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The genomic DNA molecule of tomato yellow leaf curl virus (TYLCV), a whitefly-transmitted geminivirus, was amplified from total DNA extracts of TYLCV-infected tomato (Lycopersicon esculentum) by the use of loop-mediated isothermal...
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The genomic DNA molecule of tomato yellow leaf curl virus (TYLCV), a whitefly-transmitted geminivirus, was amplified from total DNA extracts of TYLCV-infected tomato (Lycopersicon esculentum) by the use of loop-mediated isothermal amplification (LAMP). The procedure was also used to amplify TYLCV DNA from total DNA extracts of individual whiteflies (Bemisia tabaci) that had fed on TYLCV-infected plants. One of the characteristics of the LAMP method is its ability to synthesize an extremely large amount of DNA. Accordingly, a large amount of by-product, pyrophosphate ion, is produced yielding a white precipitate of magnesium pyrophosphate in the reaction mixture. The presence or absence of this white precipitate allows easy detection of amplification of TYLCV genomic DNA without gel electrophoresis.
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Kyuri green mottle mosaic virus (KGMMV) which is a major soil-borne virus, causes severe yield reduction in cucumber in Japan. We developed immunocapture reverse transcription loop-mediated isothermal amplification (Ic/RT-LAMP) me...
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Kyuri green mottle mosaic virus (KGMMV) which is a major soil-borne virus, causes severe yield reduction in cucumber in Japan. We developed immunocapture reverse transcription loop-mediated isothermal amplification (Ic/RT-LAMP) method to detect KGMMVin soil. Degenerate primer set from KGMMV C strain (KGMMV-C) and Yodo strain (KGMMV-Yodo) was designed for the detection of both strains. To optimize the reaction condition, the RT-LAMP reaction using the degenerate primer set was performed at differenttemperature (60-65°C). The reaction at 60°C gave the best results. The result on specificity test using 7 cucumber pathogenic viral isolates indicated that the RT-LAMP assay established in this study had no cross reactions. The detection limit of the RT-LAMP assay was 10 pg of purified KGMMV. The sensitivity of RT-LAMP was 10 times higher than that of RT-PCR. Detection limit of DAS-ELISA and Ic/RT-LAMP was compared by using of the dilution of purified KGMMV-C in soil. Ic/RT-LAMP could detect 0.5 ng ofKGMMV from 100 mg of soil sample and the sensitivity of Ic/RT-LAMP was 200 times higher than that of DAS-ELISA. The Ic/RT-LAMP assay could successfully detect KGMMV in soil samples from 10 fields where KGMMV had been detected in a previous cultivation.The present Ic/RT-LAMP method showed to be a rapid, simple and available detection method of KGMMV in soil.
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Denitrification hotspots in riparian aquifers often develop in a relatively narrow zone at the uplandriparian interface, where nitrate-rich ground water of upland origin interacts with available soil organic carbon. In riparian pa...
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Denitrification hotspots in riparian aquifers often develop in a relatively narrow zone at the uplandriparian interface, where nitrate-rich ground water of upland origin interacts with available soil organic carbon. In riparian paddy fields, denitrification in the aquifer has received less attention than that in the surface water and soil. This study aimed to determine the in situ activity of the denitrification hotspot formed at the vertical interface between the organic alluvial and the nitrate-rich diluvial aquifers around the depth of 2.0 m below the upland perimeter of riparian paddy, where vertical upwelling dominates the ground water recharge. The mass balances of water and solutes were approximately calculated from the one-dimensional vertical pressure head and water quality profiles with help of the stable isotopes analyses of water. The confined ground water of adjacent diluvial upland origin, with a high nitrate concentration of 1.72 pl 0.42 mmol L1, mixed with the nitrate-deficient unconfined ground water at the alluviumdiluvium interface, and 63% of nitrate was removed by denitrification at a rate of 33 mg N m2 d1 and a nitrogen isotope fractionation factor of 0.988. The increase in bicarbonate concentration with the decrease in nitrate concentration suggested a heterotrophic denitrification with a stoichiometry of C:N = 5:4. These results are the first to demonstrate the quantitative importance of denitrification in the aquifer below a riparian paddy in the removal of nitrate from the ground water of upland origin and emphasize the necessity of including this process in models for predicting watershed-scale surface water and ground water qualities.
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To introduce the characters of extremely early flowering and unique flower shape of Dianthus x isensis Hirahata et Kitamura into Dianthus caryophyllus L., reciprocal interspecific crosses were carried out between seven lines of D....
