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.

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.

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.

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.

Morphological and starch characteristics of the Japonica rice mutant variety Seolgaeng for dry-milled flour.

Producing good-quality, fine rice flour is more difficult than wheat flour because the rice grain is harder. The non-glutinous Japonica-type variety Seolgaeng, derived from N-methyl-N-nitrosourea (MNU) mutagenesis, and four other varieties, representing a range of amylose contents, were evaluated in this study. Dry-milled Seolgaeng rice flour exhibited an average particle size that is <70 µm, a more uniform particle-size proportion than other varieties. Moreover, we noted significant differences in the damaged starch content in flour from Seolgaeng compared to the other varieties (p<0.05). Seolgaeng flour showed a round starch structure, which would lead to better friability, finer particle size, and less damage to the endosperm during dry milling. Indeed, among all varieties evaluated in this study, dry-milled Seolgaeng flour had the finest particle size (averaging <70 µm) and exhibited less damaged starch. With its round starch granules, Seolgaeng is a suitable candidate for drymilled rice flour.

Nuclear and chloroplast diversity and phenotypic distribution of rice (Oryza sativa L.) germplasm from the democratic people's republic of Korea (DPRK; North Korea).

BACKGROUND: Rice accounts for 43% of staple food production in the Democratic People's Republic of Korea (DPRK). The most widely planted rice varieties were developed from a limited number of ancestral lines that were repeatedly used as parents in breeding programs. However, detailed pedigrees are not publicly available and little is known about the genetic, phenotypic, and geographical variation of DPRK varieties. RESULTS: We evaluated 80 O. sativa accessions from the DPRK, consisting of 67 improved varieties and 13 landraces. Based on nuclear SSR analysis, we divide the varieties into two genetic groups: Group 1 corresponds to the temperate japonica subpopulation and represents 78.75% of the accessions, while Group 2 shares recent ancestry with indica varieties. Interestingly, members of Group 1 are less diverse than Group 2 at the nuclear level, but are more diverse at the chloroplast level. All Group 2 varieties share a single Japonica maternal-haplotype, while Group 1 varieties trace maternal ancestry to both Japonica and Indica. Phenotypically, members of Group 1 have shorter grains than Group 2, and varieties from breeding programs have thicker and wider grains than landraces. Improved varieties in Group 1 also show similar and/or better levels of cold tolerance for most traits, except for spikelet number per panicle. Finally, geographic analysis demonstrates that the majority of genetic variation is located within regions that have the most intensive rice cultivation, including the Western territories near the capital city Pyungyang. This is consistent with the conscious and highly centralized role of human selection in determining local dispersion patterns of rice in the DPRK. CONCLUSIONS: Diversity studies of DPRK rice germplasm revealed two genetic groups. The most widely planted group has a narrow genetic base and would benefit from the introduction of new genetic variation from cold tolerant landraces, wild accessions, and/or cultivated gene pools to enhance yield potential and performance.

Molecular mapping of a gene 'ld(t)' controlling cleistogamy in rice.

Cleistogamy is the self-pollination within closed spikelets and is expected to be a useful genetic tool for prevention of possible gene transfer in transgenic crops, for maintenance of genetic purity in autogamous crops, and for increased tolerance to biotic and abiotic stresses. Mapping of the gene ld(t), which is responsible for lack of lodicules inside spikelets and causes cleistogamy, was carried out using F2 and F3 populations derived from a cleistogamous (CL) mutant CL-SNU x Milyang 23 cross. A number of STS markers along chromosomes were developed and bulked segregant analysis was adopted for preliminary mapping. The results showed that the ld(t) was located at the end region of chromosome 1L, flanked by S01178b (an STS marker developed for the locus at 178 cM based on the rice genetic map reported by Japanese Rice Genome Project) at 0.8 cM and co-segregated with S01181a and S01181b (an STS marker developed for the locus at 181 cM).

Rapid, large-scale generation of Ds transposant lines and analysis of the Ds insertion sites in rice.

Rapid, large-scale generation of a Ds transposant population was achieved using a regeneration procedure involving tissue culture of seed-derived calli carrying Ac and inactive Ds elements. In the F(2) progeny from genetic crosses between the same Ds and Ac starter lines, most of the crosses produced an independent germinal transposition frequency of 10-20%. Also, many Ds elements underwent immobilization even though Ac was expressed. By comparison, in a callus-derived regenerated population, over 70% of plants carried independent Ds insertions, indicating transposition early in callus formation. In the remaining population, the majority of plants carried only Ac. Most of the new Ds insertions were stably transmitted to a subsequent generation. An exceptionally high proportion of independent transposants in the regenerated population means that selection markers for transposed Ds and continual monitoring of Ac/Ds activities may not necessarily be required. By analyzing 1297 Ds-flanking DNA sequences, a genetic map of 1072 Ds insertion sites was developed. The map showed that Ds elements were transposed onto all of the rice chromosomes, with preference not only near donor sites (36%) but also on certain physically unlinked arms. Populations from both genetic crossing and tissue culture showed the same distribution patterns of Ds insertion sites. The information of these mapped Ds insertion sites was deposited in GenBank. Among them, 55% of Ds elements were on predicted open-reading frame (ORF) regions. Thus, we propose an optimal strategy for the rapid generation of a large population of Ds transposants in rice.

Reprogramming of the activity of the activator/dissociation transposon family during plant regeneration in rice.

Many aspects of epigenetic phenomena have been elucidated via studies of transposable elements. An active transposable element frequently loses its ability to mobilize and goes into an inactive state during development. In this study, we describe the cyclic activity of a maize transposable element dissociation (Ds) in rice. In rice genome, Ds undergoes the spontaneous loss of mobility. However, an inactive state of Ds can be changed into an active state during tissue culture. The recovery of mobility accompanies not only changes in the methylation patterns of the terminal region of Ds, but also alteration in the steady state level of the activator (Ac) mRNA that is expressed by a constitutive CaMV 35S promoter. Furthermore, the Ds-reactivation process is not random, but stage-specific during plantlet regeneration. Our findings have expanded previous observations on Ac reactivation in the tissue culture of maize.