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Abstract Rice (Oryza sativa L.) is a widely studied domesticated model plant. Seed awning is an unfavorable trait during rice harvesting and processing. Hence, loss of awn was one of the target characters selected during domestication. However, the genetic mechanisms underlying awn development in rice are not well understood. In this study, we analyzed and characterized the genes for awn development using a mapping population derived from a cross between the Korean indica cultivar ‘Milyang23’ and a near-isogenic line NIL4/9 derived from a cross between ‘Hwaseong’ and Oryza minuta. Two quantitative trait loci (QTLs), qAwn4 and qAwn9, mapped on chromosomes 4 and 9, respectively, increased awn length in an additive manner. Through comparative sequencing analyses of the parental lines, LABA1 was determined as the causal gene underlying qAwn4. qAwn9 was mapped to a 199-kb physical region between markers RM24663 and RM24679. Within this interval, 27 annotated genes were identified, and five genes, including a basic leucine zipper transcription factor 76 (OsbZIP76), were considered as candidate genes for qAwn9 based on their functional annotations and sequence variations. Haplotype analysis using the candidate gene revealed tropical-japonica specific sequence variants in the qAwn9 region, which partly explains the non-detection of qAwn9 in previous studies that used progenies from interspecific crosses. This provides further evidence that OsbZIP76 is possibly a causal gene for qAwn9. The O. minuta qAwn9 allele was identified as a major QTL, providing an important molecular target for understanding the genetic control of awn development in rice. Our results lay the foundation for further cloning of the awn gene underlying qAwn9.

Abstract Iris ruthenica Ker Gawl. and I. uniflora Pall. ex Link, which are rare and endangered species in Korea, possess considerable horticultural and medicinal value among Korean irises. However, discrimination of the species is hindered by extensive morphological similarity. Thus, the aim of the present study was to identify discriminating features by comparing the species’ complete plastid genome (i.e., plastome) sequences and micromorphological features, including leaf margins, stomatal complex distribution (hypostomatic vs. amphistomatic leaves), anther stomata density, and tepal epidermal cell patterns. Plastome comparison revealed slightly divergent regions within intergenic spacer regions, and the most variable sequences, which were distributed in non-coding regions, could be used as molecular markers for the discrimination of I. ruthenica and I. uniflora. Phylogenetic analysis of the Iris species revealed that I. ruthenica and I. uniflora formed a well-supported clade. The comparison of plastomes and micromorphological features performed in this study provides useful information for elucidating taxonomic, phylogenetic, and evolutionary relationships in Iridaceae. Further studies, including those based on molecular cytogenetic approaches using species specific markers, will offer insights into species delimitation of the two closely related Iris species.

Phytophthora root and stem rot (PRSR) disease results in substantial losses in soybean production worldwide. The occurrence of PRSR caused by Phytophthora sojae Kaufmann & Gerdemann has become increasingly important for soybean production in the Republic of Korea, but domestic soybean–P. sojae interaction has been less studied. The disease has been managed by developing varieties harboring resistance to the Phytophthora sojae (Rps) gene. The present study aimed to identify a major gene locus conferring resistance to new P. sojae isolate 2858 in the recombinant inbred line population derived from a cross between parental lines ‘Daepung’ (susceptible) and ‘Saedanbaek’ (resistant). Seventy-three recombination inbred lines (RILs) were evaluated for resistance to P. sojae isolate 2858. A resistance locus was identified in the approximate 3.3–4.3 megabase pair region on chromosome 3 using both single-marker and linkage analyses. The Rps of Saedanbaek (RpsSDB) was located on the well-known Rps gene/allele cluster region, which also partially overlapped with a locus previously identified in the Korean soybean variety, ‘Daewon’, resistant to another P. sojae isolate 2457 (RpsDW). Approximately 402 kilobase pairs of the interval region overlapped, including six nucleotide-binding site-leucine-rich repeat (NBS-LRR)-coding genes. Additional phenotypic assays revealed that Saedanbaek was susceptible to isolate 2457 and that Daewon was susceptible to isolate 2858, indicating that RpsSDB and RpsDW are different genes or alleles that confer race-specific resistance to the two P. sojae isolates. These results provide information that will be helpful for breeders developing P. sojae-resistant cultivars.

Chlorophyll biosynthesis is a crucial biological process in plants, and chlorophyll content is one of the most important traits in rice breeding programs. In this study, we identified a lethal, chlorophyll-deficient, yellow seedling (YS) phenotype segregating in progeny of CR5055-21, an F2 plant derived from a backcross between Korean japonica variety ‘Hwaseong’ (Oryza sativa) and CR5029, which is mostly Hwaseong with a small amount of Oryza grandiglumis chromosome segments. The segregation of the mutant phenotype was consistent with a single gene recessive mutation. Light microscopy of YS leaf cross-sections revealed loosely arranged mesophyll cells and sparse parenchyma in contrast to wildtype. In addition, transmission electron microscopy showed that chloroplasts did not develop in the mesophyll cells of the YS mutant. Quantitative trait loci (QTL)-seq analysis did not detect any significant QTL, however, examination of the individual delta-SNP index identified a 2-bp deletion (AG) in the OsCHLI gene, a magnesium (Mg)-chelatase subunit. A dCAPs marker was designed and genotyping of a segregating population (n = 275) showed that the mutant phenotype co-segregated with the marker. The 2-bp deletion was predicted to result in a frameshift mutation generating a premature termination. The truncated protein likely affects formation and function of Mg-chelatase, which consists of three different subunits that together catalyze the first committed step of chlorophyll biosynthesis. Transcriptome analysis showed that photosynthesis and carbohydrate metabolism pathways were significantly altered although expression of OsCHLI was not. Chlorophyll- and carotenoid-related genes were also differentially expressed in the YS mutant. Our findings demonstrated that OsCHLI plays an important role in leaf pigment biosynthesis and leaf structure development in rice.

