Ji-Ung Jeung

High-resolution mapping of two rice brown planthopper resistance genes, Bph20(t) and Bph21(t), originating from Oryza minuta

Brown planthopper (BPH) is one of the most destructive insect pests of rice. Wild species of rice are a valuable source of resistance genes for developing resistant cultivars. A molecular marker-based genetic analysis of BPH resistance was conducted using an F2 population derived from a cross between an introgression line, ‘IR71033-121-15’, from Oryza minuta (Accession number 101141) and a susceptible Korean japonica variety, ‘Junambyeo’. Resistance to BPH (biotype 1) was evaluated using 190 F3 families. Two major quantitative trait loci (QTLs) and two significant digenic epistatic interactions between marker intervals were identified for BPH resistance. One QTL was mapped to 193.4-kb region located on the short arm of chromosome 4, and the other QTL was mapped to a 194.0-kb region on the long arm of chromosome 12. The two QTLs additively increased the resistance to BPH. Markers co-segregating with the two resistance QTLs were developed at each locus. Comparing the physical map positions of the two QTLs with previously reported BPH resistance genes, we conclude that these major QTLs are new BPH resistance loci and have designated them as Bph20(t) on chromosome 4 and Bph21(t) on chromosome 12. This is the first report of BPH resistance genes from the wild species O. minuta. These two new genes and markers reported here will be useful to rice breeding programs interested in new sources of BPH resistance.

Identification and analysis of QTLs controlling cold tolerance at the reproductive stage and validation of effective QTLs in cold-tolerant genotypes of rice (Oryza sativa L.)

Low temperature or cold stress is one of the major constraints of rice production and productivity in temperate rice-growing countries and high-altitude areas in the tropics. Even though low temperature affects the rice plant in all stages of growth, the percent seed set is damaged severely by cold and this reduces the yield potential of cultivars significantly. In this study, a new source of cold-tolerant line, IR66160-121-4-4-2, was used as a donor parent with a cold-sensitive cultivar, Geumobyeo, to produce 153 F8 recombinant inbred lines (RILs) for quantitative trait locus (QTL) analysis. QTL analysis with 175 polymorphic simple sequence repeat (SSR) markers and composite interval mapping identified three main-effect QTLs (qPSST-3, qPSST-7, and qPSST-9) on chromosomes 3, 7, and 9. The SSR markers RM569, RM1377, and RM24545 were linked to the identified QTLs for cold tolerance with respect to percent seed set using cold-water (18–19°C) irrigation in the field and controlled air temperature (17°C) in the greenhouse. The total phenotypic variation for cold tolerance contributed by the three QTLs was 27.4%. RILs with high percent seed set under cold stress were validated with linked DNA markers and by haplotype analysis that revealed the contribution of progenitor genomes from the tropical japonica cultivar Jimbrug (Javanica) and temperate japonica cultivar Shen-Nung89-366. Three QTLs contributed by the cold-tolerant parent were identified which showed additive effect on percent seed set under cold treatment. This study demonstrated the utility of a new phenotyping method as well as the identification of SSR markers associated with QTLs for selection of cold-tolerant genotypes to improve temperate rice production.

Development of breeding lines with three pyramided resistance genes that confer broad-spectrum bacterial blight resistance and their molecular analysis in rice

BackgroundThe development of resistant cultivars has been the most effective and economical strategy to control bacterial leaf blight (BB) disease of rice caused by Xanthomonas oryzae pv. oryzae (Xoo). Molecular markers have made it possible to identify and pyramid valuable genes of agronomic importance in resistance rice breeding. In this study, three resistance genes (Xa4 + xa5 + Xa21) were transferred from an indica donor (IRBB57), using a marker-assisted backcrossing (MAB) breeding strategy, into a BB-susceptible elite japonica rice cultivar, Mangeumbyeo, which is high yielding with good grain quality.ResultsOur analysis led to the development of three elite advanced backcross breeding lines (ABL) with three resistance genes by foreground and phenotypic selection in a japonica genetic background without linkage drag. The background genome recovery of the ABL expressed more than 92.1% using genome-wide SSR marker analysis. The pathogenicity assays of three resistance-gene-derived ABL were conducted under glasshouse conditions with the 18 isolates of Xoo prevalent in Korea. The ABL exhibited very small lesion lengths, indicating a hypersensitive reaction to all 18 isolates of Xoo, with agronomic and grain quality traits similar to those of the recurrent parent. Pyramiding the resistance genes Xa4, xa5 and Xa21 provided a higher resistance to Xoo than the introduction of the individual resistance genes. Additionally, the combination of two dominant and one recessive BB resistance gene did not express any negative effect on agronomic traits in the ABL.ConclusionsThe strategy of simultaneous foreground and phenotypic selection to introduce multiple R genes is very useful to reduce the cost and the time required for the isolation of desirable recombinants with target resistance genes in rice. The resistance-gene-derived ABL have practical breeding value without a yield penalty by providing broad-spectrum resistance against most of the existing isolates of BB in South Korea and will have a high impact on the yield stability and sustainability of rice productivity.

Identification of fungal (Magnaporthe grisea) stress-induced genes in wild rice (Oryza minuta)

To identify fungal stress-related genes in wild rice, Oryza minuta, we constructed a subtracted library using suppression subtractive hybridization in combination with mirror orientation selection. DNA chips containing 960 randomly selected cDNA clones were applied by reverse Northern analysis to eliminate false positive clones from the library and to prescreen differentially expressed genes. In total, 377 cDNA clones were selected on the basis of their signal intensities and expression ratios. Sequence analyses of these 377 cDNA fragments revealed that 180 of them (47.7%) represented unique genes. Of these180 cDNAs, 89 clones (49.6%) showed significant homologies to previously known genes, while the remaining 91 did not match any known sequences. The putative functions of the 180 unique ESTs were categorized by aligning them with MIPS data. They were classified into seven different groups using microarray data-derived expression patterns and verified by Northern blotting.

