Genetic analysis and molecular mapping of low amylose gene du12(t) in rice (Oryza sativa L.).

KEY MESSAGE: We obtained interesting results for genetic analysis and molecular mapping of the du12(t) gene. Control of the amylose content in rice is the major strategy for breeding rice with improved quality. In this study, we conducted genetic analysis and molecular mapping to identify the dull gene in the dull rice, Milyang262. A single recessive gene, tentatively designated as du12(t), was identified as the dull gene that leads to the low amylose character of Milyang262. To investigate the inheritance of du12(t), genetic analysis on an F2 population derived from a cross between the gene carrier, Milyang262, and a moderate amylose content variety, Junam, was conducted. A segregation ratio of 3:1 (χ (2) = 1.71, p = 0.19) was observed, suggesting that du12(t) is a single recessive factor that controls the dull character in Milyang262. Allelism tests confirmed that du12(t) is not allelic to other low amylose controlling genes, wx or du1. Recessive class analysis was performed to localize the du12(t) locus. Mapping of du12(t) was conducted on F2 and F3 populations of Baegokchal/Milyang262 cross. Linkage analysis of 120 F2 plants revealed that RM6926 and RM3509 flank du12(t) at a 2.38-Mb region. To refine the du12(t) locus position, 986 F2 and 289 F3 additional normal plants were screened by the flanking markers. Twenty-six recombinant plants were identified and later genotyped with four additional adjacent markers located between RM6926 and RM3509. Finally, du12(t) was mapped to an 840-kb region on the distal region of the long arm of chromosome 6, delimited by SSR markers RM20662 and RM412, and co-segregated by RM3765 and RM176.

Fine mapping and identification of candidate rice genes associated with qSTV11(SG), a major QTL for rice stripe disease resistance.

Rice stripe disease, caused by rice stripe virus (RSV) is a serious constraint to rice production in subtropical regions of East Asia. We performed fine mapping of a RSV resistance QTL on chromosome 11, qSTV11 ( SG ), using near-isogenic lines (NILs, BC(6)F(4)) derived from a cross between the highly resistant variety, Shingwang, and the highly susceptible variety, Ilpum, using 11 insertion and deletion (InDel) markers. qSTV11 ( SG ) was localized to a 150-kb region between InDel 11 (17.86 Mbp) and InDel 5 (18.01 Mbp). Among the two markers in this region, InDel 7 is diagnostic of RSV resistance in 55 Korean japonica and indica rice varieties. InDel 7 could also distinguish the allele type of Nagdong, Shingwang, Mudgo, and Pe-bi-hun from Zenith harboring the Stv-b ( i ) allele. As a result, qSTV11 ( SG ) is likely to be the Stv-b ( i ) allele. There were 21 genes in the 150-kb region harboring the qSTV11 ( SG ) locus. Three of these genes, LOC_Os11g31430, LOC_Os11g31450, and LOC_Os11g31470, were exclusively expressed in the susceptible variety. These expression profiles were consistent with the quantitative nature along with incomplete dominance of RSV resistance. Sequencing of these genes showed that there were several amino acid substitutions between susceptible and resistant varieties. Putative functions of these candidate genes for qSTV11 (SG) are discussed.

A Method for Inoculation and Evaluation of Rice Sheath Blight Disease.

Sheath blight of rice, caused by Rhizoctonia solani, is one of the most important rice diseases worldwide; however, no rice cultivar has been found to be completely resistant to this fungus. To facilitate detailed analysis of sheath blight resistance at genetic, molecular, biochemical, and functional genomic levels, new methods were developed for effective and uniform infection and accurate evaluation of the disease. The efficiency of R. solani infection was tested on two resistant (Tetep and Jasmine 85) and two susceptible (Chucheongbyeo, Junambyeo) cultivars using three different inoculum types (agar block, liquid cultured mycelia ball, and mycelia suspension). By covering the inoculated sheaths with aluminum foil to maintain humidity, 100% infection rate was achieved in this study. Liquid cultured mycelia balls caused significantly longer lesions (5.4 cm) than other types of inoculum, including agar block (2.4 cm) and mycelia suspension (1.6 cm). An improved method for evaluating sheath blight disease was selected by comparing two methods for evaluating disease severity among three partially resistant cultivars and five susceptible cultivars inoculated with liquid cultured mycelia balls. In addition, a new formula was developed to calculate the disease susceptibility index. Lesion length and the susceptibility index generally were correlated in each leaf, but there were discrepancies between the two evaluation methods due to differences in plant architecture among the cultivars. The susceptibility index calculated using the new formula was the most accurate method for evaluating sheath blight disease across all cultivars. The effect of heading date and panicle number also was evaluated in relation to sheath blight resistance. Cultivars with late heading dates generally were more resistant to sheath blight than those with early heading dates.

