Miniature inverted-repeat transposable elements (MITEs) are abundant genomic components in many plants including rice. In this study, in order to determine whether rice MITEs are rich in sequence polymorphism and useful for developing PCR-based markers, we compared the genomic sequences of a japonica rice variety, Asominori, and an indica rice variety, IR24, in terms of MITE and MITE-less regions. Fifteen rice loci that had been previously reported to include MITEs (Gaijin, Castaway, Ditto, Wanderer, Explorer and Stowaway) were selected. Genomic sequences including MITEs at the loci were determined and compared between Asominori and IR24. Genomic sequences of Asominori and IR24 for the regions corresponding to seven RFLP markers (C81, R2303, S10019, S10602, S12564, G291 and G2155) were used to provide data for MITE-less regions. It was demonstrated that the degree of sequence polymorphism between the two varieties was much higher in the MITE regions than in the MITE-less regions. Sequence polymorphism was detected in fourteen MITE regions out of the fifteen regions tested. We also selected six rice sequences showing a high homology with a Wanderer family from a public database and surveyed sequence polymorphism between Asominori and IR24 for the regions. Some degree of polymorphism was observed in each of the six family members of Wanderer. As a result, twenty PCR-based markers in total were developed utilizing MITEs in rice and mapped on a linkage map, indicating that PCR-based markers for genetic studies and breeding programs could be developed efficiently by using MITEs.
We characterized five lesion-mimic mutants of rice (Oryza sativa L.), spotted leaf5-2 (spl5-2), Spl12, spl13, spl14 and Spl15, all of which have broad-spectrum resistance to the rice blast fungus Magnaporthe grisea and the bacterial blight pathogen Xanthomonas campestris pv. oryzae. First, we analyzed the influence of gene dosage on disease resistance and lesion-mimicry. Although the frequency of lesion-mimicry was lower in heterozygous Spl12 plants than in homozygous plants, the heterozygotes had almost the same levels of resistance to rice blast and bacterial blight as the homozygotes. Second, we analyzed whether developmental regulation of disease resistance differed in dominant and recessive mutants. We inoculated leaves at different leaf positions at the heading stage with bacterial blight, and found that spl5-2 and Spl12 mutants displayed enhanced resistance in lower and upper leaves but the level of resistance was higher in the upper leaves. Furthermore, we clarified the disease resistance and lesion-mimicry by inoculating the top expanded leaves at various developmental stages with bacterial blight. The dominant mutants (Spl12 and Spl15) showed lesion-mimicry from an earlier stage than did the recessive mutants (spl5-2, spl13 and spl14); the lesion-mimicry was more severe, and their resistance was rather greater, than in the recessive mutants. Finally, we studied the phenotypes of the progeny of crosses between the spl mutants. Although the lesion-mimicry of the Spl12spl14 double mutant was additive, the resistance phenotype of the double mutant equaled that of spl14. These results demonstrate that the mechanism by which SPL genes activate cell death and defense differs among dominant and recessive mutants.
Purple-fleshed sweet potato (Ipomoea batatas) plants containing anthocyanin pigments display a potent antioxidative activity. An 80 % ethanol extract from purple-fleshed sweet potato cultivars harvested in 2000 showed a 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity of 8.6 to 49.0 μmol expressed as Trolox equivalent/g of fresh weight. Although this DPPH radical-scavenging assay is convenient for the direct measurement of the radical-scavenging activity, a simpler and faster method should be developed for estimating the activity of a large number of breeding lines. For this purpose, the characterization of the UV-vis spectrum was performed in an 80 % ethanol extract from the purple-fleshed sweet potato. The extract exhibited absorption maxima near 325 nm and 530 nm due to the presence of cinnamic acids (or their related compounds) and anthocyanins, respectively. The absorbance at 325 nm was more highly correlated with the DPPH radical-scavenging activity than that at 530 nm and showed a correlation coefficient of 0.925. Multiple linear regression analysis using the absorbance of the extract at 325 nm and 530 nm elevated the value of the correlation coefficient to 0.979. When a prediction using this calibration curve was applied to extracts of purple-fleshed sweet potatoes harvested in 2001, a high correlation coefficient of 0.977 was obtained with lower SEP values of 1.686. Measurement of the absorbance of extracts from purple-fleshed sweet potatoes at 325 nm and 530 nm enable to obtain estimates of their DPPH radical-scavenging activity simply and rapidly. This assay could be suitable for selecting breeding lines of purple-fleshed sweet potato with a high DPPH radical-scavenging activity.