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To introduce the characters of extremely early flowering and unique flower shape of Dianthus x isensis Hirahata et Kitamura into Dianthus caryophyllus L., reciprocal interspecific crosses were carried out between seven lines of D. caryophyllus and one line of D. x isensis. When D. caryophyllus was used as the maternal parent, 79 progeny were obtained from only four lines of D. caryophyllus. Flow cytometric analysis revealed that they consisted of 30 diploid hybrid plants and 49 diploid maternal-type progeny. When D. x isensis was used as the maternal parent, five progeny including three diploid hybrids, one triploid hybrid and one maternal-type were obtained by pollination with only two lines of D. caryophyllus. The ploidy level of these hybrids was confirmed by counting chromosome numbers in root-tip cells. Flow cytometric analysis also revealed that the triploid hybrid had two genomes of D. x isensis and one genome of D. caryophyllus, suggesting that D. x isensis had produced an unreduced female gamete. Since the diploid interspecific hybrids were fertile and have some of the profitable characters of D. x isensis such as early flowering and a unique flower shape, they are expected to be used for future breeding in carnation..
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Inter-specific crosses were carried out between two lines of Dianthus x isensis Hirahata et Kitam., and one line of D. japonicus Thunb. Germinating seeds were obtained from only one line of D. x isensis used as the seed parent, an...
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Inter-specific crosses were carried out between two lines of Dianthus x isensis Hirahata et Kitam., and one line of D. japonicus Thunb. Germinating seeds were obtained from only one line of D. x isensis used as the seed parent, and a total of 46 progeny from this cross were all confirmed to be hybrids by RAPD analysis and flow cytometry. The hybrids had intermediate characters of both parents with respect to leaf-width and flower-size, but flowered at the same time as each other with a uniform flower colour, purplish-pink. All 46 hybrids initially had sterile pollen and seeds, but two later produced some flowers of larger size with fertile pollen. Flow cytometric analysis revealed that flowers with restored pollen fertility originated from a chimaeric tetraploid branch induced by natural chromosome-doubling. Amphidiploid progeny were obtained from seeds produced by self-pollination of flowers with restored pollen fertility. Since these amphidiploids had some desirable characters, such as the early flowering of D. x isensis, and the vigorous Summer growth, robust upright stems and broad leaves of D. japonicus, they can be used in carnation breeding programmes to incorporate useful traits from these two species such as disease resistance, heat tolerance and unique flower morphologies..
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An immunocapture reverse transcription loop-mediated isothermal amplification (IC/RT-LAMP) was developed for the detection of tomato spotted wilt virus (TSWV) from chrysanthemum. This method enabled sensitive, reproducible and spe...
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An immunocapture reverse transcription loop-mediated isothermal amplification (IC/RT-LAMP) was developed for the detection of tomato spotted wilt virus (TSWV) from chrysanthemum. This method enabled sensitive, reproducible and specific detection of TSWV from chrysanthemum plants. In the RT-LAMP method, TSWV genomic RNA could be amplified under isothermal (65 degrees C) conditions within 1h. The resulting amplicons were detected by the measurement or observation of the turbidity of the reaction mixture without gel electrophoresis. IC/RT-LAMP was 100 times more sensitive than IC/RT-PCR.
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Reciprocal interspecific crosses were carried out between six lines of Dianthus caryophyllus L. and one line of Dianthus japonicus Thunb. Although no seed was set when D. japonicus was used as the seed parent, six seedlings were s...
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Reciprocal interspecific crosses were carried out between six lines of Dianthus caryophyllus L. and one line of Dianthus japonicus Thunb. Although no seed was set when D. japonicus was used as the seed parent, six seedlings were successfully obtained from 2,380 immature ovules by applying the embryo-rescue technique. However, they showed seed parent-like morphology and no evidence for the hybridity by flow cytometry and RAPD analyses. When six lines of D. caryophyllus were used as seed parents, a total of 192 seedlings were successfully obtained without using the embryo-rescue technique. Among these seedlings, 12 out of 25 progenies obtained from the carnation line '98sp1651' were confirmed to be the hybrids. The remaining 13 progenies of this line, and the total 167 progenies obtained from the other carnation lines, had carnation-like morphology without any evidence of hybridity by flow cytometry and RAPD analyses. The progenies confirmed as hybrids had intermediate characters of the parents with respect to leaf width and flower size, but they had a uniform flower color, reddish purple, which was different from that of either parent. Since the hybrids obtained in the present study have some profitable characters such as vigorous growth in summer time, upright robust stem, broad leaves and early flowering, they are expected to be used for the breeding of carnation which is suitable for growing under the Japanese climate.
