A simplified genomic DNA extraction protocol for pre-germination genotyping in rice.

Genotyping is a critical step for molecular marker-assisted selection in rice. Rice genomic DNA samples for genotyping are typically isolated from living tissues such as seedlings. This requires the germination of all candidate seeds and extraction of DNA from the seedlings. Currently, an ideal individual is selected from a very large number of plants, which is time- and labor-consuming, requiring several transplantations of materials and sampling processes. In this study, we developed a simplified genomic DNA extraction protocol in rice by using amylase to treat half-seeds. The yields of genomic DNA from a half-seed of Indica and Japonica rice were greater than 203.8 ± 32.5 and 143.2 ± 25.5 ng, respectively, and the 260/280 nm absorbance ratio was 1.75-2.10. The DNA was confirmed to be sufficient for polymerase chain reaction amplification and can be used in a marker-assisted selection program.

Genetic diversity analysis of tree peony germplasm using iPBS markers.

We examined the genetic diversity of 10 wild species (populations) and 55 varieties of tree peony using inter-primer binding site (iPBS) markers. From a total of 36 iPBS primers, 16 were selected based on polymorphic amplification. The number of bands amplified by each primer ranged from 9 to 19, with an average of 12.88 bands per primer. The length of bands ranged from 100 to 2000 bp, concentrated at 200 to 1800 bp. Sixteen primers amplified 206 bands in total, of which 173 bands were polymorphic with a polymorphism ratio of 83.98%. Each primer amplified 10.81 polymorphic bands on average. The data were then used to construct a phylogenetic tree using unweighted pair group method with arithmetic mean methods. Clustering analysis showed that the genetic relationships among the varieties were not only related to the genetic background or geographic origin, but also to the flowering phase, flower color, and flower type. Our data also indicated that iPBS markers were useful tools for classifying tree peony germplasms and for tree peony breeding, and the specific bands were helpful for molecular identification of tree peony varieties.

A novel synthetic Cry1Ab gene resists rice insect pests.

A few insect control genes of Bacillus thuringiensis have been modified successfully to increase the expression in plants by replacing rare codons, increasing GC content, and avoiding the DNA elements that could cause premature transcription termination, mRNA instability, and potential methylation. However, the modification process was intricate and often confused researchers. In this study, we adopted a simple method to modify Cry1Ab only by individually replacing its amino acid sequence with corresponding rice-preferred codons based on analysis of 92,188 coding DNA sequences. Unexpectedly, all elements of A+T richness, which terminate or destabilize transcription in plants, were avoided in the newly designed mCry1Ab. However, mCry1Ab had 2 notable features: less synonymous codons and high GC content. mCry1Ab only employed 22 of the 61 codons to encode protein and had an enhanced GC content of 65%. The increase in GC content caused abundant potential methylation signals to emerge in mCry1Ab. To test whether mCry1Ab could be expressed in rice, we transferred it into Oryza japonica variety Wanjing97. Insect bioassays revealed that transgenic plants harboring this gene driven by 2 promoters, CaMV35S and OsTSP I, were highly resistant to rice leaffolder (Cnaphalocrocis medinalis). Analysis of R0 to R2 generation plants indicated that the mCry1Ab was inherited stably by the progeny. Our study provided a simple modified method for expressing exogenous genes in rice and confirmed that less synonymous codons and high GC content do not affect transgene expression in rice.

A high-throughput, high-quality plant genomic DNA extraction protocol.

The isolation of high-quality genomic DNA (gDNA) is a crucial technique in plant molecular biology. The quality of gDNA determines the reliability of real-time polymerase chain reaction (PCR) analysis. In this paper, we reported a high-quality gDNA extraction protocol optimized for real-time PCR in a variety of plant species. Performed in a 96-well block, our protocol provides high throughput. Without the need for phenol-chloroform and liquid nitrogen or dry ice, our protocol is safer and more cost-efficient than traditional DNA extraction methods. The method takes 10 mg leaf tissue to yield 5-10 µg high-quality gDNA. Spectral measurement and electrophoresis were used to demonstrate gDNA purity. The extracted DNA was qualified in a restriction enzyme digestion assay and conventional PCR. The real-time PCR amplification was sufficiently sensitive to detect gDNA at very low concentrations (3 pg/µL). The standard curve of gDNA dilutions from our phenol-chloroform-free protocol showed better linearity (R(2) = 0.9967) than the phenol-chloroform protocol (R(2) = 0.9876). The results indicate that the gDNA was of high quality and fit for real-time PCR. This safe, high-throughput plant gDNA extraction protocol could be used to isolate high-quality gDNA for real-time PCR and other downstream molecular applications.