Analysis of differentially expressed transcripts from planthopper-infested wild rice (Oryza minuta)

A subtracted library was constructed from planthopper-infested wild rice (Oryza minuta) by suppression subtractive hybridization in combination with mirror orientation selection. To screen the differentially expressed transcripts in the library, we applied a cDNA microarray containing 960 random clones in a reverse Northern blot analysis using cDNA probes prepared from the mRNAs of control and planthopper-infested samples. On the basis of the signal intensities and expression ratios obtained from experiments performed in triplicate, we selected 383 clones. The elevated expression levels and overall profiles over time were verified by Northern blot analysis. Although Southern blot analysis showed similar copy numbers of the screened genes in O. minuta and O. sativa, it also revealed that the expression profiles had a different pattern . Functional categorization placed the identified transcripts in the categories of subcellular localization, metabolism, and protein fate. The presence of these expressed sequence tags implies that resistance of O. minuta to insect infestation can be achieved not only by an elevated expression of defense-related genes but also by enhanced metabolic activities.

Comparative analysis of 5,211 leaf ESTs of wild rice (Oryza minuta)

The expressed sequence tags (ESTs) presented in this report are the first transcriptomes of wild rice. A cDNA library was constructed from 4-week-old leaf samples of greenhouse-grown Oryza minuta. The 5,211 cDNA clones of O. minuta represent 3,401 unique sequences, consisting of 2,787 singletons and 614 assembled sequences. Database comparisons of the cDNAs in GenBank’s non-redundant databases using BLAST revealed that 4,957 of the 5,211 cDNAs (95.1%) showed a high degree of sequence homology to genes from other organisms. Most of the transcripts identified were genes related to metabolism, energy, protein biosynthesis and subcellular localization. The metabolism and energy categories of the O. minuta ESTs showed a considerably higher gene expression level than those of O. sativa ESTs. These data and genes can be utilized in rice breeding.

Effects of allelic variations in starch synthesis-related genes on grain quality traits of Korean nonglutinous rice varieties under different temperature conditions

Influences of allelic variations in starch synthesis-related genes (SSRGs) on rice grain quality were examined. A total of 187 nonglutinous Korean rice varieties, consisting of 170 Japonica and 17 Tongil-type varieties, were grown in the field and in two greenhouse conditions. The percentages of head rice and chalky grains, amylose content, alkali digestion value, and rapid visco-analysis characteristics were evaluated in the three different environments. Among the 10 previously reported SSRG markers used in this study, seven were polymorphic, and four of those showed subspecies-specific allele distributions. Six out of the seven polymorphic SSRG markers were significantly associated with at least one grain quality trait (R2 > 0.1) across the three different environments. However, the association level and significance were markedly lower when the analysis was repeated using only the 170 Japonica varieties. Similarly, the significant associations between SSRG allelic variations and changes in grain quality traits under increased temperature were largely attributable to the biased allele frequency between the two subpopulations. Our results suggest that within Korean Japonica varieties, these 10 major SSRG loci have been highly fixed during breeding history and variations in grain quality traits might be influenced by other genetic factors.

Resistance Genes and Their Effects to Blast in Korean Rice Varieties (Oryza sativa L.)

A total of 98 varieties consisting of 88 japonica and 10 Tongil-type of rice were analyzed to resistant genes and their effects to blast. The 13 major blast resistance (R) genes against Magnaporthe oryzae were screened in a number of Korean rice varieties using molecular markers. Twenty-eight (28.6%) were found to contain the Pia gene originating from Japanese japonica rice genotypes. The Pib gene from BL1 and BL7 was incorporated into 39 Korean japonica varieties, whereas this same gene from the IRRI-bred indica varieties was detected in all Tongil-type variety. The Pii gene was found in 17 of the japonica varieties. The Pii gene in Korean rice varieties originates from the Korean japonica variety Nongbaeg, and Japanese japonica varieties Hitomebore, Inabawase, and Todorokiwase. The Pi5 gene, which clusters with Pii on chromosome 9, was identified only in an Tongil-type, Taebaeg. Thirty-four varieties were found to contain alleles of the resistance gene Pita or Pia-2. The Pita gene in japonica varieties was inherited from the Japanese japonica genotype Shimokita, and the Pita-2 gene was from Fuji280 and Sadominori. Seventeen japonica and one Tongil-type varieties contained the Piz gene, which in the japonica varieties originates from Fukuhikari and 54BC-68. The Piz-t gene contained in three Tongil-type varieties was derived from IRRI-bred indica rice varieties. The Pi9(t) gene locus that is present in Korean japonica and Tongil-type varieties was not inherited from the original Pi9 gene from wild rice Oryza minuta. The Pik-multiple allele genes Pik, Pik-m, and Pik-p were identified in 24 of the varieties tested. The Pit gene inherited from the indica rice K59 was not found in any of the Korean rice varieties tested. In haplotype analysis for the loci related to two QTLs as well as five major resistant genes and, two

Development of multi-resistant lines to brown planthopper, bacterial blight, and rice stripe virus using anther culture in rice.

R

Development of breeding lines for early maturing glutinous japonica rice carrying bacterial blight resistance genes, Xa2, Xa3, xa5, and Xa21.

genes Piz and Pita showed stable resistant effects to blast nurseries and isolates in~Korea.