Map-Based Cloning and Characterization of a Major QTL Gene, FfR1, Which Confers Resistance to Rice Bakanae Disease.

Bakanae disease (BD), caused by the fungal pathogen Fusarium fujikuroi, is a serious threat to rice production worldwide. Breeding elite rice varieties resistant to BD requires the identification of resistance genes. Previously, we discovered a resistant quantitative trait locus (QTL), qFfR1, in a Korean japonica rice variety, Nampyeong. In this study, we fine-mapped qFfR1 with a Junam*4/Nampyeong BC3F3 population and delimited its location to a 37.1 kb region on chromosome 1. Complementation experiments with seven candidate genes in this region revealed that OsI_02728 is the gene for qFfR1. This gene encodes a protein with a typical leucine-rich repeat (LRR) receptor-like protein structure. RNA-sequencing-based transcriptomic analysis revealed that FfR1 induces the transcription of defense genes, including lignin and terpenoid biosynthesis genes, pathogenesis-related genes, and thionin genes. These results may facilitate investigations into the molecular mechanisms underlying BD resistance, including molecular patterns of Fusarium fujikuroi interacting with FfR1 and players working in signal transduction pathways downstream of FfR1, and the breeding of new BD-resistant varieties by providing a BD resistance gene with its precise selection marker. This will contribute to efficient control of BD, which is becoming more prevalent according to temperature rises due to climate change.

A rice seed-specific glycine-rich protein OsDOR1 interacts with GID1 to repress GA signaling and regulates seed dormancy.

Seed dormancy is an important agronomic trait under the control of complex genetic and environmental interactions, which have not been yet comprehensively understood. From the field screening of rice mutant library generated by a Ds transposable element, we identified a pre-harvest sprouting (PHS) mutant dor1. This mutant has a single insertion of Ds element at the second exon of OsDOR1 (LOC_Os03g20770), which encodes a novel seed-specific glycine-rich protein. This gene successfully complemented the PHS phenotype of dor1 mutant and its ectopic expression enhanced seed dormancy. Here, we demonstrated that OsDOR1 protein binds to the GA receptor protein, OsGID1 in rice protoplasts, and interrupts with the formation OsGID1-OsSLR1 complex in yeast cells. Co-expression of OsDOR1 with OsGID1 in rice protoplasts attenuated the GA-dependent degradation of OsSLR1, the key repressor of GA signaling. We showed the endogenous OsSLR1 protein level in the dor1 mutant seeds is significantly lower than that of wild type. The dor1 mutant featured a hypersensitive GA-response of α-amylase gene expression during seed germination. Based on these findings, we suggest that OsDOR1 is a novel negative player of GA signaling operated in the maintenance of seed dormancy. Our findings provide a novel source of PHS resistance.

CTP synthase is essential for early endosperm development by regulating nuclei spacing.

Cereal grain endosperms are an important source of human nutrition. Nuclear division in early endosperm development plays a major role in determining seed size; however, this development is not well understood. We identified the rice mutant endospermless 2 (enl2), which shows defects in the early stages of endosperm development. These phenotypes arise from mutations in OsCTPS1 that encodes a cytidine triphosphate synthase (CTPS). Both wild-type and mutant endosperms were normal at 8 h after pollination (HAP). In contrast, at 24 HAP, enl2 endosperm had approximately 10-16 clumped nuclei while wild-type nuclei had increased in number and migrated to the endosperm periphery. Staining of microtubules in endosperm at 24 HAP revealed that wild-type nuclei were evenly distributed by microtubules while the enl2-2 nuclei were tightly packed due to their reduction in microtubule association. In addition, OsCTPS1 interacts with tubulins; thus, these observations suggest that OsCTPS1 may be involved in microtubule formation. OsCTPS1 transiently formed macromolecular structures in the endosperm during early developmental stages, further supporting the idea that OsCTPS1 may function as a structural component during endosperm development. Finally, overexpression of OsCTPS1 increased seed weight by promoting endosperm nuclear division, suggesting that this trait could be used to increase grain yield.

