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.

Teosinte Branched 1 modulates tillering in rice plants.

Tillering is an important trait of cereal crops that optimizes plant architecture for maximum yield. Teosinte Branched 1 (TB1) is a negative regulator of lateral branching and an inducer of female inflorescence formation in Zea mays (maize). Recent studies indicate that TB1 homologs in Oryza sativa (rice), Sorghum bicolor and Arabidopsis thaliana act downstream of the auxin and MORE AUXILIARY GROWTH (MAX) pathways. However, the molecular mechanism by which rice produces tillers remains unknown. In this study, transgenic rice plants were produced that overexpress the maize TB1 (mTB1) or rice TB1 (OsTB1) genes and silence the OsTB1 gene through RNAi-mediated knockdown. Because lateral branching in rice is affected by the environmental conditions, the phenotypes of transgenic plants were observed in both the greenhouse and the paddy field. Compared to wild-type plants, the number of tillers and panicles was reduced and increased in overexpressed and RNAi-mediated knockdown OsTB1 rice plants, respectively, under both environmental conditions. However, the effect was small for plants grown in paddy fields. These results demonstrate that both mTB1 and OsTB1 moderately regulate the tiller development in rice.

Shotgun proteomic analysis for detecting differentially expressed proteins in the reduced culm number rice.

To survey protein expression patterns in the reduced culm number (RCN) rice, a comparative shotgun proteomic analysis was conducted. For large-scale protein identification, multidimensional protein identification technology (MudPIT) coupled with pre-fractionation of plant shoot proteins led to the identification of 3004 non-redundant rice proteins. By statistically comparing relative amounts of 1353 reproducibly identified proteins between the RCN rice and the wild-type rice, 44 differentially expressed proteins were detected, where 42 proteins were increased and 2 proteins were decreased in the RCN rice. These proteins appear to have roles in glycolysis, trichloroacetic acid cycle, secondary metabolism, nutrient recycling, and nucleotide metabolism and repair. Consequently, we hypothesized that the RCN rice might fail to maintain sugar nutrient homeostasis. This was confirmed with the observation that the sucrose concentration was increased significantly in the RCN rice compared with the wild-type rice. Also, the RCN rice showed a hypersensitive response to exogenous sucrose treatment.

Single nucleotide polymorphisms and haplotype diversity in rice sucrose synthase 3.

Rice sucrose synthase 3 (RSUS3) is expressed predominantly in rice seed endosperm and is thought to play an important role in starch filling during the milky stage of rice seed ripening. Because the genetic diversity of this locus is not known yet, the full sequence of RSUS3 from 43 rice varieties was amplified to examine the distribution of DNA polymorphisms. A total of 254 sequence variants, including SNPs and insertion/deletions, were successfully identified in the 7733 bp sequence that comprises the promoter, exons and introns, and 3' downstream nontranscribed region (NTR). Eleven haplotypes were distinguished among the 43 rice varieties based on nucleotide variation in the 3 defined regions (5' NTR, transcript, and 3' NTR). The promoter region showed evidence of a base change on a cis-element that might influence the functional role of the motif in seed-specific expression. The genetic diversity of the RSUS3 gene sequences in the rice germplasm used in this study appears to be the result of nonrandom processes. Analysis of polymorphism sites indicated that at least 11 recombinations have occurred, primarily in the transcribed region. This finding provides insight into the development of a cladistic approach for establishing future genetic association studies of the RSUS3 locus.

SPL28 encodes a clathrin-associated adaptor protein complex 1, medium subunit micro 1 (AP1M1) and is responsible for spotted leaf and early senescence in rice (Oryza sativa).

