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We previously identified a cluster of yield-related quantitative trait loci (QTLs) including plant height in CR4379, a near-isogenic line from a cross between Oryza sativa spp. japonica cultivar ‘Hwaseong’ and the wild relative Oryza rufipogon. Map-based cloning and transgenic approaches revealed that APX9, which encodes an l-ascorbate peroxidase 4, is associated with this cluster. A 3 bp InDel was observed leading to the addition of a valine in Hwaseong compared with O. rufipogon. APX9-overexpressing transgenic plants in the Hwaseong background were taller than Hwaseong. Consistent with these results, APX9 T-DNA insertion mutants in the japonica cultivar Dongjin were shorter. These results confirm that APX9 is the causal gene for the QTL cluster. Sequence analysis of APX9 from 303 rice accessions revealed that the 3 bp InDel clearly differentiates japonica (APX9HS) and O. rufipogon (APX9OR) alleles. indica accessions shared both alleles, suggesting that APX9HS was introgressed into indica followed by crossing. The finding that O. rufipogon accessions with different origins carry APX9OR suggests that the 3 bp insertion was specifically selected in japonica during its domestication. Our findings demonstrate that APX9 acts as a major regulator of plant development by controlling a valuable suite of agronomically important traits in rice., DNA marker-based smart breeding revealed that the ascorbate peroxidase gene APX9 of the wild rice relative Oryza rufipogon with low yield can make cultivated rice more productive and stress-tolerant.

Genetic analysis of genes that regulate the color pigmentation of sterile lemma and apiculus has been conducted. “Josaengjado” has small and round grains with purple leaf, sterile lemma and apiculus. In the F2 population from a cross between Josaengjado and Daeribbyeo 1, 246 and 182 plants exhibited purple and straw-white sterile lemma, respectively. It fitted a 9:7 segregation ratio indicating that two complementary genes control the pigmentation in sterile lemma and apiculus. Genetic analysis was performed using the F2:3 and KASP (Kompetitive Allele-Specific PCR) markers. Genes for the coloration of leaf sheath, ligule, sterile lemma, and apiculus were detected on chromosomes 1 and 6. Sequence comparison showed a single nucleotide substitution C (Josaengjado) to A (Daeribbyeo 1) in the second exon of the Rd gene on chromosome 1 leading to a premature stop in Daeribbyeo 1. In C1, a 3-bp deletion in the second exon was detected in Daeribbyeo 1. Haplotype analysis was performed in the Rd and C1 genes of the 78 rice accessions. 78 accessions were divided into 14 groups. A total of 11 and 1 mutation sites were detected in OsC1 and Rd, respectively. The haplotype analysis also confirmed that two complementary genes, Rd and OsC1 are necessary to express anthocyanin pigmentation in sterile lemma and apiculus. To our knowledge, this is the first report to identify genes for the coloration of sterile lemma in rice.

A novel hybrid weakness gene, DTE9, associated with a dark tip embryo (DTE) trait, was observed in CR6078, an introgression line derived from a cross between the Oryza sativa spp. japonica “Hwayeong” (HY) and the wild relative Oryza rufipogon. CR6078 seeds exhibit protruding embryos and flowers have altered inner floral organs. DTE9 was also associated with several hybrid weakness symptoms including decreased grain weight. Map-based cloning and transgenic approaches revealed that DTE9 is an allele of OsMADS8, a MADS-domain transcription factor. Genetic analysis indicated that two recessive complementary genes were responsible for the expression of the DTE trait. No sequence differences were observed between the two parental lines in the OsMADS8 coding region; however, numerous single nucleotide polymorphisms were detected in the promoter and intronic regions. We generated overexpression (OX) and RNA interference (RNAi) transgenic lines of OsMADS8 in HY and CR6078, respectively. The OsMADS8-OX lines showed the dark tip embryo phenotype, whereas OsMADS8-RNAi recovered the normal embryo phenotype. Changes in gene expression, including of ABCDE floral homeotic genes, were observed in the OsMADS8-OX and OsMADS8-RNAi lines. Overexpression of OsMADS8 led to decreased expression of OsEMF2b and ABA signaling-related genes including OsVP1/ABI3. HY seeds showed higher ABA content than CR6078 seeds, consistent with OsMADS8/DTE9 regulating the expression of genes related ABA catabolism in CR6078. Our results suggest that OsMADS8 is critical for floral organ determination and seed germination and that these effects are the result of regulation of the expression of OsEMF2b and its role in ABA signaling and catabolism.