The OsERF115/AP2EREBP110 Transcription Factor Is Involved in the Multiple Stress Tolerance to Heat and Drought in Rice Plants.

The AP2/EREBP family transcription factors play important roles in a wide range of stress tolerance and hormone signaling. In this study, a heat-inducible rice ERF gene was isolated and functionally characterized. The OsERF115/AP2EREBP110 was categorized to Group-IIIc of the rice AP2/EREBP family and strongly induced by heat and drought treatment. The OsERF115/AP2EREBP110 protein targeted to nuclei and suppressed the ABA-induced transcriptional activation of Rab16A promoter in rice protoplasts. Overexpression of OsERF115/AP2EREBP110 enhanced thermotolerance of seeds and vegetative growth stage plants. The OsERF115/AP2EREBP110 overexpressing (OE) plants exhibited higher proline level and increased expression of a proline biosynthesis P5CS1 gene. Phenotyping of water use dynamics of the individual plant indicates that the OsERF115/AP2EREBP110-OE plant exhibited better water saving traits under heat and drought combined stress. Our combined results suggest the potential use of OsERF115/AP2EREBP110 as a candidate gene for genetic engineering approaches to develop heat and drought stress-tolerant crops.

QTL mapping for pre-harvest sprouting resistance in japonica rice varieties utilizing genome re-sequencing.

Pre-harvest sprouting (PHS) leads to serious economic losses because of reductions in yield and quality. To analyze the quantitative trait loci (QTLs) for PHS resistance in japonica rice, PHS rates on panicles were measured in 160 recombinant inbred lines (RILs) derived from a cross between the temperate japonica varieties Odae (PHS resistant) and Unbong40 (PHS susceptible) under two different environmental conditions-field (summer) and greenhouse (winter) environments. Genome re-sequencing of the parental varieties detected 266,773 DNA polymorphisms including 248,255 single nucleotide polymorphisms and 18,518 insertions/deletions. We constructed a genetic map comprising 239 kompetitive allele-specific PCR and 49 cleaved amplified polymorphic sequence markers. In the field environment, two major QTLs, qPHS-3FD and qPHS-11FD, were identified on chromosomes 3 and 11, respectively, whereas three major QTLs, qPHS-3GH, qPHS-4GH, and qPHS-11GH, were identified on chromosomes 3, 4, and 11, respectively, in the greenhouse environment. qPHS-11GH and qPHS-11FD had similar locations on chromosome 11, suggesting the existence of a gene conferring stable PHS resistance effects under different environmental conditions. The QTLs identified in this study can be used to improve the PHS resistance of japonica varieties, and they may improve our understanding of the genetic basis of PHS resistance.

High-throughput phenotyping platform for analyzing drought tolerance in rice.

MAIN CONCLUSION: A new imaging platform was constructed to analyze drought-tolerant traits of rice. Rice was used to quantify drought phenotypes through image-based parameters and analyzing tools. Climate change has increased the frequency and severity of drought, which limits crop production worldwide. Developing new cultivars with increased drought tolerance and short breeding cycles is critical. However, achieving this goal requires phenotyping a large number of breeding populations in a short time and in an accurate manner. Novel cutting-edge technologies such as those based on remote sensors are being applied to solve this problem. In this study, new technologies were applied to obtain and analyze imaging data and establish efficient screening platforms for drought tolerance in rice using the drought-tolerant mutant osphyb. Red-Green-Blue images were used to predict plant area, color, and compactness. Near-infrared imaging was used to determine the water content of rice, infrared was used to assess plant temperature, and fluorescence was used to examine photosynthesis efficiency. DroughtSpotter technology was used to determine water use efficiency, plant water loss rate, and transpiration rate. The results indicate that these methods can detect the difference between tolerant and susceptible plants, suggesting their value as high-throughput phenotyping methods for short breeding cycles as well as for functional genetic studies of tolerance to drought stress.