To expand our understanding of cell death in plant defense responses, we isolated a novel rice (Oryza sativa) spotted leaf mutant (spl28) that displays a lesion mimic phenotype in the absence of pathogen attack through treatment of Hwacheongbyeo (an elite Korean japonica cultivar) with N-methyl-N-nitrosourea (MNU). Early stage development of the spl28 mutant was normal. However, after flowering, spl28 mutants exhibited a significant decrease in chlorophyll content, soluble protein content, and photosystem II efficiency, and high concentrations of reactive oxygen species (ROS), phytoalexin, callose, and autofluorescent phenolic compounds that localized in or around the lesions. The spl28 mutant also exhibited significantly enhanced resistance to rice blast and bacterial blight. Using a map-based cloning approach, we determined that SPL28 encodes a clathrin-associated adaptor protein complex 1, medium subunit micro 1 (AP1M1), which is involved in the post-Golgi trafficking pathway. A green fluorescent protein (GFP) fusion protein of SPL28 (SPL28::GFP) localized to the Golgi apparatus, and expression of SPL28 complemented the membrane trafficking defect of apm1-1 Delta yeast mutants. SPL28 was ubiquitously expressed and contained a highly conserved adaptor complex medium subunit (ACMS) family domain. SPL28 appears to be involved in the regulation of vesicular trafficking, and SPL28 dysfunction causes the formation of hypersensitive response (HR)-like lesions, leading to the initiation of leaf senescence.

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.

Inactivation of the UGPase1 gene causes genic male sterility and endosperm chalkiness in rice (Oryza sativa L.).

A rice genic male-sterility gene ms-h is recessive and has a pleiotropic effect on the chalky endosperm. After fine mapping, nucleotide sequencing analysis of the ms-h gene revealed a single nucleotide substitution at the 3'-splice junction of the 14th intron of the UDP-glucose pyrophosphorylase 1 (UGPase1; EC2.7.7.9) gene, which causes the expression of two mature transcripts with abnormal sizes caused by the aberrant splicing. An in vitro functional assay showed that both proteins encoded by the two abnormal transcripts have no UGPase activity. The suppression of UGPase by the introduction of a UGPase1-RNAi construct in wild-type plants nearly eliminated seed set because of the male defect, with developmental retardation similar to the ms-h mutant phenotype, whereas overexpression of UGPase1 in ms-h mutant plants restored male fertility and the transformants produced T(1) seeds that segregated into normal and chalky endosperms. In addition, both phenotypes were co-segregated with the UGPase1 transgene in segregating T(1) plants, which demonstrates that UGPase1 has functional roles in both male sterility and the development of a chalky endosperm. Our results suggest that UGPase1 plays a key role in pollen development as well as seed carbohydrate metabolism.

Map-based cloning of the ERECT PANICLE 3 gene in rice.

Panicle architecture in rice can have a strong influence on yield. Using N-methyl-N-nitrosourea mutagenesis, we isolated an erect panicle mutant, Hep, from Hwasunchalbyeo, a glutinous japonica rice cultivar. Genetic analysis revealed that the erect panicle phenotype was controlled by a single recessive mutation designated erect panicle 3 (ep3). Genetic mapping revealed that the ep3 mutation was located on the short arm of chromosome 2 in a 0.1 cM region delimited by the STS markers STS5803-5 and STS5803-7. The ep3 locus corresponded to 46.8 kb region and contained six candidate genes. Comparison of the DNA sequences of the candidate genes from wild-type and erect panicle plants revealed a single base-pair change in the second exon of LOC_Os02g15950, which is predicted to result in a nonsense mutation. LOC_Os02g15950 encodes a putative F-box protein containing 515 amino acids and is expressed throughout the plant during all growth stages. A line carrying a T-DNA insertion in LOC_ Os02g15950 was obtained and shown to have the same phenotype as the ep3 mutant, thus confirming the identification of LOC_Os02g15950 as the ERECT PANICLE 3 (EP3) gene. The ep3 mutation causes a significant increase in the number of small vascular bundles as well as the thickness of parenchyma in the peduncle, which results in the erect panicle phenotype.

High-resolution mapping of two rice brown planthopper resistance genes, Bph20(t) and Bph21(t), originating from Oryza minuta.