Quantitative detection of the rice false smut pathogen Ustilaginoidea virens by real-time PCR.

Rice false smut (RFS) is an important rice disease that is caused by the pathogen Ustilaginoidea virens. In this study, we developed a real-time polymerase chain reaction (PCR) assay to detect U. virens and to estimate the level of disease. The genomic DNAs of U. virens and rice were extracted together from the rice samples. Real-time PCR assays were performed and compared to conventional nested-PCR assays. The real-time PCR assay presented a consistent linearity of the standard curve (R(2) = 0.9999). The detection limit could be as low as 40 fg U. virens DNA with a rice genomic DNA background on using the real-time PCR assay, which showed significantly higher sensitivity than the conventional nested-PCR assay. We conclude that the real-time PCR quantitative assay is a useful tool for detecting U. virens and for early defense and control of RFS.

First Report of Leaf Spot Disease of Peony Caused by Seimatosporium botan in China.

Tree peony (Paeonia suffruticosa Andrews), a perennial ligneous deciduous shrub in the Paeoniaceae family, is known for its beautiful and charming flowers. It is regarded as the flower symbol of China and is cultivated throughout the country. In August 2008, a previously unknown leaf spot was observed on peony cultivated in the Mountain Peony Garden located in the Luoyang area of Henan Province, China. In 2009, the leaf spot disease was observed in some gardens in the city of Luoyang, China. Initial symptoms appeared as small, round or irregular, brown, necrotic lesions in the middle of leaves. These lesions gradually enlarged up to 1 cm in diameter and were circular or irregular, brown to dark brown, and brown on the margins. In a humid atmosphere, black, sessile, discoid acervuli developed on the lesions, and the lesions sometimes became waxy-like, eventually coalesced, and nearly covered the entire leaf. Conidia produced in acervuli had two morphologically different types. One type had a single basal appendage, ellipsoid to fusiform, transversely three septate, 16 to 20 × 5 to 7 µm, smooth, basal cell obconic with a truncate base, subhyaline, 3 to 5 µm long; two central cells subcylindrical to dolioform, brown to dark brown, 8 to 10 µm long, apical cell conical with rounded apex, concolorous with the central cells, 4 to 5 µm long, basal appendage filiform, unbranched, excentric, 4 to 8 µm long. The other type had a single appendage at both ends, fusiform to subcylindrical, transversely three septate, 16 to 20 × 4 to 5 µm, smooth; basal cell obconic with a truncate base, subhyaline, 4 to 5 µm long; two central cells subcylindrical to dolioform, pale brown, 8 to 11 µm long; apical cell conical with an acute apex, hyaline to subhyaline, 4 to 5 µm long; basal appendage filiform, unbranched, excentric, 4 to 8 µm long; apical appendage filiform, unbranched, 4 to 8 µm long. Single conidial isolates of both types of conidia yielded identical colonies, which produced both types of conidia on potato dextrose agar (PDA), thus showing that both types of conidia belonged to the same fungus. Colonies on PDA were slimy in appearance, yellow to villous with an irregular taupe margin; reverse brown to grayish brown. Cultural and conidial characteristics of the isolates were similar to those of Seimatosporium botan (1). The DNA sequence for the fungus showed internal transcribed spacer region (ITS1-5.8S-ITS2) sequences (GenBank Accession No. HM067840) with 93% sequence identity to S. discosioides (Accession Nos. EF600970.1 and EF600969.1). This is the first submission of a S. botan sequence to GenBank. To determine pathogenicity, 20 healthy leaves of P. suffruticosa were inoculated by spraying a conidial suspension of S. botan onto the foliage. Ten leaves were sprayed with sterile water and served as controls. Plants were covered with plastic for 24 h to maintain high relative humidity. After 15 days, the symptoms described above were observed on leaves in all inoculated plants, whereas symptoms did not develop on the control plants. The pathogen was reisolated from inoculated leaves, fulfilling Koch's postulates. On the basis of morphology and ITS region sequences, we conclude that S. botan is the causal agent of leaf spots of P. suffruticosa. There is a report of S. botan on P. suffruticosa stems in Japan (1), but to our knowledge, this is the first report of leaf spot disease of peony caused by S. botan in China. References: (1) S. Hatakeyama et al. Mycoscience 45:106, 2004.

First Report of Pilidium concavum on Paeonia suffruticosa in China.