High Throughput Phenotyping for Various Traits on Soybean Seeds Using Image Analysis.

Data phenotyping traits on soybean seeds such as shape and color has been obscure because it is difficult to define them clearly. Further, it takes too much time and effort to have sufficient number of samplings especially length and width. These difficulties prevented seed morphology to be incorporated into efficient breeding program. Here, we propose methods for an image acquisition, a data processing, and analysis for the morphology and color of soybean seeds by high-throughput method using images analysis. As results, quantitative values for colors and various types of morphological traits could be screened to create a standard for subsequent evaluation of the genotype. Phenotyping method in the current study could define the morphology and color of soybean seeds in highly accurate and reliable manner. Further, this method enables the measurement and analysis of large amounts of plant seed phenotype data in a short time, which was not possible before. Fast and precise phenotype data obtained here may facilitate Genome Wide Association Study for the gene function analysis as well as for development of the elite varieties having desirable seed traits.

Rice Genome Resequencing Reveals a Major Quantitative Trait Locus for Resistance to Bakanae Disease Caused by Fusarium fujikuroi.

Bakanae disease (BD), caused by the fungal pathogen Fusarium fujikuroi, has become a serious threat in rice-cultivating regions worldwide. In the present study, quantitative trait locus (QTL) mapping was performed using F2 and F3 plants derived after crossing a BD-resistant and a BD-susceptible Korean japonica rice variety, 'Samgwang' and 'Junam', respectively. Resequencing of 'Junam' and 'Samgwang' genomes revealed 151,916 DNA polymorphisms between the two varieties. After genotyping 188 F2 plants, we constructed a genetic map comprising 184 markers, including 175 kompetitive allele-specific PCR markers, eight cleaved amplified polymorphic sequence (CAPS) markers, and a derived CAPS (dCAPS) marker. The degree of BD susceptibility of each F2 plant was evaluated on the basis of the mortality rate measured with corresponding F3 progeny seedlings by in vitro screening. Consequently, qFfR9, a major QTL, was discovered at 30.1 centimorgan (cM) on chromosome 9 with a logarithm of the odds score of 60.3. For the QTL interval, 95% probability lay within a 7.24-7.56 Mbp interval. In this interval, we found that eight genes exhibited non-synonymous single nucleotide polymorphisms (SNPs) by comparing the 'Junam' and 'Samgwang' genome sequence data, and are possibly candidate genes for qFfR9; therefore, qFfR9 could be utilized as a valuable resource for breeding BD-resistant rice varieties.

Mapping of a major quantitative trait locus for bakanae disease resistance in rice by genome resequencing.

Bakanae disease (BD) has emerged as a serious threat in almost all rice cultivation regions worldwide. Nampyeong is a Korean japonica rice variety known to be resistant to BD. In this study, quantitative trait locus (QTL) mapping was performed with F2 and F3 plants derived from a cross between the Nampyeong variety and a susceptible Korean japonica line, DongjinAD. First, resequencing of Nampyeong and DongjinAD was performed, which identified 171,035 single nucleotide polymorphisms (SNPs) between the two parental varieties. Using these SNPs, 161 cleaved amplified polymorphic sequence (CAPS) markers and six derived CAPS markers were developed; then, a genetic map was constructed from the genotypes of 180 plants from the DongjinAD/Nampyeong F2 plants. The total length of the constructed genetic map was 1386 cM, with an average interval of 8.9 cM between markers. The BD mortality rates of each F3 family were measured by testing 40 F3 progenies using in vitro seedling screening method. QTL analysis based on the genetic map and mortality rate data revealed a major QTL, qFfR1, on rice chromosome 1. qFfR1 was located at 89.8 cM with a logarithm of the odds (LOD) score of 22.7. Further, there were three markers at this point: JNS01033, JNS01037, and JNS01041. A total of 15 genes were identified with annotations related to defense against plant diseases among the 179 genes in the qFfR1 interval at 95% probability, thereby providing potential candidate genes for qFfR1. qFfR1 and its closely linked markers will be useful in breeding rice varieties resistant to BD.

