued by introducing functional nuclear restorer-of-fertility (Rf) gene. The Tetep-CMS/Rf lines were developed through successive inter-subspecific backcrosses between indica and japonica rice accessions. Phenotypic characterization of Tetep-CMS lines revealed abnormal anther dehiscence and the inability to release, while possessing functional pollen. Transverse sections of developing anthers collected from CMS plants showed connective tissue deformities and aberrant dehydration of endothecium and epidermis. Fine mapping of Rf-Tetep using a series of segregating populations, delimited the candidate region to an approximately 109 kb genomic interval between M2099 and FM07 flanking markers. Nanopore long-read sequencing and genome assembly, proceeded by gene prediction and annotation revealed 11 open reading frames (ORFs) within the candidate region, and suggest ORF6 annotated as pentatricopeptide repeat motif containing gene 1 (PPR1), as a possible candidate gene responsible for fertility restoration. This study suggests that tissue-specific abnormalities in anthers are responsible for indehiscence-based sterility, and propose that the functional Rf gene is derived from allelic variation between inter-subspecies in rice.

Evidence for evolution and selection of drought-resistant genes based on high-throughput resequencing in weedy rice.

Weedy rice (Oryza sativa f. spontanea) is a relative of cultivated rice that propagates in paddy fields and has strong drought resistance. In this study, we used 501 rice accessions to reveal the selection mechanism of drought resistance in weedy rice through a combination of selection analysis, genome-wide association studies, gene knockout and overexpression analysis, and Ca2+ and K+ ion flux assays. The results showed that the weedy rice species investigated have gene introgression with cultivated rice, which is consistent with the hypothesis that weedy rice originated from de-domestication of cultivated rice. Regions related to tolerance have particularly diversified during de-domestication and three drought-tolerance genes were identified. Of these, Os01g0800500 was also identified using an assay of the degree of leaf withering under drought, and it was named as PAPH1, encoding a PAP family protein. The drought-resistance capacity of PAPH1-knockout lines was much lower than that of the wild type, while that of overexpression lines was much higher. Concentrations of Ca2+ and K+ were lower in the knockout lines and higher in the overexpression lines compared with those of the wild type, suggesting that PAPH1 plays important roles in coping with drought stress. Our study therefore provides new insights into the genetic mechanisms underlying adaptive tolerance to drought in wild rice and highlights potential new resistance genes for future breeding programs in cultivated rice.

Sequencing and de novo assembly of the Koshihikari genome and identification of the genomic region related to the eating quality of cooked rice.

The japonica rice (Oryza sativa L.) cultivar Koshihikari is considered an important breeding material with good eating quality (EQ). To effectively utilize Koshihikari in molecular breeding programs, determining its whole genome sequence including cultivar-specific segment is crucial. Here, the Koshihikari genome was sequenced using Nanopore and Illumina platforms, and de novo assembly was performed. A highly contiguous Koshihikari genome sequence was compared with Nipponbare, the reference genome of japonica. Genome-wide synteny was observed, as expected, without large structural variations. However, several gaps in alignment were detected on chromosomes 3, 4, 9, and 11. It was notable that previously identified EQ-related QTLs were found in these gaps. Moreover, sequence variations were identified in chromosome 11 at a region flanking the P5 marker, one of the significant markers of good EQ. The Koshihikari-specific P5 region was found to be transmitted through the lineage. High EQ cultivars derived from Koshihikari possessed P5 sequences; on the other hand, Koshihikari-derived low EQ cultivars didn't contain the P5 region, which implies that the P5 genomic region affects the EQ of Koshihikari progenies. The EQ of near-isogenic lines (NILs) of Samnam (a low EQ cultivar) genetic background harboring the P5 segment was improved compared to that of Samnam in Toyo taste value. The structure of the Koshihikari-specific P5 genomic region associated with good EQ was analyzed, which is expected to facilitate the molecular breeding of rice cultivars with superior EQ. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01335-3.