Analysis of intragenic Ds transpositions and excision events generating novel allelic variation in rice.

Even though Ac/Ds gene-tagging systems have been established in many higher plants, maize is the only major plant in which short-distance transposition of Ac/Ds has been utilized to probe gene function. This study was performed to evaluate the efficiency of obtaining new alleles and functional revertants from Ds insertion loci in rice. By analyzing 1,580 plants and the progeny of selected lines, the insertion sites and orientations of Ds elements within 16 new heritable alleles of three rice loci were identified and characterized. Intragenic transposition was detected in both directions from the original insertion sites. The closest interval was 35 bp. Three of the alleles had two Ds elements in cis configuration in the same transcription units. We also analyzed the excision footprints of intragenic and extragenic transpositions in Ds-inserted alleles at 5 loci. The 134 footprints obtained from different plants revealed predominant patterns. Ds excision at each locus left a predominant footprint at frequencies of 30-75%. Overall, 66% of the footprints were 7-bp additions. In addition, 16% of the excisions left 0-, 3-, 6-, and 9-bp additions with the potential of conserving reading frame.

Morphological and molecular characterization of a new frizzy panicle mutant, "fzp-9(t)", in rice (Oryza sativa L.).

The spikelet identity gene "fzp" (frizzy panicle) is required for transformation of the floral meristems to inflorescent shoots. In fzp mutants, spikelets are replaced by branches and spikelet meristems produce massive numbers of branch meristems. We have isolated and characterized a new fzp mutant derived from anther culture lines in rice and designated as fzp-9(t). The fzp-9(t) mutant showed retarded growth habit and developed fewer tillers than those of the wild-type plant. The primary and secondary rachis branches of fzp-9(t) appeared to be normal, but higher-order branches formed continuous bract-like structures without developing spikelets. The genetic segregation of fzp-9(t) showed a good fit to the expected ratio of 3: 1. The sequence analysis of fzp-9(t) revealed that there is a single nucleotide base change upstream of the ERF (ethylene-responsive element-binding factor) domain compare to wild-type plant. The mutation point of fzp-9(t) (W66G) was one of the six amino acids of the ERF domain that contributed to GCC box-specific binding. The premature formation of a stop codon at the beginning of the ERF domain might cause a non-functional product.

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.

Saponarin from barley sprouts inhibits NF-κB and MAPK on LPS-induced RAW 264.7 cells.

Saponarin (SA), a natural flavonoid, is known for its antioxidant and hepatoprotective activities. SA is the predominant compound (1142.7 ± 0.9 mg per 100 g) in barley sprouts, constituting 72% of the total polyphenol content. We investigated, for the first time, the effects of SA from barley sprouts on cellular anti-inflammatory responses. In lipopolysaccharide (LPS)-induced RAW 264.7 macrophages, SA suppressed the activation of NF-κB, as evidenced by the inhibition of NF-κB DNA binding, nuclear translocation, IκBα phosphorylation, and reporter gene expression, and it downregulated the expression of the pro-inflammatory mediator IL-6. Furthermore, SA reduced the transcription of NF-κB target molecules COX2 and FLIP inhibited the phosphorylation of mitogen-activated protein kinases ERK and p38. These results suggest that SA isolated from barley sprouts exerts anti-inflammatory effects in LPS-induced RAW 264.7 macrophages via inhibition of NF-κB, ERK and p38 signaling. Thus, SA may be a promising natural anti-inflammatory agent.