Low-temperature germinability (LTG) is an important trait for rice direct seeding at temperate and high-altitude region. Previously, five QTLs (quantitative trait loci) for LTG were detected using an interspecific cross population between the Korea japonica cultivar Hwaseong and Oryza rufipogon (IRGSP#105491). O. rufipogon alleles at all loci increased the germination rate at the 13℃ condition. In this study, we tried to confirm and fine-map qLTG1 located on the short arm of chromosome 1. To map the qLTG1, two introgression lines, TR5 and TR20 were crossed to Hwaseong to develop F2:3 populations. QTL analysis confirmed the existence of the qLTG1 and it explained 55.5% and 29.9% of phenotypic variation in two populations, respectively. Substitution mapping using informative recombinant lines indicated that qLTG1 was located in 167-kb region between two SSR markers RM10310 and RM10326. This segment harbored 18 genes with nine of them were annotated with specific gene function. In addition, the O. rufipogon introgression in this region was associated with an increase in spikelets per panicle in the Hwaseong background. The results strongly indicate that the O. rufipogon alleles will be a valuable source of genes in improving japonica rice for low-temperature germinability and yield. To our knowledge, this is the first report to fine-map qLTG1 associated with LTG in rice considering that no QTL for LTG has not been reported near this QTL region from other biparental populations.

Leaf senescence is the final stage of plant development. Many internal and external factors affect the senescence process in rice (Oryza sativa L.). In this study, we identified qCC2, a major quantitative trait locus (QTL) for chlorophyll content using a population derived from an interspecific cross between O. sativa (cv. Hwaseong) and Oryza grandiglumis. The O. grandiglumis allele at qCC2 increased chlorophyll content and delayed senescence. GW2 encoding E3 ubiquitin ligase in the qCC2 region was selected as a candidate for qCC2. To determine if GW2 is allelic to qCC2, a gw2-knockout mutant (gw2-ko) was examined using a dark-induced senescence assay. gw2-ko showed delayed leaf senescence in the dark with down-regulated expression of senescence-associated genes (SAGs) and chlorophyll degradation genes (CDGs). The association of the GW2 genotype with the delayed senescence phenotype was confirmed in an F2 population. RNA-seq analysis was conducted to investigate 30-day-old leaf transcriptome dynamics in Hwaseong and a backcross inbred line&mdash, CR2002&mdash, under dark treatment. This resulted in the identification of genes involved in phytohormone signaling and associated with senescence. These results suggested that transcriptional regulation was associated with delayed senescence in CR2002, and RING-type E3 ubiquitin ligase GW2 was a positive regulator of leaf senescence in rice.

BackgroundRice (Oryza sativaL.) is generally sensitive to low temperatures, and in production systems that use direct-seeding, low-temperature germinability (LTG) is a desired trait. Previously, the QTLs,qLTG1andqLTG3,that control LTG, were mapped using the BC4F8population, which is a cross of Korean elite cultivar Hwaseong andO. rufipogon(IRGC 105491). We have characterized and analyzed the interaction between the two QTLs, by crossing TR20 that hasO. rufipogonalleles atqLTG1andqLTG3in a Hwaseong background, with Hwaseong, to develop an F2population.ResultsThe F2plants with bothqLTG1andqLTG3alleles fromO. rufipogonshowed higher LTG scores, than the plants with onlyqLTG1orqLTG3. No significant interaction between theqLTG1andqLTG3was observed, indicating that they may regulate LTG via different pathways. Based on its location,qLTG3appears to be allelic withqLTG3–1, a major QTL known to control LTG. To investigate the genetic differences between the two parents, that were controlling LTG, we compared theirqLTG3–1sequences. In the coding region, three sequence variations leading to amino acid changes were identified between the Hwaseong andO. rufipogon. Of these, a non-synonymous substitution at the 62nd amino acid site, had not previously been reported. To understand the cause of the LTG variations between the parents, we genotyped three sequence variations ofqLTG3–1, that were identified in 98 Asian cultivated rice accessions (Oryza sativaL.). The 98 accessions were classified into 5 haplotypes, based on three variations and a 71-bp deletion. Mean low-temperature germination rates were compared among the haplotypes, and haplotype 5 (O. rufipogon-type) showed a significantly higher germination rate than haplotype 2 (Nipponbare-type), and haplotype 3 (Italica Livorno-type).ConclusionsTheO. rufipogon qLTG3–1allele can be utilized for the improvement of LTG in rice breeding programs. Nearly isogenic lines harboring bothqLTG1andqLTG3–1alleles fromO. rufipogon,showed higher LTG scores than the NILs withqLTG1orqLTG3–1alone, and the two QTLs regulate LTG via different pathways. To our knowledge, this is the first report to detect a newqLTG3–1allele and analyze the interaction of the two LTG QTLs in a nearly isogenic background.