Transcriptional Changes in the Developing Rice Seeds Under Salt Stress Suggest Targets for Manipulating Seed Quality.

Global sea-level rise, the effect of climate change, poses a serious threat to rice production owing to saltwater intrusion and the accompanying increase in salt concentration. The reclaimed lands, comprising 22.1% of rice production in Korea, now face the crisis of global sea-level rise and a continuous increase in salt concentration. Here, we investigated the relationship between the decrease in seed quality and the transcriptional changes that occur in the developing rice seeds under salt stress. Compared to cultivation on normal land, the japonica rice cultivar, Samgwang, grown on reclaimed land showed a greatly increased accumulation of minerals, including sodium, magnesium, potassium, and sulfur, in seeds and a reduced yield, delayed heading, decreased thousand grain weight, and decreased palatability and amylose content. Samgwang showed phenotypical sensitivity to salt stress in the developing seeds. Using RNA-seq technology, we therefore carried out a comparative transcriptome analysis of the developing seeds grown on reclaimed and normal lands. In the biological process category, gene ontology enrichment analysis revealed that the upregulated genes were closely associated with the metabolism of biomolecules, including amino acids, carboxylic acid, lignin, trehalose, polysaccharide, and chitin, and to stress responses. MapMan analysis revealed the involvement of upregulated genes in the biosynthetic pathways of abscisic acid and melatonin and the relationship of trehalose, raffinose, and maltose with osmotic stress. Interestingly, many seed storage protein genes encoding glutelins and prolamins were upregulated in the developing seeds under salt stress, indicating the negative effect of the increase of storage proteins on palatability. Transcription factors upregulated in the developing seeds under salt stress included, in particular, bHLH, MYB, zinc finger, and heat shock factor, which could act as potential targets for the manipulation of seed quality under salt stress. Our study aims to develop a useful reference for elucidating the relationship between seed response mechanisms and decreased seed quality under salt stress, providing potential strategies for the improvement of seed quality under salt stress.

The Effect of Water Level in Rice Cropping System on Phosphorus Uptake Activity of Pup1 in a Pup1+Sub1 Breeding Line.

Pyramiding useful QTLs into an elite variety is a promising strategy to develop tolerant varieties against multiple abiotic stresses. However, some QTLs may not be functionally compatible when they are introgressed into the same variety. Here, we tested the functional compatibility of Pup1 and Sub1, major QTLs for tolerance to phosphorus (P)-deficiency and submergence conditions, respectively. Phenotypic analysis revealed that IR64-Pup1+Sub1 (IPS) plants harboring both Pup1 and Sub1 QTLs show significant tolerance to submerged conditions, similarly to IR64-Sub1, while IPS failed to tolerate P deficiency and mild drought conditions; only IR64-Pup1 showed P deficiency tolerance. In submerged conditions, Sub1A and OsPSTOL1, major genes for Sub1 and Pup1 QTLs, respectively, were expressed at the same levels as in IPS and IR64-Sub1 and in IPS and IR64-Pup1, respectively. On the other hand, in P-non-supplied condition, crown root number, root length, and OsPSTOL1 expression level were significantly lower in IPS compared to those of IR64-Pup1. However, there was no significant difference in P content between IPS and IR64-Pup1. These results imply that Pup1 does not compromise Sub1 function in submerged condition, while Sub1 suppresses Pup1 function in P-non-supplied condition, possibly by regulating the transcript level of Pup1. In conclusion, Pup1 and Sub1 are regarded as functionally compatible under submergence condition but not under P-non-supplied condition. Further study is needed to elucidate the functional incompatibility of Pup1 and Sub1 QTLs in IPS under P-non-supplied condition.

OsCOP1 regulates embryo development and flavonoid biosynthesis in rice (Oryza sativa L.).