Identification and evaluation of flavone-glucosides isolated from barley sprouts and their inhibitory activity against bacterial neuraminidase.

Neuraminidase (NA) is one of the key enzymes responsible for bacterial infection and pathogenesis. This study aimed to gain deeper insights into the inhibitory effects of flavone-glucosides (1-9) isolated from barley sprouts (BS) on neuraminidase activity. The isolated compounds were identified as, lutonarin (1), saponarin (2), isoorientin (3), orientin (4), isovitexin (5), isoscoparin-7-O-[6-sinapoyl]-glucoside (6), isoscoparin-7-O-[6-feruloyl]-glucoside (7), isovitexin-7-O-[6-sinapoyl]-glucoside (8), and isovitexin-7-O-[6-feruloyl]-glucoside (9). Among them, compounds 1-5 exhibited neuraminidase-inhibitory activities in a dose-dependent manner, with IC50 values ranging from 20.1 to 32.7 µM, in a non-competitive inhibition mode according to kinetic studies. Moreover, the individual flavone-glucoside levels differed notably, in particular, lutonarin (1) and saponarin (2) were shown to be present in the greatest amounts, according to UPLC analysis. Consequently, our results suggest that BS may be utilized as an effective NA inhibitor in human health food, additives, and feed.

Overexpression of OsMYB4P, an R2R3-type MYB transcriptional activator, increases phosphate acquisition in rice.

R2R3 MYB transcription factors play regulatory roles in plant responses to various environmental stresses and nutrient deficiency. In this study, we isolated and designated OsMYB4P, an R2R3 MYB transcription factor, from rice (Oryza sativa L. 'Dongjin') under phosphate-deficient conditions. OsMYB4P was localized in the nucleus and acted as a transcriptional activator. Transcriptional levels of OsMYB4P in cell suspension, shoots, and roots of rice increased under phosphate-deficient conditions. Shoots and roots of OsMYB4P-overexpressing plants grew well in high- and phosphate-deficient conditions. In addition, root system architecture was altered considerably as a result of OsMYB4P overexpression. Under both phosphate-sufficient and -deficient conditions, more Pi accumulated in shoots and roots of OsMYB4P-overexpressing plants than in the wild type. Overexpression of OsMYB4P led to greater expression of Pi transporter-family proteins OsPT1, OsPT2, OsPT4, OsPT7, and OsPT8 in shoots, and to decreased or unchanged expression of these proteins in roots, with the exception of OsPT8. These results demonstrate that OsMYB4P may be associated with efficient utilization of Pi in rice through transcriptional activation of Pi homeostasis-related genes.

Effect of the growth stage and cultivar on policosanol profiles of barley sprouts and their adenosine 5'-monophosphate-activated protein kinase activation.

Adenosine 5'-monophosphate-activated protein kinase (AMPK) is an intracellular sensor that can regulate glucose levels within the cell. For this reason, it is well-known to be a target for drugs against diabetes and obesity. AMPK was activated significantly by the hexane extract of barley sprouts. This AMPK activation emerges across the growth stages of the sprout, becoming most significant (3 times above the initial stages) 10 days after sprouting. After this time, the activation decreased between 13 and 20 days post-sprouting. Analysis of the hexane extracts by gas chromatography-mass spectrometry showed that the amounts of policosanols (PCs, which are linear, primary aliphatic alcohols with 20-30 carbons) in the plant dramatically increased between 5 days (109.7 mg/100 g) and 10 days (343.7 mg/100 g) post-sprouting and then levels fell back down, reaching 76.4 mg/100 g at 20 days post-sprouting. This trend is consistent with PCs being the active ingredient in the barley plants. We validate this by showing that hexacosanol is an activator of AMPK. The richest cultivar for PCs was found to be the Daejin cultivar. Cultivars had a significant effect on the total PC content (113.2-183.5 mg/100 g) within the plant up to 5 days post-sprouting. However this dependence upon the cultivar was not so apparent at peak stages of PC production (10 days post-sprouting). The most abundant PC in barley sprout, hexacosanol, contributed 62-80% of the total PC content at every stage. These results are valuable to determine the optimal times of harvest to obtain the highest yield of PCs.