Brown planthopper (BPH) is one of the most destructive insect pests of rice. Wild species of rice are a valuable source of resistance genes for developing resistant cultivars. A molecular marker-based genetic analysis of BPH resistance was conducted using an F(2) population derived from a cross between an introgression line, 'IR71033-121-15', from Oryza minuta (Accession number 101141) and a susceptible Korean japonica variety, 'Junambyeo'. Resistance to BPH (biotype 1) was evaluated using 190 F(3) families. Two major quantitative trait loci (QTLs) and two significant digenic epistatic interactions between marker intervals were identified for BPH resistance. One QTL was mapped to 193.4-kb region located on the short arm of chromosome 4, and the other QTL was mapped to a 194.0-kb region on the long arm of chromosome 12. The two QTLs additively increased the resistance to BPH. Markers co-segregating with the two resistance QTLs were developed at each locus. Comparing the physical map positions of the two QTLs with previously reported BPH resistance genes, we conclude that these major QTLs are new BPH resistance loci and have designated them as Bph20(t) on chromosome 4 and Bph21(t) on chromosome 12. This is the first report of BPH resistance genes from the wild species O. minuta. These two new genes and markers reported here will be useful to rice breeding programs interested in new sources of BPH resistance.

Sugary Endosperm is Modulated by Starch Branching Enzyme IIa in Rice (Oryza sativa L.).

BACKGROUND: Starch biosynthesis is one of the most important pathways that determine both grain quality and yield in rice (Oryza sativa L.). Sugary endosperm, sugary-1 (sug-1), is a mutant trait for starch biosynthesis. Rice plants carrying sug-1 produce grains that accumulate water-soluble carbohydrates instead of starch, even after maturity. Although this trait enhances the diversity of grain quality, sugary endosperm rice has hardly been commercialized due to the severely wrinkled grains and subsequent problems in milling. This study was conducted to identify the genes responsible for the sug-h phenotype through a map-based cloning technology. RESULTS: We induced a mild sugary mutant, sugary-h (sug-h) through the chemical mutagenesis on the Korean japonica cultivar Hwacheong. Grains of the sug-h mutant were translucent and amber-colored, and the endosperm appeared less wrinkled than sug-1, whereas the soluble sugar content was fairly high. These characteristics confer greater marketability to the sug-h mutant. Genetic analyses indicated that the sug-h mutant phenotype was controlled by a complementary interaction of two recessive genes, Isoamylase1 (OsISA1), which was reported previously, and Starch branching enzyme IIa (OsBEIIa), which was newly identified in this study. Complementation tests indicated that OsBEIIa regulated the properties of sugary endosperm. CONCLUSIONS: Complementary interactions between the starch biosynthesis genes OsISA1 and OsBEIIa determine the mild sugary endosperm mutant, sugary-h, in rice. Our finding may facilitate the breeding of sugaryendosperm rice for commercial benefit.

Isolation and characterization of a dominant dwarf gene, d-h, in rice.

Plant height is an important agronomic trait that affects grain yield. Previously, we reported a novel semi-dominant dwarf mutant, HD1, derived from chemical mutagenesis using N-methyl-N-nitrosourea (MNU) on a japonica rice cultivar, Hwacheong. In this study, we cloned the gene responsible for the dwarf mutant using a map-based approach. Fine mapping revealed that the mutant gene was located on the short arm of chromosome 1 in a 48 kb region. Sequencing of the candidate genes and rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) analysis identified the gene, d-h, which encodes a protein of unknown function but whose sequence is conserved in other cereal crops. Real-time (RT)-PCR analysis and promoter activity assays showed that the d-h gene was primarily expressed in the nodes and the panicle. In the HD1 plant, the d-h gene was found to carry a 63-bp deletion in the ORF region that was subsequently confirmed by transgenic experiments to be directly responsible for the gain-of-function phenotype observed in the mutant. Since the mutant plants exhibit a defect in GA response, but not in the GA synthetic pathway, it appears that the d-h gene may be involved in a GA signaling pathway.