Dry-milled flour rice 'Seolgaeng' harbors a mutated fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase2.

'Seolgaeng', an opaque-endosperm rice (Oryza sativa) mutant, is used to prepare high-quality dry-milled rice flour. The mutation causing its opaque-endosperm phenotype was unknown. Map-based cloning identified a missense mutation in the gene FRUCTOSE-6-PHOSPHATE 2-KINASE/FRUCTOSE-2,6-BISPHOSPHATASE 2 (OsF2KP2) in Seolgaeng. Transfer DNA insertion and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-induced f2kp2 mutants exhibited opaque endosperm. Rice harbors another F2KP gene, OsF2KP1. CRISPR/Cas9-induced double mutants of OsF2KP1 and OsF2KP2 (f2kp-d) possessed more opaque endosperm compared to f2kp2 single mutants, whereas the endosperm of the f2kp1 single mutant was normal. Grain hardness and damaged starch content were significantly reduced in f2kp2 mutants compared to the wild type and f2kp1. Amylose content was lower than normal in f2kp2 mutants but not f2kp1. Grain hardness and amylose content were much lower in f2kp-d than in f2kp2. Starch polymerization analysis revealed altered amylopectin structure in f2kp2 and f2kp-d mutants. F2KP activity was lower in f2kp2 and much lower in the double mutants when compared to the wild types, but f2kp1 showed no significant difference. In coleoptiles, hypoxia induced OsF2KP2 expression but downregulated OsF2KP1. These results suggest that OsF2KP2 functions as the main F2KP isoform in endosperm experiencing hypoxia, but OsF2KP1 may partially compensate for the absence of OsF2KP2. We propose that F2KP has a crucial role in inorganic pyrophosphate-utilizing energy metabolism for starch biosynthesis in rice endosperm.

QTL Mapping of Tiller Number in Korean Japonica Rice Varieties.

Tiller number is an important trait associated with yield in rice. Tiller number in Korean japonica rice was analyzed under greenhouse conditions in 160 recombinant inbred lines (RILs) derived from a cross between the temperate japonica varieties Odae and Unbong40 to identify quantitative trait loci (QTLs). A genetic map comprising 239 kompetitive allele-specific PCR (KASP) and 57 cleaved amplified polymorphic sequence markers was constructed. qTN3, a major QTL for tiller number, was identified at 132.4 cm on chromosome 3. This QTL was also detected under field conditions in a backcross population; thus, qTN3 was stable across generations and environments. qTN3 co-located with QTLs associated with panicle number per plant and culm diameter, indicating it had pleiotropic effects. The qTN3 regions of Odae and Unbong40 differed in a known functional variant (4 bp TGTG insertion/deletion) in the 5' UTR of OsTB1, a gene underlying variation in tiller number and culm strength. Investigation of variation in genotype and tiller number revealed that varieties with the insertion genotype had lower tiller numbers than those with the reference genotype. A high-resolution melting marker was developed to enable efficient marker-assisted selection. The QTL qTN3 will therefore be useful in breeding programs developing japonica varieties with optimal tiller numbers for increased yield.

Characterization and QTL Mapping of a Major Field Resistance Locus for Bacterial Blight in Rice.

