Identification of QTL Combinations that Cause Spikelet Sterility in Rice Derived from Interspecific Crosses.

BACKGROUND: The exploitation of useful genes through interspecific and intersubspecific crosses has been an important strategy for the genetic improvement of rice. Postzygotic reproductive isolation routinely occurs to hinder the growth of pollen or embryo sacs during the reproductive development of the wide crosses. RESULT: In this study, we investigated the genetic relationship between the hybrid breakdown of the population and transferred resistance genes derived from wide crosses using a near-isogenic population composed of 225 lines. Five loci (qSS12, qSS8, qSS11, ePS6-1, and ePS6-2) associated with spikelet fertility (SF) were identified by QTL and epistatic analysis, and two out of five epistasis interactions were found between the three QTLs (qSS12, qSS8 and qSS11) and background marker loci (ePS6-1 and ePS6-2) on chromosome 6. The results of the QTL combinations suggested a genetic model that explains most of the interactions between spikelet fertility and the detected loci with positive or negative effects. Moreover, the major-effect QTLs, qSS12 and qSS8, which exhibited additive gene effects, were narrowed down to 82- and 200-kb regions on chromosomes 12 and 8, respectively. Of the 13 ORFs present in the target regions, Os12g0589400 and Os12g0589898 for qSS12 and OS8g0298700 for qSS8 induced significantly different expression levels of the candidate genes in rice at the young panicle stage. CONCLUSION: The results will be useful for obtaining a further understanding of the mechanism causing the hybrid breakdown of a wide cross and will provide new information for developing rice cultivars with wide compatibility.

Improving the Glossiness of Cooked Rice, an Important Component of Visual Rice Grain Quality.

BACKGROUND: Rice is one of the few cereals consumed as a whole grain, and therefore the appearance of the final milled product, both before and after cooking, strongly influences the consumer's perception of product quality. Matching consumer preference for rice grain quality is a key component of rice variety development programs, as the quality drives demand, which in turn drives variety adoption, market price, and profitability. The quality of cooked rice is normally evaluated indirectly, through measurement of key elements driving quality as well as more directly by sensory evaluation, but remains a complex trait conditioned by the genetic complexity of factors driving quality, changes wrought by environment, and the complexity of consumer preferences. RESULT: In this study, we evaluated 17 traits, including the taste value obtained by glossiness of cooked rice (TV), to explain rice eating quality by statistical methods and identified QTLs associated with TV. To explain the correlation among traits, exploratory factor analysis was performed for 2 years. The overall eating quality (OE) was correlated with TV and protein content loading at the same factor (PA1) in 2017, and there was a relationship between the OE (PA1) and the TV (PA2) in 2018 (PA1:PA2, r = 0.3). In QTL analysis using 174 RILs, three QTLs for TV derived from Wandoaengmi6 were detected on chromosomes 4, 6, and 9. The QTL qTV9 delimited within Id9007180 and 9,851,330 on chromosome 9 was detected in both years, explaining approximately 17% of the variation, on average. Through the use of fine mapping, qTV9 was delimited to an approximately 34-Kbp segment flanked by the DNA markers CTV9_9 and CTV9_13, and nine ORFs were listed in the target region as candidate genes associated with TV. In the evaluation of qTV9's effect on OE, the lines with qTV9 showed a significant increase in correlation coefficiency compared to the negative lines. These data will apply to functional analysis on the glossiness and the MAS breeding program to improve the eating quality of japonica as a donor line. CONCLUSION: In this paper we report a number of QTL associated with changes in glossiness of cooked rice, and these may have utility in the development of MAS in breeding programs with a specific focus on cooked grain 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 breeding lines with three pyramided resistance genes that confer broad-spectrum bacterial blight resistance and their molecular analysis in rice.

BACKGROUND: The 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. RESULTS: Our 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. CONCLUSIONS: 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 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.

Exploiting leaf starch synthesis as a transient sink to elevate photosynthesis, plant productivity and yields.

