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.

Genomic selection for growth characteristics in Korean red pine (Pinus densiflora Seibold & Zucc.).

Traditionally, selective breeding has been used to improve tree growth. However, traditional selection methods are time-consuming and limit annual genetic gain. Genomic selection (GS) offers an alternative to progeny testing by estimating the genotype-based breeding values of individuals based on genomic information using molecular markers. In the present study, we introduced GS to an open-pollinated breeding population of Korean red pine (Pinus densiflora), which is in high demand in South Korea, to shorten the breeding cycle. We compared the prediction accuracies of GS for growth characteristics (diameter at breast height [DBH], height, straightness, and volume) in Korean red pines under various conditions (marker set, model, and training set) and evaluated the selection efficiency of GS compared to traditional selection methods. Training the GS model to include individuals from various environments using genomic best linear unbiased prediction (GBLUP) and markers with a minor allele frequency larger than 0.05 was effective. The optimized model had an accuracy of 0.164-0.498 and a predictive ability of 0.018-0.441. The predictive ability of GBLUP against that of additive best linear unbiased prediction (ABLUP) was 0.86-5.10, and against the square root of heritability was 0.19-0.76, indicating that GS for Korean red pine was as efficient as in previous studies on forest trees. Moreover, the response to GS was higher than that to traditional selection regarding the annual genetic gain. Therefore, we conclude that the trained GS model is more effective than the traditional breeding methods for Korean red pines. We anticipate that the next generation of trees selected by GS will lay the foundation for the accelerated breeding of Korean red pine.

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.

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.

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.

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.

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.

Long-lasting Corolla Cultivars in Japanese Azaleas: A Mutant AP3/DEF Homolog Identified in Traditional Azalea Cultivars from More Than 300 Years Ago.

Floral shape in higher plants typically requires genetic regulation through MADS transcription factors. In Japan, hundreds of azalea cultivars including flower shape mutations have been selected from the diversity of endogenous species and natural hybrids since the early 17th century, the Edo era (1603-1867). The long-lasting trait, known as "Misome-sho" in Japanese, has been identified in several species and cultivar groups of evergreen azaleas (Rhododendron L.) from three hundred years ago in Japan. However, the natural mutation conferring the long-lasting trait in azalea remains unknown. Here, we showed MADS-box gene mutations in long-lasting flowers, R. kaempferi 'Nikko-misome,' R. macrosepalum 'Kocho-zoroi,' R. indicum 'Chojyu-ho,' and R. × hannoense 'Amagi-beni-chojyu.' All of the long-lasting flowers exhibited small-sized corollas with stomata during long blooming. In the long-lasting flowers, transcript of the APETALA3 (AP3)/DEFICIENS (DEF) homolog was reduced, and an LTR-retrotransposon was independently inserted into exons 1, 2, and 7 or an unknown sequence in exon 1 in gDNA of each cultivar. This insertion apparently abolished the normal mRNA sequence of the AP3/DEF homolog in long-lasting flowers. Also, long-lasting flowers were shown from F2 hybrids that had homozygous ap3/def alleles. Therefore, we concluded that the loss of function of the AP3/DEF homolog through a transposable element insertion may confer a stable long-lasting mutation in evergreen azaleas.