KEY MESSAGE: Novel mutations of OsCOP1 were identified to be responsible for yellowish pericarp and embryo lethal phenotype, which revealed that OsCOP1 plays a crucial role in flavonoid biosynthesis and embryogenesis in rice seed. Successful production of viable seeds is a major component of plant life cycles, and seed development is a complex, highly regulated process that affects characteristics such as seed viability and color. In this study, three yellowish-pericarp embryo lethal (yel) mutants, yel-hc, yel-sk, and yel-cc, were produced from three different japonica cultivars of rice (Oryza sativa L). Mutant seeds had yellowish pericarps and exhibited embryonic lethality, with significantly reduced grain size and weight. Morphological aberrations were apparent by 5 days after pollination, with abnormal embryo development and increased flavonoid accumulation observed in the yel mutants. Genetic analysis and mapping revealed that the phenotype of the three yel mutants was controlled by a single recessive gene, LOC_Os02g53140, an ortholog of Arabidopsis thaliana CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1). The yel-hc, yel-sk, and yel-cc mutants carried mutations in the RING finger, coiled-coil, and WD40 repeat domains, respectively, of OsCOP1. CRISPR/Cas9-targeted mutagenesis was used to knock out OsCOP1 by targeting its functional domains, and transgenic seed displayed the yel mutant phenotype. Overexpression of OsCOP1 in a homozygous yel-hc mutant background restored pericarp color, and the aberrant flavonoid accumulation observed in yel-hc mutant was significantly reduced in the embryo and endosperm. These results demonstrate that OsCOP1 is associated with embryo development and flavonoid biosynthesis in rice grains. This study will facilitate a better understanding of the functional roles of OsCOP1 involved in early embryogenesis and flavonoid biosynthesis in rice seeds.

Identification of a Candidate Gene for the Novel Cytoplasmic Male Sterility Derived from Inter-Subspecific Crosses in Rice (Oryza sativa L.).

Tetep-cytoplasmic male sterility (CMS) was developed through successive backcrosses between subspecies indica and japonica in rice (Oryza sativa L.), which showed abnormal anther dehiscence phenotypes. Whole genome sequencing and de novo assembly of the mitochondrial genome identified the chimeric gene orf312, which possesses a transmembrane domain and overlaps with two mitotype-specific sequences (MSSs) that are unique to the Tetep-CMS line. The encoded peptide of orf312 was toxic to Escherichia coli and inhibited cell growth compared to the control under isopropyl-ß-D-1-thiogalactopyranoside (IPTG) induction. The peptide of orf312 contains COX11-interaction domains, which are thought to be a main functional domain for WA352c in the wild abortive (WA-CMS) line of rice. A QTL for Rf-Tetep (restorer-of-fertility gene(s) originating from Tetep) was identified on chromosome 10. In this region, several restorer genes, Rf1a, Rf1b, and Rf4, have previously been reported. Collectively, the interactions of orf312, a candidate gene for Tetep-CMS, and Rf-Tetep, a restorer QTL, confer male sterility and fertility restoration, respectively, which enables a hybrid rice breeding system. Further studies on orf312 and isolation of Rf-Tetep will help to identify the underlying molecular mechanism of mitochondrial ORFs with the COX11-interaction domains.

Major QTLs, qARO1 and qARO9, Additively Regulate Adaxial Leaf Rolling in Rice.

Moderate leaf rolling is considered optimal for the ideal plant type in rice (Oryza sativa L.), as it improves photosynthetic efficiency and, consequently, grain yield. Determining the genetic basis of leaf rolling via the identification of quantitative trait loci (QTLs) could facilitate the development of high-yielding varieties. In this study, we identified three stable rice QTLs, qARO1, qARO5, and qARO9, which control adaxial leaf rolling in a recombinant inbred line (RIL) population derived from a cross between Tong 88-7 (T887) and Milyang 23 (M23), using high-density SNP markers. These QTLs controlled the rolling phenotype of both the flag leaf (FL) and secondary leaf (SL), and different allelic combinations of these QTLs led to a wide variation in the degree of leaf rolling. Additive gene actions of qARO1 and qARO9 on leaf rolling were observed in a backcross population. In addition, qARO1 (markers: 01id4854718 and 01asp4916781) and qARO9 (markers: 09id19650402 and 09id19740436) were successfully fine-mapped to approximately 60- and 90-kb intervals on chromosomes 1 and 9, respectively. Histological analysis of near-isogenic lines (NILs) revealed that qARO1 influences leaf thickness across the small vein, and qARO9 affects leaf thickness in the entire leaf and bulliform cell area, thus leading to adaxial leaf rolling. The results of this study advance our understanding of the genetic and molecular bases of adaxial leaf rolling, and this information can be used for the development of rice varieties with the ideal plant type.