Manipulation of two α-endo-ß-1,4-glucanase genes, AtCel6 and GmCel7, reduces susceptibility to Heterodera glycines in soybean roots.

Plant endo-ß-1,4-glucanases (EGases) include cell wall-modifying enzymes that are involved in nematode-induced growth of syncytia (feeding structures) in nematode-infected roots. EGases in the α- and ß-subfamilies contain signal peptides and are secreted, whereas those in the γ-subfamily have a membrane-anchoring domain and are not secreted. The Arabidopsis α-EGase At1g48930, designated as AtCel6, is known to be down-regulated by beet cyst nematode (Heterodera schachtii) in Arabidopsis roots, whereas another α-EGase, AtCel2, is up-regulated. Here, we report that the ectopic expression of AtCel6 in soybean roots reduces susceptibility to both soybean cyst nematode (SCN; Heterodera glycines) and root knot nematode (Meloidogyne incognita). Suppression of GmCel7, the soybean homologue of AtCel2, in soybean roots also reduces the susceptibility to SCN. In contrast, in studies on two γ-EGases, both ectopic expression of AtKOR2 in soybean roots and suppression of the soybean homologue of AtKOR3 had no significant effect on SCN parasitism. Our results suggest that secreted α-EGases are likely to be more useful than membrane-bound γ-EGases in the development of an SCN-resistant soybean through gene manipulation. Furthermore, this study provides evidence that Arabidopsis shares molecular events of cyst nematode parasitism with soybean, and confirms the suitability of the Arabidopsis-H. schachtii interaction as a model for the soybean-H. glycines pathosystem.

Identification of QTLs for seed germination capability after various storage periods using two RIL populations in rice.

Seed germination capability of rice is one of the important traits in the production and storage of seeds. Quantitative trait loci (QTL) associated with seed germination capability in various storage periods was identified using two sets of recombinant inbred lines (RILs) which derived from crosses between Milyang 23 and Tong 88-7 (MT-RILs) and between Dasanbyeo and TR22183 (DT-RILs). A total of five and three main additive effects (QTLs) associated with seed germination capability were identified in MT-RILs and DT-RILs, respectively. Among them, six QTLs were identified repeatedly in various seed storage periods designated as qMT-SGC5.1, qMT-SGC7.2, and qMT-SGC9.1 on chromosomes 5, 7, and 9 in MT-RILs, and qDT-SGC2.1, qDT-SGC3.1, and qDT-SGC9.1 on chromosomes 2, 3, and 9 in DT-RILs, respectively. The QTL on chromosome 9 was identified in both RIL populations under all three storage periods, explaining up to 40% of the phenotypic variation. Eight and eighteen pairs additive × additive epistatic effect (epistatic QTL) were identified in MT-RILs and DT-RILs, respectively. In addition, several near isogenic lines (NILs) were developed to confirm six repeatable QTL effects using controlled deterioration test (CDT). The identified QTLs will be further studied to elucidate the mechanisms controlling seed germination capability, which have important implications for long-term seed storage.

PCR marker-based evaluation of the eating quality of japonica rice ( Oryza sativa L.).

Evaluation of eating quality in early breeding generations of rice is critical to developing varieties with better palatability. This paper reports DNA markers associated with eating quality of temperate japonica rice and an evaluation method aided by multiple regression analysis. A total of 30 markers comprising STSs, SNPs, and SSRs were tested for their association with palatability using 22 temperate japonica varieties with different palatability values. Eating quality-related traits of the 22 varieties were also measured. Of the 30 markers, 18 were found to be significantly associated with palatability and, consequently, a model regression equation with an R(2) value of 0.99 was formulated to estimate the palatability by the marker data set. Validation of the model equation using selected breeding lines indicated that the marker set and the equation are highly applicable to evaluation of the palatability of cooked rice in temperate japonica varieties.