The Effect of Seed-borne Mycoflora from Sorghum and Foxtail Millet Seeds on Germination and Disease Transmission.

The seed-borne mycoflora of sorghum and foxtail millet collected from different growing areas in South Korea were isolated and taxonomically identified using dry inspection, standard blotter and the agar plate method. We investigated the in vitro and in vivo germination rates of disinfected and non-disinfected seeds of sorghum and foxtail millet using sterilized and unsterilized soil. The percent recovery of seed-borne mycoflora from the seed components of sorghum and foxtail millet seeds was determined and an infection experiment using the dominant species was evaluated for seedling emergence and mortality. A higher number of seed-borne fungi was observed in sorghum compared to that of foxtail millet. Eighteen fungal genera with 34 fungal species were identified from the seeds of sorghum and 13 genera with 22 species were identified from the seeds of foxtail millet. Five dominant species such as Alternaria alternata, Aspergillus flavus, Curvularia lunata, Fusarium moniliforme and Phoma sp. were recorded as seed-borne mycoflora in sorghum and 4 dominant species (Alternaria alternata, Aspergillus flavus, Curvularia lunata, Fusarium moniliforme) were observed in foxtail millet. The in vitro and in vivo germination rates were higher using disinfected seeds and sterilized soil. More seed-borne fungi were recovered from the pericarp compared to the endosperm and seed embryo. The percent recovery of seed-borne fungi ranged from 2.22% to 60.0%, and Alternaria alternata, Curvularia lunata and 4 species of Fusarium were isolated from the endosperm and embryo of sorghum and foxtail millet. Inoculation of the dominant seed-borne fungi showed considerable mortality of seedlings. All the transmitted seed-borne fungi might well be a primary source of infection of sorghum and foxtail millet crops.

Increased expression of OsPT1, a high-affinity phosphate transporter, enhances phosphate acquisition in rice.

Most high-affinity phosphate transporter genes (OsPTs) in rice were highly induced in roots when phosphate was depleted. OsPT1, however, was highly expressed in primary roots and leaves regardless of external phosphate concentrations. This finding was confirmed histochemically using transgenic rice plants that express the GUS reporter gene under the control of the OsPT1 promoter, which exhibited high GUS activity even in the phosphate sufficient condition. Furthermore, transgenic rice plants overexpressing the OsPT1 gene accumulated almost twice as much phosphate in the shoots as did wild-type plants. As a result, transgenic plants had more tillers than did wild-type plants, which is a typical physiological indicator for phosphate status in rice.

Molecular tagging of the Bph1 locus for resistance to brown planthopper (Nilaparvata lugens Stål) through representational difference analysis.

During brown planthopper (BPH) feeding on rice plants, we employed a modified representational difference analysis (RDA) method to detect rare transcripts among those differentially expressed in SNBC61, a BPH resistant near-isogenic line (NIL) carrying the Bph1 resistance gene. This identified 3 RDA clones: OsBphi237, OsBphi252 and OsBphi262. DNA gel-blot analysis revealed that the loci of the RDA clones in SNBC61 corresponded to the alleles of the BPH resistant donor Samgangbyeo. Expression analysis indicated that the RDA genes were up-regulated in SNBC61 during BPH feeding. Interestingly, analysis of 64 SNBC NILs, derived from backcrosses of Samgangbyeo with a BPH susceptible Nagdongbyeo, using a cleaved amplified polymorphic sequence (CAPS) marker indicated that OsBphi252, which encodes a putative lipoxygenase (LOX), co-segregates with BPH resistance. Our results suggest that OsBphi252 is tightly linked to Bph1, and may be useful in marker-assisted selection (MAS) for resistance to BPH.

A phosphate starvation-induced acid phosphatase from Oryza sativa: phosphate regulation and transgenic expression.