Improvements in plant productivity (biomass) and yield have centered on increasing the efficiency of leaf CO(2) fixation and utilization of products by non-photosynthetic sink organs. We had previously demonstrated a correlation between photosynthetic capacity, plant growth, and the extent of leaf starch synthesis utilizing starch-deficient mutants. This finding suggested that leaf starch is used as a transient photosynthetic sink to recycle inorganic phosphate and, in turn, maximize photosynthesis. To test this hypothesis, Arabidopsis thaliana and rice (Oryza sativa L.) lines were generated with enhanced capacity to make leaf starch with minimal impact on carbon partitioning to sucrose. The Arabidopsis engineered plants exhibited enhanced photosynthetic capacity; this translated into increased growth and biomass. These enhanced phenotypes were displayed by similarly engineered rice lines. Manipulation of leaf starch is a viable alternative strategy to increase photosynthesis and, in turn, the growth and yields of crop and bioenergy plants.

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.

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 BC(3)F(4-6) in the Suweon365 background, a low eating quality variety, and some BC(1)F(3) 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.

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 R(2) 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.

Development of near-isogenic Japonica rice lines with enhanced resistance to Magnaporthe grisea.

Thirteen near-isogenic lines (NILs) of japonica rice were developed via a backcross method using the recurrent parent Chucheong, which is of good eating quality but is susceptible to Magnaporthe grisea, and three blast resistant japonica donors, Seolak, Daeseong and Bongkwang. The agro-morphological traits of these NILs, such as heading date, culm length, and panicle length, were similar to those of Chucheong. In a genome-wide scan using 158 SSR markers, chromosome segments of Chucheong were identified in most polymorphic regions of the 13 NIL plants, and only a few chromosome segments were found to have been substituted by donor alleles. The genetic similarities of the 13 NILs to the recurrent parent Chucheong averaged 0.961, with a range of 0.932-0.984. Analysis of 13 major blast resistance (R) genes in these lines using specific DNA markers showed that each NIL appeared to contain some combination of the four R genes, Pib, Pii, Pik-m and Pita-2, with the first three genes being present in each line. Screening of nine M. grisea isolates revealed that one NIL M7 was resistant to all nine isolates; the remaining NILs were each resistant to between three and seven isolates, except for NIL M106, which was resistant to only two isolates. In a blast nursery experiment, all the NILs proved to be more resistant than Chucheong. These newly developed NILs have potential as commercial rice varieties because of their increased resistance to M. grisea combined with the desirable agronomic traits of Chucheong. They also provide material for studying the genetic basis of blast resistance.

Characterization of rice mutants with enhanced susceptibility to rice blast.

As a first step towards identifying genes involving in the signal transduction pathways mediating rice blast resistance, we isolated 3 mutants lines that showed enhanced susceptibility to rice blast KJ105 (91-033) from a T-DNA insertion library of the japonica rice cultivar, Hwayeong. Since none of the susceptible phenotypes co-segregated with the T-DNA insertion we adapted a map-based cloning strategy to isolate the gene(s) responsible for the enhanced susceptibility of the Hwayeong mutants. A genetic mapping population was produced by crossing the resistant wild type Hwayeong with the susceptible cultivar, Nagdong. Chi-square analysis of the F2 segregating population indicated that resistance in Hwayeong was controlled by a single major gene that we tentatively named Pi-hy. Randomly selected susceptible plants in the F2 population were used to build an initial map of Pi-hy. The SSLP marker RM2265 on chromosome 2 was closely linked to resistance. High resolution mapping using 105 F2 plants revealed that the resistance gene was tightly linked, or identical, to Pib, a resistance gene with a nucleotide binding sequence and leucine-rich repeats (NB-LRR) previously isolated. Sequence analysis of the Pib locus amplified from three susceptible mutants revealed lesions within this gene, demonstrating that the Pi-hy gene is Pib. The Pib mutations in 1D-22-10-13, 1D-54-16-8, and 1C-143-16-1 were, respectively, a missense mutation in the conserved NB domain 3, a nonsense mutation in the 5th LRR, and a nonsense mutation in the C terminus following the LRRs that causes a small deletion of the C terminus. These findings provide evidence that NB domain 3 and the C terminus are required for full activity of the plant R gene. They also suggest that alterations of the resistance gene can cause major differences in pathogen specificity by affecting interactions with an avirulence factor.