Arachis hypogaea resveratrol synthase 3 alters the expression pattern of UDP-glycosyltransferase genes in developing rice seeds.

The resveratrol-producing rice (Oryza sativa L.) inbred lines, Iksan 515 (I.515) and Iksan 526 (I.526), developed by the expression of the groundnut (Arachis hypogaea) resveratrol synthase 3 (AhRS3) gene in the japonica rice cultivar Dongjin, accumulated both resveratrol and its glucoside, piceid, in seeds. Here, we investigated the effect of the AhRS3 transgene on the expression of endogenous piceid biosynthesis genes (UGTs) in the developing seeds of the resveratrol-producing rice inbred lines. Ultra-performance liquid chromatography (UPLC) analysis revealed that I.526 accumulates significantly higher resveratrol and piceid in seeds than those in I.515 seeds and, in I.526 seeds, the biosynthesis of resveratrol and piceid reached peak levels at 41 days after heading (DAH) and 20 DAH, respectively. Furthermore, RNA-seq analysis showed that the expression patterns of UGT genes differed significantly between the 20 DAH seeds of I.526 and those of Dongjin. Quantitative real-time PCR (RT-qPCR) analyses confirmed the data from RNA-seq analysis in seeds of Dongjin, I.515 and I.526, respectively, at 9 DAH, and in seeds of Dongjin and I.526, respectively, at 20 DAH. A total of 245 UGTs, classified into 31 UGT families, showed differential expression between Dongjin and I.526 seeds at 20 DAH. Of these, 43 UGTs showed more than 2-fold higher expression in I.526 seeds than in Dongjin seeds. In addition, the expression of resveratrol biosynthesis genes (PAL, C4H and 4CL) was also differentially expressed between Dongjin and I.526 developing seeds. Collectively, these data suggest that AhRS3 altered the expression pattern of UGT genes, and PAL, C4H and 4CL in developing rice seeds.

Genetic Basis of Tiller Dynamics of Rice Revealed by Genome-Wide Association Studies.

A tiller number is the key determinant of rice plant architecture and panicle number and consequently controls grain yield. Thus, it is necessary to optimize the tiller number to achieve the maximum yield in rice. However, comprehensive analyses of the genetic basis of the tiller number, considering the development stage, tiller type, and related traits, are lacking. In this study, we sequence 219 Korean rice accessions and construct a high-quality single nucleotide polymorphism (SNP) dataset. We also evaluate the tiller number at different development stages and heading traits involved in phase transitions. By genome-wide association studies (GWASs), we detected 20 significant association signals for all traits. Five signals were detected in genomic regions near known candidate genes. Most of the candidate genes were involved in the phase transition from vegetative to reproductive growth. In particular, HD1 was simultaneously associated with the productive tiller ratio and heading date, indicating that the photoperiodic heading gene directly controls the productive tiller ratio. Multiple linear regression models of lead SNPs showed coefficients of determination (R2) of 0.49, 0.22, and 0.41 for the tiller number at the maximum tillering stage, productive tiller number, and productive tiller ratio, respectively. Furthermore, the model was validated using independent japonica rice collections, implying that the lead SNPs included in the linear regression model were generally applicable to the tiller number prediction. We revealed the genetic basis of the tiller number in rice plants during growth, By GWASs, and formulated a prediction model by linear regression. Our results improve our understanding of tillering in rice plants and provide a basis for breeding high-yield rice varieties with the optimum the tiller number.