Paeonia suffruticosa Andrews, a deciduous perennial shrub, is known for its beautiful and charming flowers. It is regarded as the flower symbol of China and cultivated throughout the country. Since 2006, large, brown necrotic spots have been observed on numerous P. suffruticosa plants in gardens in Luoyang, China. Spots appeared each year and were observed on more than 50% of the plants, sometimes affecting more than half of the leaf. Initial symptoms appeared as small, round, water-soaked lesions in the middle or on the margin of leaves. These areas enlarged up to 1 to 3 cm in diameter and were circular or irregular, brown to dark brown, and pale brown on the margins. In a humid atmosphere, black, sessile, discoid conidiomata developed on the spots and exuded a pink spore mass that turned brown with age. Conidiophores were hyaline, unicellular, cylindrical, and fusiform and 5.0 to 8.0 µm long and 1.4 to 2.0 µm wide. Pure cultures were obtained by plating the spores on potato dextrose agar (PDA) medium. In culture, the fungus produced a gray-to-brown colony with whitish aerial mycelium. The morphology and size of conidia were comparable with previous descriptions of Pilidium concavum (Desm.) Höhn. (1). The ITS1-5.8S-ITS2 region of the isolate was amplified by PCR with primers ITS1 and ITS4 and sequenced. The 472-nt sequence was 100% identical to that of the Pilidium concavum specimen voucher BPI 1107275 (GenBank Accession No. AY487094). To validate Koch's postulates, pathogenicity was tested by inoculating 10 leaves of P. suffruticosa with mycelia plugs from a colony growing on PDA; leaves inoculated with the plugs of PDA medium only served as the control. Leaves were covered with plastic for 24 h to maintain high relative humidity. After 7 days, 100% of the mycelium-inoculated leaves showed symptoms identical to those observed on P. suffruticosa leaves affected in the field, whereas all leaves inoculated with PDA medium only remained free of symptoms. Reisolation of the fungus from leaf lesions confirmed that the causal agent was Pilidium concavum. Thus, we concluded that Pilidium concavum is the causal agent of leaf spots of P. suffruticosa. This disease has been reported to be frequently occurring on P. suffruticosa stems imported from Japan (1), but to our knowledge, this is the first report of Pilidium concavum on P. suffruticosa in China. References: (1) M. E. Palm. Mycologia 83:787, 1991.

A comparative study of the H + FO (v = 0, j = 0) --> (OH + F)/(HF + O) reaction from QM and QCT methods.

The dynamics of the title reaction are investigated using both the time-dependent quantum wave packet and the quasi-classical trajectory methods and employing a recently developed adiabatic ground 1(3)A'' potential energy surface [Gomez-Carrasco et al. J. Chem. Phys. 2004, 121, 4605]. By comparison to the quantum J = 0 reaction probabilities, the QCT method is first validated for the title reaction and further employed to produce the integral cross sections and rate constants. No resonance structures have been observed in both the QCT J = 0 and the quantum reaction probabilities of OH + F as well as in the QCT integral cross sections of both product channels, while there are some undulations in the calculated quantum reaction probabilities of HF + O. It is also found that Coriolis coupling effects play a significant role in the quantum calculation and that formation of the OH product is favored over the HF product in the reactive system.

Synaptic transmission of chaotic spike trains between primary afferent fiber and spinal dorsal horn neuron in the rat.

Primary sensory neurons can generate irregular burst firings in which the existence of significant deterministic behaviors of chaotic dynamics has been proved with nonlinear time series analysis. But how well the deterministic characteristics and neural information of presynaptic chaotic spike trains were transmitted into postsynaptic spike trains is still an open question. Here we investigated the synaptic transmission of chaotic spike trains between primary Adelta afferent fiber and spinal dorsal horn neuron. Two kinds of basic stimulus unit, brief burst and single pulse, were employed by us to comprise chaotic stimulus trains. For time series analysis, we defined "events" as the longest sequences of spikes with all interspike intervals less than or equal to a certain threshold and extracted the interevent intervals (IEIs) from spike trains. Return map analysis of the IEI series showed that the main temporal structure of chaotic input trains could be detected in postsynaptic output trains, especially under brief-burst stimulation. Using correlation dimension and nonlinear prediction methods, we found that synaptic transmission could influence the nonlinear characteristics of chaotic trains, such as fractal dimension and short-term predictability, with greater influence made under single-pulse stimulation. By calculating the mutual information between input and output trains, we found the information carried by presynaptic spike trains could not be completely transmitted at primary afferent synapses, and that brief bursts could more reliably transmit the information carried by chaotic input trains across synapses. These results indicate that although unreliability exists during synaptic transmission, the main deterministic characteristics of chaotic burst trains can be transmitted across primary afferent synapses. Moreover, brief bursts that come from the periphery can more reliably transmit neural information between primary afferent fibers and spinal dorsal horn neurons.

