Young-Chan Cho

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.

Molecular Aspect of Good Eating Quality Formation in Japonica Rice

The composition of amylopectin is the determinant of rice eating quality under certain threshold of protein content and the ratio of amylose and amylopectin. In molecular biology level, the fine structure of amylopectin is determined by relative activities of starch branching enzyme (SBE), granule-bound starch synthase (GBSS), and soluble starch synthase (SSS) in rice grain under the same ADP-Glucose level. But the underlying mechanism of eating quality in molecular biology level remains unclear. This paper reports the differences on major parameters such as SNP and insertion-deletion sites, RNA expressions, and enzyme activities associated with eating quality of japonica varieties. Eight japonica rice varieties with significant differences in various eating quality parameters such as palatability and protein content were used in this experiment. Association analysis between nucleotide polymorphism and eating quality showed that S12 and S13 loci in SBE1, S55 in SSS1, S58 in SSS2A were significantly associated with apparent amylose content, alkali digestion value, setback viscosity, consistency viscosity, pasting temperature, which explained most of the variation in apparent amylose content, setback viscosity, and consistency viscosity; and explained almost all variations in alkali digestion value and pasting temperature. Thirty-five SNPs and insertion-deletions from SBE1, SBE3, GBSS1, SSS1, and SSS2A differentiated high or intermediate palatability rice varieties from low palatability rice varieties. Correlation analysis between enzyme activities and eating quality properties revealed that SBE25 and SSS15/W15 were positively correlated with palatability, whereas GBSS10 and GBSS15 were negatively correlated. Gene expressions showed that SBE1 and SBE3 expressions in high palatability varieties tended to be higher than middle and low palatability varieties. Collectively, SBE1, SBE3, SSS1, and SSS2A, especially SBE1 and SBE3 could improve eating quality, but GBSS1 decreased eating quality. The results indicated the possibility of developing high palatability cultivars through modification of key genes related to japonica rice eating quality formation in starch biosynthesis.

PCR Marker-Based Evaluation of the Eating Quality of Japonica Rice (Oryza sativa L.)

Evaluation of eating quality in early breeding generations of rice is critical to developing varieties with better palatability. This paper reports DNA markers associated with eating quality of temperate japonica rice and an evaluation method aided by multiple regression analysis. A total of 30 markers comprising STSs, SNPs, and SSRs were tested for their association with palatability using 22 temperate japonica varieties with different palatability values. Eating quality-related traits of the 22 varieties were also measured. Of the 30 markers, 18 were found to be significantly associated with palatability and, consequently, a model regression equation with an R2 value of 0.99 was formulated to estimate the palatability by the marker data set. Validation of the model equation using selected breeding lines indicated that the marker set and the equation are highly applicable to evaluation of the palatability of cooked rice in temperate japonica varieties.

Identification of Quantitative Trait Loci Associated with Rice Eating Quality Traits Using a Population of Recombinant Inbred Lines Derived from a Cross between Two Temperate japonica Cultivars

Improved eating quality is a major breeding target in japonica rice due to market demand. In this study, we performed genetic analysis to identify quantitative trait loci (QTLs) that control rice eating quality traits using 192 recombinant inbred lines (RILs) derived from a cross between two japonica cultivars, ‘Suweon365’ and ‘Chucheongbyeo’. We evaluated the stickiness (ST) and overall evaluation (OE) of cooked rice using a sensory test, the glossiness of cooked rice (GCR) using a Toyo-taste meter, and measured the amylose content (AC), protein content (PC), alkali digestion value (ADV), and days to heading (DH) of the RILs in the years 2006 and 2007. Our analysis revealed 21 QTLs on chromosomes 1, 4, 6, 7, 8, and 11. QTLs on chromosomes 6, 7, and 8 were detected for three traits related to eating quality in both years. QTLs for ST and OE were identified by a sensory test in the same region of the QTLs for AC, PC, ADV, GCR and DH on chromosome 8. QTL effects on the GCR were verified using QTL-NILs (near-isogenic lines) of BC3F4–6 in the Suweon365 background, a low eating quality variety, and some BC1F3 lines. Chucheongbyeo alleles at QTLs on chromosomes 7 and 8 increased the GCR in the NILs and backcrossed lines. The QTLs identified by our analysis will be applicable to future marker-assisted selection (MAS) strategies for improving the eating quality of japonica rice.