The DROOPING LEAF (DR) gene encoding GDSL esterase is involved in silica deposition in rice (Oryza sativa L.).

Leaf morphology is one of the most important agronomic traits in rice breeding because of its contribution to crop yield. The drooping leaf (dr) mutant was developed from the Ilpum rice cultivar by ethyl methanesulfonate (EMS) mutagenesis. Compared with the wild type, dr plants exhibited drooping leaves accompanied by a small midrib, short panicle, and reduced plant height. The phenotype of the dr plant was caused by a mutation within a single recessive gene on chromosome 2, dr (LOC_Os02g15230), which encodes a GDSL esterase. Analysis of wild-type and dr sequences revealed that the dr allele carried a single nucleotide substitution, glycine to aspartic acid. RNAi targeted to LOC_Os02g15230 produced same phenotypes to the dr mutation, confirming LOC_Os02g15230 as the dr gene. Microscopic observations and plant nutrient analysis of SiO2 revealed that silica was less abundant in dr leaves than in wild-type leaves. This study suggests that the dr gene is involved in the regulation of silica deposition and that disruption of silica processes lead to drooping leaf phenotypes.

Identification and characterization of the stunted sterile (ss) mutant in rice.

BACKGROUND: Proper organ development is pivotal for normal rice growth and production. Many genes are involved in this process, and these genes provide a basis for rice breeding. OBJECTIVE: To identify a novel mutation causing developmental defects in rice. METHODS: The phenotype of a rice mutant, stunted sterile (ss), identified from the japonica rice cultivar Samkwang treated with N-methyl-N-nitrosourea, was characterized, including anatomical and pollen activity analyses. A genetic analysis and fine mapping were performed to identify a candidate locus, followed by a sequence analysis to determine the causal mutation for the phenotype. RESULTS: Compared with wild-type plants, the mutant exhibited a 34% reduction in height, 46% reduction in flag leaf width, and complete panicle sterility. Cell proliferation in the leaf and pollen viability were significantly inhibited in the mutant. The mutant phenotypes were controlled by a single recessive gene that was fine-mapped to an 84 kb region between two SNP markers on the short arm of chromosome 5. A candidate gene analysis determined that the mutant carries an 11 bp insertion in the coding region of LOC_Os05g03550, which encodes a protein containing two SANT domains, resulting in a premature termination codon before the conserved domain. CONCLUSIONS: We identified a novel rice gene, Stunted sterile, involved in the regulation of various developmental processes. Our findings improve our understanding of the role of chromatin remodeling in organ development and have implications for breeding owing to the broad effects of the gene on plant growth.

Characterization of the Common Japonica-Originated Genomic Regions in the High-Yielding Varieties Developed from Inter-Subspecific Crosses in Temperate Rice (Oryza sativa L.).

The inter-subspecific crossing between indica and japonica subspecies in rice have been utilized to improve the yield potential of temperate rice. In this study, a comparative study of the genomic regions in the eight high-yielding varieties (HYVs) was conducted with those of the four non-HYVs. The Next-Generation Sequencing (NGS) mapping on the Nipponbare reference genome identified a total of 14 common genomic regions of japonica-originated alleles. Interestingly, the HYVs shared japonica-originated genomic regions on nine chromosomes, although they were developed through different breeding programs. A panel of 94 varieties was classified into four varietal groups with 38 single nucleotide polymorphism (SNP) markers from 38 genes residing in the japonica-originated genomic regions and 16 additional trait-specific SNPs. As expected, the japonica-originated genomic regions were only present in the japonica (JAP) and HYV groups, except for Chr4-1 and Chr4-2. The Wx gene, located within Chr6-1, was present in the HYV and JAP variety groups, while the yield-related genes were conserved as indica alleles in HYVs. The japonica-originated genomic regions and alleles shared by HYVs can be employed in molecular breeding programs to further develop the HYVs in temperate rice.