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A differential detection method for three wheat viruses: Wheat yellow mosaic virus (WYMV), Japanese soil-borne mosaic virus (JSBWMV) and Chinese wheat mosaic virus (CWMV) using reverse transcription loop-mediated isothermal amplif...
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A differential detection method for three wheat viruses: Wheat yellow mosaic virus (WYMV), Japanese soil-borne mosaic virus (JSBWMV) and Chinese wheat mosaic virus (CWMV) using reverse transcription loop-mediated isothermal amplification (RT-LAMP) reaction was developed. All three primer sets, which were designed from the genome sequences of WYMV, JSBWMV and CWMV respectively, worked most efficiently at 65 degrees C and could detect each virus RNA within 10 min by fluorescence monitoring using an isothermal DNA amplification and fluorescence detection device. Furthermore, these primer sets showed unique annealing curves. The peak denaturing temperatures of WYMV, JSBWMV and CWMV primer sets were 87.6 degrees C, 84.8 degrees C and 86.4 degrees C, respectively and were clearly distinguished by the isothermal DNA amplification and fluorescence detection device. The RT-LAMP assay including all three primer sets was found to be 100 times more sensitive than RT-PCR for WYMV and JSBWMV and as sensitive as RT-PCR for CWMV. The RT-LAMP method was validated for the simultaneous detection of these viruses in wheat and barley leaves.
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In this study a selection experiment was carried out in order to investigate the effectiveness of index selection including oviposition time as a selection trait for persistency of laying strain building in laying hens. The select...
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In this study a selection experiment was carried out in order to investigate the effectiveness of index selection including oviposition time as a selection trait for persistency of laying strain building in laying hens. The selection was applied byusing an index consisting of age at first egg (SM), egg weight at 270 days of age (EW) and the rate of lay from 151 to 270 days of age between 06.00 and 11.00 h (6-11 EP) under 14 hour light-10 hour dark cycle (photo period; 06.00 to 20.00 h). After sixgenerations of index selection, 6-11 EP showed significant change, therefore short-term rate of lay from 151 to 270 days of age (SEP) and long-term rate of lay from 151 to 400 days of age (LEP) both improved significantly. Realized genetic gains per generation (P < 0.01) were: 6-11 EP, 3.0 percent; SEP, 0.9 percent; LEP, 1.3 percent; rate of lay from 151 to 270 days of age between 11.00 and 20.00 h (11-20 EP), -2.1 percent; residual-term rate of lay from 271 to 400 days of age (REP), 1.6 percent. Realized genetic gains were higher in 6-11 EP than in SEP and LEP. However, improvement was not obtained in SM and there was not a marked genetic change in EW. The pooled heritability estimates for LEP and SEP were similar. 0.19 and 0.15 respectively, howeverthe heritability estimate for 6-11 EP was higher at 0.41. The genetic correlations pooled over 6 generations between LEP and egg production traits were all high and positive except 11-20 EP: 6-11 EP, 0.51; SEP, 0.71; REP, 0.94; 11-20 EP, -0.25. These results indicated that rate of lay including oviposition time are useful as selection traits. Therefore, the index selection consisted of 6-11 EP was successful in persistency of laying strain building.
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A loop-mediated isothermal amplification (LAMP) reaction with a primer set designed from the rDNA ITS sequence of P. aphanidermatum was developed. Results of a specificity test using 57 strains of Pythium spp. indicated that the L...
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A loop-mediated isothermal amplification (LAMP) reaction with a primer set designed from the rDNA ITS sequence of P. aphanidermatum was developed. Results of a specificity test using 57 strains of Pythium spp. indicated that the LAMP assay gave no cross reactions in other 39 Pythium species, 11 strains of Phytophthora spp. and eight other soil borne pathogens. The detection limit was 10 fg of genomic DNA, which was ten times the sensitivity of the polymerase chain reaction. The LAMP assay was applied to hydroponic solution samples from tomato fields, and the results were compared to those of the conventional plating method. LAMP was observed to be effective for the specific detection of P. aphanidermatum. Furthermore, P. aphanidermatum was detected directly in tomato roots infected with P. aphanidermatum without DNA extraction. The LAMP method established in this study is a simple, sensitive and rapid tool for the detection of P. aphanidermatum.
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