Internode elongation of the rice culm begins almost simultaneously with the change from the vegetative phase to the reproductive phase. Although this synchronism suggests the existence of an interaction between the panicle development and culm internode elongation, little information is available about panicle and internode interaction. In this study, we examined 18 mutants from cv. Taichung 65 that were defective in panicle and/or spikelet development. The 18 mutants were classified into three groups by the organ in which the major mutant phenotypes were observed; (1) mutants with aberrant rachis/branches, (2) mutants with aberrant spikelets/flowers, (3) mutants with aberration in both rachis and spikelets. Most of the mutants with aberrant rachis/branches showed an abnormal culm elongation. In contrast, those defective in only spikelet development showed an almost normal culm elongation. The correlation analysis showed that panicle traits were significantly correlated with the length of the distal one or two internodes. Principal component analysis of the culm internode length showed that the mutants defective in rachis/branch development were positioned far from the wild type, while those with normal panicles and aberrant spikelets were distributed near the wild type. These analyses revealed that culm elongation, especially for the upper internodes, was affected by the early developmental mode of panicles. Although the mechanism by which panicle development regulates the elongation of upper internodes has not yet been identified, the present study indicates that the elucidation of early panicle development is essential for a comprehensive understanding of culm internode elongation.
α-Amylase activity was measured in distal half-grains of four wheat lines, RL4137, OS21-5, Shirogane-komugi (Shirogane) and Hokkai250, after 72 h soaking at 20°C. The half-grains of RL4137 and Hokkai250 at the maturity stage (about 15 % water content) showed a significantly (P < 0.01) higher α-amylase activity after 72 h soaking (3.95 mU/mg, and 2.14 mU/mg, respectively) than those of OS21-5 and Shirogane (1.05 mU/mg and 0.91 mU/mg). During grain development, 30 to 50 days postanthesis (DPA), soaking also induced the production of α-amylase in the half-grains of RL4137 after 72 h. The α-amylases induced consisted of heat-labile α-AMY2 (low pI) type of enzymes. They were similar to α-AMY2 which was reported to be induced in the developing pericarp in the absence of stimulus from gibberellic acid (GA), but different from heat-resistant α-AMY2 expressed in the aleurone tissue after stimulation by GA. It is suggested that the RL4137 aleurone tissue has a potential for the synthesis of heat-labile α-AMY2, although the level of induced α-amylase activity was very low compared with that of the enzyme synthesized in the embryo and endosperm at the time of germination.
Miniature inverted repeat transposable elements (MITEs) are a major component of interspersed repetitive sequences in the rice genome. These elements serve as excellent tools for fine genomic analysis throughout the genome. Using the rice genome database, we evaluated MITEs present in a 200-kb region surrounding the rice waxy locus and selected four MITE subfamilies for MITE-transposon display (MITE-TD) analyses. MITE-TD, which is an AFLP-related technique based on MITE sequences, was applied to detect polymorphisms among the AA-genome Oryza species using the four selected MITEs. The MITE-TD used here enabled the most efficient detection of polymorphisms of all the molecular marker techniques applied to date in the Oryza species. Of the four MITEs, Mashu, a new MITE family, was found to be the best system for detecting the polymorphisms, with a detection frequency 3-1.5 times higher than that of the other three MITEs. The MITE-TDs also revealed information about genetic variations within the AA-genome species, and the complexity of the genetic relationships between O. sativa and O. rufipogon.
Improvement of the quality and quantity of soybean seed constituents is one of the most important objectives in soybean breeding. Although the quality of seed constituents has been studied extensively, information on the quantity is still limited. In order to analyze the genetic basis of these traits, recombinant inbred lines (RILs) derived from a cross between Glycine max (L.) Merrill variety Misuzudaizu and variety Moshidou Gong 503 were planted in two environments and evaluated for seed protein and lipid contents. Protein and lipid contents were determined by NIR transmittance spectroscopy using an intact single seed. The broad sense heritability of the traits ranged from 0.73 to 0.79 in our RIL population. Single-factor analysis of variance, interval mapping and composite interval mapping were used to detect significant associations between the traits and genetic markers. A total of 17 QTLs, 10 for proteins and 7 for lipids, which were significant in at least one environment were identified. Each QTL explained the total phenotypic variation for protein and lipid contents in the range from 3.4 % to 29.7 % and 6.1 % to 10.1 %, respectively. Among all the detected QTLs, three for the protein content and three for the lipid content were detected in both environments. The negative correlation between protein and lipid contents was also confirmed. Epistatic interactions were detected in this study, as another source of genetic variation in our population. The results obtained in our study may serve as a base for analyzing the genetic control of protein and lipid contents and may eventually enable to change the seed constituents.