A phosphate starvation-induced acid phosphatase cDNA was cloned from the rice, Oryza sativa. The cDNA encoding O. sativa acid phosphatase (OsACP1) has 1100 bp with an open reading frame of 274 amino acid residues. The deduced amino acid sequence of OsACP1 cDNA showed 53% identity to tomato acid phosphatase and 46-50% identity to several other plant phosphatases. OsACP1 expression was up-regulated in the rice plant and in cell culture in the absence of phosphate (Pi). The induced expression of OsACP1 was a specific response to Pi starvation, and was not affected by the deprivation of other nutrients. OsACP1 expression was responsive to the level of Pi supply, with transcripts of OsACP1 being abundant in Pi-deprived root. The OsACP1 cDNA was expressed as a 30 kDa polypeptide in baculovirus-infected insect Sf9 cells. In addition, the OsACP1 gene was introduced into Arabidopsis via Agrobacterium-mediated transformation. Functional expression of the OsACP1 gene in the transgenic Arabidopsis lines was confirmed by Northern blot and Western blot analyses, as well as phosphatase activity assays. These results suggest that the OsACP1 gene can be used to develop new transgenic dicotyledonous plants able to adapt to Pi-deficient conditions.

The identification of candidate rice genes that confer resistance to the brown planthopper (Nilaparvata lugens) through representational difference analysis.

The development of rice varieties (Oryza sativa L.) that are resistant to the brown planthopper (BPH; Nilaparvata lugens Stål) is an important objective in current breeding programs. In this study, we generated 132 BC(5)F(5) near-isogenic rice lines (NILs) by five backcrosses of Samgangbyeo, a BPH resistant indica variety carrying the Bph1 locus, with Nagdongbyeo, a BPH susceptible japonica variety. To identify genes that confer BPH resistance, we employed representational difference analysis (RDA) to detect transcripts that were exclusively expressed in one of our BPH resistant NIL, SNBC61, during insect feeding. The chromosomal mapping of the RDA clones that we subsequently isolated revealed that they are located in close proximity either to known quantitative trait loci or to an introgressed SSR marker from the BPH resistant donor parent Samgangbyeo. Genomic DNA gel-blot analysis further revealed that loci of all RDA clones in SNBC61 correspond to the alleles of Samgangbyeo. Most of the RDA clones were found to be exclusively expressed in SNBC61 and could be assigned to functional groups involved in plant defense. These RDA clones therefore represent candidate defense genes for BPH resistance.

Analysis of gene-trap Ds rice populations in Korea.

Insertional mutagen-mediated gene tagging populations have been essential resources for analyzing the function of plant genes. In rice, maize transposable elements have been successfully utilized to produce transposant populations. However, many generations and substantial field space are required to obtain a sufficiently sized transposant population. In rice, the japonica and indica subspecies are phenotypically and genetically divergent. Here, callus cultures with seeds carrying Ac and Ds were used to produce 89,700 lines of Dongjin, a japonica cultivar, and 6,200 lines of MGRI079, whose genome is composed of a mixture of the genetic backgrounds of japonica and indica. Of the more than 3,000 lines examined, 67% had Ds elements. Among the Ds-carrying lines, 81% of Dongjin and 63% of MGRI079 contained transposed Ds, with an average of around 2.0 copies. By examining more than 15,000 lines, it was found that 12% expressed the reporter gene GUS during the early-seedling stage. GUS was expressed in root hairs and crown root initials at estimated frequencies of 0.78% and 0.34%, respectively. The 5,271 analyzed Ds loci were found to be randomly distributed over all of the rice chromosomes.

Rapid and simple procedure for homogenizing leaf tissues suitable for mini-midi-scale DNA extraction in rice.

We describe a rapid and simple procedure for homogenizing leaf samples suitable for mini/midi-scale DNA preparation in rice. The methods used tungsten carbide beads and general vortexer for homogenizing leaf samples. In general, two samples can be ground completely within 11.3+/-1.5 sec at one time. Up to 20 samples can be ground at a time using a vortexer attachment. The yields of the DNA ranged from 2.2 to 7.6 microg from 25-150 mg of young fresh leaf tissue. The quality and quantity of DNA was compatible for most of PCR work and RFLP analysis.

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.