Structure and expression of OsUBP6, an ubiquitin-specific protease 6 homolog in rice (Oryza sativa L.)

Although the possible cellular roles of several ubiquitin-specific proteases (UBPs) were identified in Arabidopsis, almost nothing is known about UBP homologs in rice, a monocot model plant. In this report, we searched the rice genome database (http://signal.salk.edu/cgi-bin/RiceGE) and identified 21 putative UBP family members (OsUBPs) in the rice genome. These OsUBP genes each contain a ubiquitin carboxyl-terminal hydrolase (UCH) domain with highly conserved Cys and His boxes and were subdivided into 9 groups based on their sequence identities and domain structures. RT-PCR analysis indicated that rice OsUBP genes are expressed at varying degrees in different rice tissues. We isolated a full-length cDNA clone for OsUBP6, which possesses not only a UCH domain, but also an N-terminal ubiquitin motif. Bacterially expressed OsUBP6 was capable of dismantling K48-linked tetraubiquitin chains in vitro. Quantitative real-time RT-PCR indicated that OsUBP6 is constitutively expressed in different tissues of rice plants. An in vivo targeting experiment showed that OsUBP6 is predominantly localized to the nucleus in onion epidermal cells. We also examined how knock-out of OsUBP6 affects developmental growth of rice plants. Although homozygous T3 osubp6 T-DNA insertion mutant seedlings displayed slower growth relative to wild type seedlings, mature mutant plants appeared to be normal. These results raise the possibility that loss of OsUBP6 is functionally compensated for by an as-yet unknown OsUBP homolog during later stages of development in rice plants.

QTL Detection Associated with Eating Quality Based on Palatability Test in Japonica Rice (Oryza sativa L.)

A recombinant inbred line (RIL) population derived from the cross between Ilpumbyeo (a Japonica of high eating quality) and Moroberekan (a tropical Japonica type of low eating quality) was used for mapping QTLs associated with grain physicochemical properties and eating quality traits in rice. A total of 182 recombinant inbred lines were evaluated for two physicochemical traits, alkali digestive value and amylose content, and five traits associated with eating quality including glossiness of cooked rice determined by Toyo-taste meter, and glossiness, stickiness, hardness and overall evaluation for taste evaluation by panels. A total of 30 QTLs associated with seven traits in 10 loci on chromosomes 1, 3, 5, 6, 10, and 12 were identified. The most critical regions for eating quality were on chromosomes 3, 5 and 6 by Ilpumbyeo alleles, whereas Moroberekan contributed a total of 6 loci on chromosomes 1, 10 and 12. The QTL region on chromosome 5 contains the novel alleles for eating quality from Ilpumbyeo. MAS using DNA markers tightly linked with those QTLs will be useful for breeding Japonica cultivars with high eating quality.

BrUGE1 transgenic rice showed improved growth performance with enhanced drought tolerance

UDP-glucose 4-epimerase (UGE) catalyzes the reversible conversion of UDP-glucose to UDP-galactose. To understand the biological function of UGE from Brassica rapa, the gene BrUGE1 was cloned and introduced into the genome of wild type rice ‘Gopum’ using the Agrobacterium-mediated transformation method. Four lines which carried a single copy gene were selected and forwarded to T3 generation. Agronomic traits evaluation of the transgenic T3 lines (CB01, CB03, and CB06) under optimal field conditions revealed enriched biomass production particularly in panicle length, number of productive tillers, number of spikelets per panicle, and filled spikelets. These remarkably improved agronomic traits were ascribed to a higher photosynthetic rate complemented with higher CO2 assimilation. Transcripts of BrUGE1 in transgenic lines continuously accumulated at higher levels after the 20% PEG6000 treatment, implying its probable role in drought stress regulation. This was paralleled by rapid accumulation of soluble sugars which act as osmoprotectants, leading to delayed leaf rolling and drying. Our findings suggest the potential of BrUGE1 in improving rice growth performance under optimal and water deficit conditions.