Adrenergic sensitivity of neurons with non-periodic firing activity in rat injured dorsal root ganglion.

In this study, we compared the sensitivity of non-periodically and periodically active neurons in chronically compressed dorsal root ganglion in rats to norepinephrine and sympathetic stimulation. Forty-nine of 58 (84.5%) neurons with non-periodic activity showed responses to norepinephrine, whereas only five of 48 (10.4%) neurons with periodic activity displayed any response. The dose-response relationship of norepinephrine to the irregular burst pattern neurons shifted towards the left significantly compared to that of the periodic activity neurons. Responses to norepinephrine became apparent in eight neurons after their periodic firing activity was transformed into the non-periodic firing activity through the increase in Ca(2+). Changes in the time-response curves indicate a higher sensitivity of irregular burst pattern neurons to sympathetic stimulation than the periodic activity neurons. Finally, deterministic dynamics contained within the interburst interval series for non-periodic activity were identified. From these results, we suggest that the non-periodic activity neurons have a higher adrenergic sensitivity than those displaying periodic activity, and that this sensitivity may depend on the deterministic chaos within its firing dynamic system.

Detection of determinism within time series of irregular burst firing from the injured sensory neuron.

Irregular burst firing is spontaneously generated by rat dorsal root ganglion neuron after chronic compression. To investigate the time series of burst firing, we recorded interspike intervals (ISIs) of single-unit firing in vitro and processed the ISIs to obtain interevent intervals (IEIs). Then, two non-linear methods were applied to detect deterministic dynamic behaviors in IEI series. No evidence for the existence of determinism was found with non-linear prediction method. Using unstable periodic orbit identification method, significant period-1 orbits were identified in all 10 data, period-2 orbits in eight, and period-3 orbits in six. The results indicate that there exist significant deterministic behaviors in the time series of irregular burst firing from the injured sensory neuron.

High resolution spectral analysis of 13CH3OH in the excited torsional states.

In this work, we have extended our previous analysis of the Hamiltonian of 13C substituted methanol to include a large number of spectral lines involving the second excited torsional state using an improved model. The data set consisted of 2529 Fourier transform and microwave transitions with the rotational angular momentum J < or = 10, K < or = 6 and n < or = 2 (with 336 MW lines). The data set was fitted with the new Hamiltonian model to derive the molecular parameters. The results indicate that the model developed for the other methanol species (CH3OH, CH3(18)OH and CH3OD) is also valid for the C-13 substituted species. The results will allow the energy levels of the molecule to be calculated for higher torsional levels above the internal rotational barrier with improved precision and allow the analysis to be carried out for more excited torsional states.

[Pattern and dynamic changes of integer multiples in spontaneous discharge of injured dorsal root ganglion neurons].

For the purpose of the present investigation, spontaneous discharges of dorsal root ganglion (DRG) neurons of the rats, which had undergone 3-10 days' chronic compression, were studied. It was found that the interspike interval (ISI) of 17 out of 156 fibers had integer multiples temporal rhythm pattern, in which all the ISI were integer multiples of a basic ISI and a return map of their ISI could be constructed as a crystal grid structure. This temporal pattern could be modified by Na+ channel and K+ channel on their membranes. These data indicated the presence of some irregular discharge trains with their intrinsic regularity.

Studies on mechanisms and blockade of carcinogenic action of female sex hormones.

Tumours of mice are induced by administration of Inj. Hydroxyprogesteroni Caproatis Co. (EP) in a practical subthreshold dose of carcinogenesis or 2.5-5 times the human contraceptive dose (simply referred to as 2.5- to 5-fold dose) combined with whole-body 0.5 Gy gamma-ray irradiation. Malignant transformation of Syrian golden hamster embryo (SHE) cells is also induced by 5-fold dose of EP combined with 0.3 Gy gamma-ray irradiation in vitro, thereby indicating that synergistic carcinogenesis can be obtained by combined use of physical and chemical carcinogens. The mechanisms of synergistic carcinogenesis have been further explained by cytogenetics, damage extent of the target cell DNA and production of free radicals. The Chinese traditional medicine with antioxidating effect (Sulekang Capsule, SC), food additive--butylated hydroxyanisole (BHA) and green tea can effectively inhibit the carcinogenic effect of EP or EP combined with gamma rays in mice. They all have marked ability to scavenge or remove the free radicals and thereby reduce the DNA damage.