Northern Vietnam is one of the most important centers of genetic diversity for cultivated rice. Over thousands of years of cultivation, natural and artificial selection has preserved many traditional rice landraces in northern Vietnam due to its geographic situation, climatic conditions, and many ethnic groups. These local landraces serve as a rich source of genetic variation—an important resource for future crop improvement. In this study, we determined the genetic diversity and population structure of 79 rice landraces collected from northern Vietnam and 19 rice accessions collected from different countries. In total, 98 rice accessions could be differentiated into japonica and indica with moderate genetic diversity and a polymorphism information content of 0.382. Moreover, we found that genetic differentiation was related to geographical regions with an overall PhiPT (analog of fixation index FST) value of 0.130. We also detected subspecies-specific markers to classify rice (Oryza sativa L.) into indica and japonica. Additionally, we detected five marker-trait associations and rare alleles that can be applied in future breeding programs. Our results suggest that rice landraces in northern Vietnam have a dynamic genetic system that can create different levels of genetic differentiation among regions, but also maintain a balanced genetic diversity between regions.

A quantitative trait locus (QTL) gw8.1 was detected in the population derived from a cross between the elite japonica cultivar, 'Hwaseong' and Oryza rufipogon (IRGC 105491). Near isogenic lines (NILs) harboring the O. rufipogon segment on chromosome 8 showed increased grain length and weight compared to those of the recurrent parent, Hwaseong. This QTL was mapped to a 175.3-kb region containing 28 genes, of which four were considered as candidates based on the presence of mutations in their coding regions and as per the RNA expression pattern during the inflorescence stage. Leaves and panicles obtained from plants harvested 5 days after heading showed differences in gene expression between Hwaseong and gw8.1-NILs. Most genes were upregulated in O. rufipogon and gw8.1-NIL than in Hwaseong. Scanning electron microscopy analysis of the lemma inner epidermal cells indicated that cell length was higher in gw8.1 NIL than in Hwaseong, indicating that gw8.1 might regulate cell elongation. Among the candidate genes, LOC_Os08g34380 encoding a putative receptor-like kinase and LOC_Os08g34550 encoding putative RING-H2 finger protein were considered as possible candidates based on their functional similarity.

Previously, five putative quantitative trait loci (QTLs) for low-temperature germination (LTG) have been detected using 96 BC3F8 lines derived from an interspecific cross between the Korean japonica cultivar &ldquo, Hwaseong&rdquo, and Oryza rufipogon. In the present study, two introgression lines, CR1517 and CR1518, were used as parents to detect additional QTLs and analyze interactions among QTLs for LTG. The F2 population (154 plants) along with parental lines, Hwaseong and O. rufipogon, were evaluated for LTG and coleoptile length under low-temperature conditions (13 &deg, C). Among five QTLs for LTG, two major QTLs, qLTG1 and qLTG3, were consistently detected at 6 and 7 days after incubation. Three minor QTLs were detected on chromosomes 8 and 10. Two QTLs, qLTG10.1 and qLTG10.2, showing linkage on chromosome 10, exerted opposite effects with the Hwaseong allele at qLTG10.2 and the O. rufipogon allele at qLTG10.1 respectively, in turn, increasing LTG. Interactions among QTLs were not significant, implying that the QTLs act in an additive manner. Near-isogenic line plants with the combination of favorable alleles from O. rufipogon and Hwaseong exhibited higher LTG than two introgression lines. With regard to coleoptile length, three QTLs observed on chromosomes 1, 3, and 8 were colocalized with QTLs for LTG, suggesting the pleiotropy of the single gene at each locus. According to the results, the introgression of favorable O. rufipogon alleles could hasten the development of rice with high LTG and high coleoptile elongation in japonica cultivars.