Evaluation of Whole-Genome Sequence, Genetic Diversity, and Agronomic Traits of Basmati Rice (Oryza sativa L.).

Basmati is considered a unique varietal group of rice (Oryza sativa L.) because of its aroma and superior grain quality. Previous genetic analyses of rice showed that most of the Basmati varieties are classified into the aromatic group. Despite various efforts, genomic relationship of Basmati rice with other varietal groups and genomic variation in Basmati rice are yet to be understood. In the present study, we resequenced the whole genome of three traditional Basmati varieties at a coverage of more than 25X using Illumina HiSeq2500 and mapped the obtained sequences to the reference genome sequences of Nipponbare (japonica rice), Kasalath (aus rice), and Zhenshan 97 (indica rice). Comparison of these sequences revealed common single nucleotide polymorphisms (SNPs) in the genic regions of three Basmati varieties. Analysis of these SNPs revealed that Basmati varieties showed fewer sequence variations compared with the aus group than with the japonica and indica groups. Gene ontology (GO) enrichment analysis indicated that SNPs were present in genes with various biological, molecular, and cellular functions. Additionally, functional annotation of the Basmati mutated gene cluster shared by Nipponbare, Kasalath, and Zhenshan 97 was found to be associated with the metabolic process involved in the cellular aromatic compound, suggesting that aroma is an important specific genomic feature of Basmati varieties. Furthermore, 30 traditional Basmati varieties were classified into three different groups, aromatic (22 varieties), aus (four varieties), and indica (four varieties), based on genome-wide SNPs. All 22 aromatic Basmati varieties harbored the fragrant-inducing Badh2 allele. We also performed comparative analysis of 13 key agronomic and grain quality traits of Basmati rice and other rice varieties. Three traits including length-to-width ratio of grain (L/W ratio), panicle length (PL), and amylose content (AC) showed significant (P < 0.05 and P < 0.01) differences between the aromatic and indica/aus groups. Comparative analysis of genome structure, based on genome sequence variation and GO analysis, revealed that the Basmati genome was derived mostly from the aus and japonica groups. Overall, whole-genome sequence data and genetic diversity information obtained in this study will serve as an important resource for molecular breeding and genetic analysis of Basmati varieties.

Analysis of evolutionary relationships provides new clues to the origins of weedy rice.

Weedy rice (WR) (Oryza sativa f. spontanea) is considered to be a pest in modern rice production systems because it competes for resources, has poor yield characteristics, and subsequently has a negative effect on rice grain yield. The evolutionary relationships among WR, landrace rice (LR), improved rice (IR) cultivars, and wild rice are largely unknown. In this study, we conducted a population genetic analysis based on neutral markers and gene haplotypes in 524 rice accessions and a comparative transcriptomic analysis using 15 representative samples. The results showed that WR populations have the highest level of genetic diversity (H e = 0.8386) and can be divided into two groups (japonica-type and indica-type). The japonica-type WR accessions from Heilongjiang province (HLJ), Jilin province (JL), Liaoning province (LN), and NX provinces clustered with the landraces grown in these same provinces. The indica-types from Jiangsu province (JS) also clustered with the indica-type landraces from JS province. Comparative transcriptome analysis of WR' IR and LR from HLJ, JL, and LN provinces showed that the WR still clustered with the LR, and that the IR lines comprise a single population. Thirty-two differentially expressed genes were shared by the IR and LR groups as well as between the IR and WR groups. Using Gene ontology (GO) analysis, we identified 19 shared GO terms in the IR and LR groups as well as between the IR and WR groups. Our results suggest that WR populations in China have diverse origins, and comparative transcriptome analysis of different types of rice from HLJ, JL, and LN provinces suggests that IR populations have become a end point in the evolution of WR, which provides a new perspective for the study of WR origins and lays a solid foundation for rice breeding.