Development of Resistant Gene-Pyramided Japonica Rice for Multiple Biotic Stresses Using Molecular Marker-Assisted Selection

Advances in plant molecular techniques have dramatically widened the applicability of gene identification and pyramiding valuable genes. This study was carried out to pyramid five resistance genes for biotic stress into the japonica rice cultivar using marker-assisted selection (MAS) and marker-assisted background analysis of selected progenies using SSR markers. The Pi40, Xa4, xa5, Xa21 and Bph18 genes were combined in Jinbubyeo, a Korean japonica rice variety using MAS. Gene specific co-dominant PCR-based markers were used to select for homozygous recombinant lines in a segregating population derived from a cross between the parental homozygous resistant gene introgression lines. We had successfully developed multiple gene pyramided breeding lines (GPLs) for bacterial blight, blast, and brown planthopper using MAS in rice. The GPLs exhibited high resistance against biotic stress and had around 93% of the genetic background of the recurrent parent Jinbubyeo based on SSR graphical mapping. The yield and agronomic traits of the GPLs were similar to those of the recurrent parent, indicating that there is no apparent agronomic trait penalty associated with the presence of the resistance genes. The 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 GPLs could be useful to enhance effective resistance for biotic stress and produce stable grain yield in japonica rice breeding programs.

SSR Analysis of Genetic Diversity and Cold Tolerance in Temperate Rice Germplasm

A total of 23 elite rice cultivars from eight countries were evaluated for cold tolerance using two screening methods at Chuncheon Substation, National Institute of Crop Science (NICS), Republic of Korea. The rice cultivars Jinbu, Mustaqillik, and Avangard showed cold tolerance and high spikelet fertility (63-79%) in cold-water irrigation screening. Under greenhouse screening, five cultivars (Giza 177, Avangard, Mustaqillik, Jinbu, and Jungan) showed high cold tolerance and high spikelet fertility (71-81%). Simple sequence repeat (SSR) marker analysis of 21 genotypes revealed two major clusters, the japonica and indica groups, with a genetic similarity of 0.69. Out of 21 rice cultivars, only four (Giza 178 from Egypt, Attey and Zakha from Bhutan, and Millin from Australia) fell under the indica cluster. The cold-tolerant varieties Jinbu, Mustaqillik, and Avangard were clustered with the japonica group, which had genetic similarity of 0.83. These varieties are considered as potential germplasm that will help diversify the japonica gene pool for cold-tolerant rice breeding. A one-way linear analysis of variance identified a significant relationship between individual alleles and traits. Three SSR markers were significantly associated with spikelet fertility under cold-water irrigation on chromosomes 1, 2, and 7. Five SSR markers were associated with spikelet fertility under a cool-environment greenhouse on chromosomes 8, 9, 10, and 12. The SSR markers associated with cold tolerance may also be useful as selection markers in indica/japonica cross combinations to improve cold tolerance.

What it Takes to Achieve Durable Resistance to Rice Blast

Based on performance record, varieties with a reputation for possessing durable blast resistance can be identified from breeding programs. For most of these varieties, a combination of major resistance genes and multiple QTL appears to be the underlying genetic basis for durability. Despite this seemingly simple observation, re-creating genotypes with durable resistance and proper agronomic characteristics remains challenging in breeding. We suggest that genetic mapping, saturation mutagenesis, and transcriptome analysis can together provide a genomewide view of the essential regions for disease resistance, enabling the creation and selection of desirable gene combinations. We further propose actions needed to sustain breeding for durable resistance. These include (a) develop breeding-ready near-isogenic lines for important major resistance (R) genes and QTL, (b) improve knowledge of neck blast through better screening and genetic analysis, (c) promote exchange of breeding materials between countries for evaluation and validation of durable resistance, and (d) resume monitoring of blast pathogen populations with functional diagnostic markers. International collaboration as well as enhanced linkages within countries are much needed to implement these actions.