Bacterial blight (BB) disease, caused by Xanthomonas oryzae pv. oryzae (Xoo), is among the major factors that can cause rice yields to decrease. To address BB disease, researchers have been looking for ways to change pesticides and cultivation methods, but developing resistant cultivars is the most effective method. However, the resistance and genetic factors of cultivars may be destroyed due to the emergence of new Xoo species caused by recent and rapid climate changes. Therefore, breeders need to identify resistance genes that can be sustained during unpredictable climate changes and utilized for breeding. Here, qBBR11, a quantitative trait locus (QTL) for resistance to BB disease, was detected in KJ (Korea Japonica varieties) 11_067 to KJ11_068 on chromosome 11 in a population derived by crossing JJ (Jeonju) 623 and HR(High resistant)27,195, which possess similar genetic backgrounds but different degrees of resistance to BB disease. qBBR11 was reduced from 18.49-18.69 Mbp of chromosome 11 to 200 kbp segment franked. In this region, 16 candidate genes were detected, and we identified 24 moderate-impact variations and four high-impact variations. In particular, high-impact variations were detected in Os11g0517800 which encode the domain region of GCN2 which is the eIF-2-alpha kinase associated with the resistance of abiotic/biotic stress in rice. In JJ623, which is moderately resistant to BB disease, a stop codon was created due to single nucleotide polymorphism (SNP). Therefore, compared with HR27195, JJ623 has weaker resistance to BB disease, though the two have similar genetic backgrounds. The results suggest that variation in the qBBR11 region regulates an important role in improving resistance to BB diseases, and qBBR11 is useful in providing an important resource for marker-assisted selection to improve mechanisms of resistance to BB disease.

Marker-Assisted Backcrossing (MABc) to Improve Eating Quality with Thin Seed Coat and Aleurone Layer of Non-Glutinous Japonica Variety in Rice.

Brown rice is composed of rice bran, pericarp, seed coat, and aleurone layers, and the rice bran layer contains a large number of substances useful for the human body, such as dietary fiber, α-tocopherol, α-tocotrienol, and vitamins. However, more than 90% of these substances are removed when polished, and white rice has the disadvantage of losing food-related ingredients, such as umami-related amino acids, when compared to the unpolished group. In this study, we tried to develop new breeding lines with a thinner seed coat and aleurone layer to provide high eating quality with softer chewing characteristics and processability in rice grain. We detected an SNP for foreground selection for the backcross population by comparing genome sequences between Samgwang and Seolgaeng and developed high eating quality brown rice breeding lines by applying marker-assisted backcrossing (MABC) breeding programs to backcross populations between Samgwang and Seolgaeng using KASP markers. SNP markers for foreground selection were identified to improve eating and processability through SNP mapping of Samgwang and Seolgaeng with SSIIa as a target gene in this study. Line selection according to genotype of KASP markers was successful in BC1F1 and BC2F1 generations, with the recurrent parent genome recovery ratio ranging from 91.22% to 98.65%. In BC2F1 seeds of the selected lines, thickness of the aleurone layer was found to range from 13.82 to 21.67 µm, which is much thinner than the 30.91 µm of the wild type, suggesting that selection by MABc could be used as an additional breeding material for the development of highly processed rice varieties. These lines will be useful to develop new brown rice varieties with softer chewing characteristics and processability in rice grain.

Development and Application of a Target Capture Sequencing SNP-Genotyping Platform in Rice.

The development of efficient, robust, and high-throughput SNP genotyping platforms is pivotal for crop genetics and breeding. Recently, SNP genotyping platforms based on target capture sequencing, which is very flexible in terms of the number of SNP markers, have been developed for maize, cassava, and fava bean. We aimed to develop a target capture sequencing SNP genotyping platform for rice. A target capture sequencing panel containing 2565 SNPs, including 1225 SNPs informative for japonica and 1339 SNPs informative for indica, was developed. This platform was used in diversity analysis of 50 rice varieties. Of the 2565 SNP markers, 2341 (91.3%) produced useful polymorphic genotype data, enabling the production of a phylogenetic tree of the 50 varieties. The mean number of markers polymorphic between any two varieties was 854. The platform was used for QTL mapping of preharvest sprouting (PHS) resistance in an F8 recombinant inbred line population derived from the cross Odae × Joun. A genetic map comprising 475 markers was constructed, and two QTLs for PHS resistance were identified on chromosomes 4 and 11. This system is a powerful tool for rice genetics and breeding and will facilitate QTL studies and gene mapping, germplasm diversity analysis, and marker-assisted selection.

Inactivation of the CTD phosphatase-like gene OsCPL1 enhances the development of the abscission layer and seed shattering in rice.