In rice, low-temperature germinability (LTG) is essential for stable stand establishment using the direct seeding method in temperate and high-altitude areas. Previously, we reported that the quantitative trait locus qLTG1 is associated with LTG. qLTG1 is also associated with tolerance to several abiotic stresses, such as salt and osmotic conditions. In this study, map-based cloning and sequence analysis indicated that qLTG1 is allelic to DWARF2 (OsD2), which encodes cytochrome P450 D2 (LOC_Os01g10040) involved in brassinosteroid (BR) biosynthesis. Sequence comparison of the two parental lines, Hwaseong and Oryza rufipogon (IRGC 105491), revealed five single nucleotide polymorphisms (SNPs) in the coding region. Three of these SNPs led to missense mutations in OsD2, whereas the other two SNPs were synonymous. We evaluated two T-DNA insertion mutants, viz., overexpression (OsD2-OE) and knockdown (OsD2-KD) mutants of OsD2, with the Dongjin genetic background. OsD2-KD plants showed a decrease in LTG and grain size. In contrast, OsD2-OE plants showed an increase in grain size and LTG. We also examined the expression levels of several BR signaling and biosynthetic genes using the T-DNA insertion mutants. Gene expression analysis and BR application experiments demonstrated that BR enhanced the seed germination rate under low-temperature conditions. These results suggest that OsD2 is associated with the regulation of LTG and improving grain size. Thus, OsD2 may be a suitable target for rice breeding programs to improve rice yield and LTG.

Four near-isogenic lines (NILs) with different allele combinations of the starch branching enzyme 3 (SBE3) and granule-bound starch synthase 1 (GBSS1) were developed by crossing the japonica rice cultivars “Dodamssal” and “Hwayeong.” The associations between sequence variations in SBE3 and GBSS1, and starch-related traits were investigated. These sequence variations led to changes in seed morphology, starch structure, starch crystallinity, amylopectin chain length distribution, digestibility, apparent amylose content (AAC), and resistant starch content (RS). SBE3 and GBSS1 showed genetic interaction in regulating AAC and RS. Gene expression profiling of panicle tissues revealed significant differences in expression levels of GBSS1, SBE3, and other starch-related genes among the four NILs, indicating that variations in GBSS1 and SBE3 changed the expression level of starch-related genes. These variations contributed to the changes observed in AAC, RS, and physico-chemical characteristics of the rice starch from the NILs.

Abstract Background Rice (Oryza sativa L.) 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, qLTG1 and qLTG3, that control LTG, were mapped using the BC4F8 population, which is a cross of Korean elite cultivar Hwaseong and O. rufipogon (IRGC 105491). We have characterized and analyzed the interaction between the two QTLs, by crossing TR20 that has O. rufipogon alleles at qLTG1 and qLTG3 in a Hwaseong background, with Hwaseong, to develop an F2 population. Results The F2 plants with both qLTG1 and qLTG3 alleles from O. rufipogon showed higher LTG scores, than the plants with only qLTG1 or qLTG3. No significant interaction between the qLTG1 and qLTG3 was observed, indicating that they may regulate LTG via different pathways. Based on its location, qLTG3 appears to be allelic with qLTG3–1, a major QTL known to control LTG. To investigate the genetic differences between the two parents, that were controlling LTG, we compared their qLTG3–1 sequences. In the coding region, three sequence variations leading to amino acid changes were identified between the Hwaseong and O. 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 of qLTG3–1, that were identified in 98 Asian cultivated rice accessions (Oryza sativa L.). 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). Conclusions The O. rufipogon qLTG3–1 allele can be utilized for the improvement of LTG in rice breeding programs. Nearly isogenic lines harboring both qLTG1 and qLTG3–1 alleles from O. rufipogon, showed higher LTG scores than the NILs with qLTG1 or qLTG3–1 alone, and the two QTLs regulate LTG via different pathways. To our knowledge, this is the first report to detect a new qLTG3–1 allele and analyze the interaction of the two LTG QTLs in a nearly isogenic background.

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.

Developing rice varieties with increased mineral element content is the most cost-effective and efficient approach for alleviating human malnutrition and nutrient deficiencies. In this study, quantitative trait loci (QTLs) were mapped for mineral element content in 96 introgression lines derived from a cross between the elite Korean Oryza sativa japonica cultivar “Hwaseong” and the wild rice Oryza rufipogon (IRGC105491). The population was grown in two locations, and Fe, Zn, Mn, and Ca contents of the brown rice were measured. Six QTLs were identified on chromosomes 6, 8, and 10, and all O. rufipogon alleles increased trait values. The positions of qFe10 and qZn10 were further defined; higher Fe and Zn contents are related to the 375-kb O. rufipogon segment between the markers RM1873 and RM25612. The combined analysis of the whole-genome sequencing data, spatiotemporal expression profile, and gene expression suggested that a transcription factor gene, namely the rice homeobox gene 9 (LOC_Os10g33960) marks as the high potential candidate associated with Fe and/or Zn regulation. This study provides valuable information on candidate genes qFe10 and qZn10 from O. rufipogon, which may be vital in developing rice varieties with increased Fe and/or Zn content without any penalty in traits of agronomic importance.

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.