Although susceptibility to seed shattering causes severe yield loss during cereal crop harvest, it is an adaptive trait for seed dispersal in wild plants. We previously identified a recessive shattering locus, sh-h, from the rice shattering mutant line Hsh that carries an enhanced abscission layer. Here, we further mapped sh-h to a 34-kb region on chromosome 7 by analyzing 240 F(2) plants and five F(3) lines from the cross between Hsh and Blue&Gundil. Hsh had a point mutation at the 3' splice site of the seventh intron within LOC_Os07g10690, causing a 15-bp deletion of its mRNA as a result of altered splicing. Two transferred DNA (T-DNA) insertion mutants and one point mutant exhibited the enhanced shattering phenotype, confirming that LOC_Os07g10690 is indeed the sh-h gene. RNA interference (RNAi) transgenic lines with suppressed expression of this gene exhibited greater shattering. This gene, which encodes a protein containing a conserved carboxy-terminal domain (CTD) phosphatase domain, was named Oryza sativa CTD phosphatase-like 1 (OsCPL1). Subcellular localization and biochemical analysis revealed that the OsCPL1 protein is a nuclear phosphatase, a common characteristic of metazoan CTD phosphatases involved in cell differentiation. These results demonstrate that OsCPL1 represses differentiation of the abscission layer during panicle development.

Transcriptomic Analysis of Rice Plants Overexpressing PsGAPDH in Response to Salinity Stress.

In plants, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a main enzyme in the glycolytic pathway. It plays an essential role in glycerolipid metabolism and response to various stresses. To examine the function of PsGAPDH (Pleurotus sajor-caju GAPDH) in response to abiotic stress, we generated transgenic rice plants with single-copy/intergenic/homozygous overexpression PsGAPDH (PsGAPDH-OX) and investigated their responses to salinity stress. Seedling growth and germination rates of PsGAPDH-OX were significantly increased under salt stress conditions compared to those of the wild type. To elucidate the role of PsGAPDH-OX in salt stress tolerance of rice, an Illumina HiSeq 2000 platform was used to analyze transcriptome profiles of leaves under salt stress. Analysis results of sequencing data showed that 1124 transcripts were differentially expressed. Using the list of differentially expressed genes (DEGs), functional enrichment analyses of DEGs such as Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were performed. KEGG pathway enrichment analysis revealed that unigenes exhibiting differential expression were involved in starch and sucrose metabolism. Interestingly, trehalose-6-phosphate synthase (TPS) genes, of which expression was enhanced by abiotic stress, showed a significant difference in PsGAPDH-OX. Findings of this study suggest that PsGAPDH plays a role in the adaptation of rice plants to salt stress.

Genomic Variation in Korean japonica Rice Varieties.

Next-generation sequencing technologies have enabled the discovery of numerous sequence variations among closely related crop varieties. We analyzed genome resequencing data from 24 Korean temperate japonica rice varieties and discovered 954,233 sequence variations, including 791,121 single nucleotide polymorphisms (SNPs) and 163,112 insertions/deletions (InDels). On average, there was one variant per 391 base-pairs (bp), a variant density of 2.6 per 1 kbp. Of the InDels, 10,860 were longer than 20 bp, which enabled conversion to markers resolvable on an agarose gel. The effect of each variant on gene function was predicted using the SnpEff program. The variants were categorized into four groups according to their impact: high, moderate, low, and modifier. These groups contained 3524 (0.4%), 27,656 (2.9%), 24,875 (2.6%), and 898,178 (94.1%) variants, respectively. To test the accuracy of these data, eight InDels from a pre-harvest sprouting resistance QTL (qPHS11) target region, four highly polymorphic InDels, and four functional sequence variations in known agronomically important genes were selected and successfully developed into markers. These results will be useful to develop markers for marker-assisted selection, to select candidate genes in map-based cloning, and to produce efficient high-throughput genome-wide genotyping systems for Korean temperate japonica rice varieties.

Development of 454 New Kompetitive Allele-Specific PCR (KASP) Markers for Temperate japonica Rice Varieties.

Temperate japonica rice varieties exhibit wide variation in the phenotypes of several important agronomic traits, including disease resistance, pre-harvest sprouting resistance, plant architecture, and grain quality, indicating the presence of genes contributing to favorable agronomic traits. However, gene mapping and molecular breeding has been hampered as a result of the low genetic diversity among cultivars and scarcity of polymorphic DNA markers. Single nucleotide polymorphism (SNP)-based kompetitive allele-specific PCR (KASP) markers allow high-throughput genotyping for marker-assisted selection and quantitative trait loci (QTL) mapping within closely related populations. Previously, we identified 740,566 SNPs and developed 771 KASP markers for Korean temperate japonica rice varieties. However, additional markers were needed to provide sufficient genome coverage to support breeding programs. In this study, the 740,566 SNPs were categorized according to their predicted impacts on gene function. The high-impact, moderate-impact, modifier, and low-impact groups contained 703 (0.1%), 20,179 (2.7%), 699,866 (94.5%), and 19,818 (2.7%) SNPs, respectively. A subset of 357 SNPs from the high-impact group was selected for initial KASP marker development, resulting in 283 polymorphic KASP markers. After incorporation of the 283 markers with the 771 existing markers in a physical map, additional markers were developed to fill genomic regions with large gaps between markers, and 171 polymorphic KASP markers were successfully developed from 284 SNPs. Overall, a set of 1225 KASP markers was produced. The markers were evenly distributed across the rice genome, with average marker density of 3.3 KASP markers per Mbp. The 1225 KASP markers will facilitate QTL/gene mapping and marker-assisted selection in temperate japonica rice breeding programs.

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.

Mutations in the microRNA172 binding site of SUPERNUMERARY BRACT (SNB) suppress internode elongation in rice.

BACKGROUND: Internode elongation is an important agronomic trait in rice that determines culm length, which is related to lodging, panicle exsertion, and biomass. sui4 (shortened uppermost internode 4) mutants show reduced internode length and a dwarf phenotype due to shortened internodes; the uppermost internode is particularly severely affected. The present study was performed to identify the molecular nature and function of the SUI4 gene during internode elongation. RESULTS: Our previous study showed that the SUI4 gene was mapped to a 1.1-Mb interval on chromosome 7 (Ji et al. 2014). In order to isolate the gene responsible for the sui4 phenotype, genomic DNA resequencing of sui4 mutants and wild-type plants and reciprocal transformation of wild-type and mutant alleles of the putative SUI4 gene was performed. The data revealed that the causative mutation of sui4 was a T to A nucleotide substitution at the microRNA172 binding site of Os07g0235800, and that SUI4 is a new allele of the previously reported gene SUPERNUMERARY BRACT (SNB), which affects flower structure. In order to understand the effect of this mutation on expression of the SUI4/SNB gene, SUI4/SNB native promoter-fuzed GUS transgenics were examined, along with qRT-PCR analysis at various developmental stages. In sui4 mutants, the SUI4/SNB gene was upregulated in the leaves, culms, and panicles, especially when internodes were elongated. In culms, SUI4/SNB was expressed in the nodes and the lower parts of elongating internodes. In order to further explore the molecular nature of SUI4/SNB during internode elongation, RNA-seq and qRT-PCR analysis were performed with RNAs from the culms of sui4 mutants and wild-type plants in the booting stage. The data showed that in sui4 mutants, genes deactivating bioactive gibberellins and cytokinin were upregulated while genes related to cell expansion and cell wall synthesis were downregulated. CONCLUSION: In summary, this paper shows that interaction between SUI4/SNB and microRNA172 could determine internode elongation during the reproductive stage in rice plants. Due to a mutation at the microRNA172 binding site in sui4 mutants, the expression of SUI4/SNB was enhanced, which lowered the activities of cell expansion and cell wall synthesis and consequently resulted in shortened internodes.