Anther culture in rice proportionally rescues microspores according to gametophytic gene effect and enhances genetic study of hybrid sterility

Background

To investigate plant hybrid sterility, we studied interspecific hybrids of two cultivated rice species, Asian rice (Oryza sativa) and African rice (O. glaberrima). Male gametes of these hybrids display complete sterility owing to a dozen of hybrid sterility loci, termed HS loci, but this complicated genetic system remains poorly understood.

Results

Microspores from these interspecific hybrids form sterile pollen but are viable at the immature stage. Application of the anther culture (AC) method caused these immature microspores to induce callus. The segregation distortion of 11 among 13 known HS loci was assessed in the callus population. Using many individual calli, fine mapping of the HS loci was attempted based on heterozygotes produced from chromosome segment substitution lines (CSSLs). Transmission ratio distortion (TRD) from microspores was detected at 6 of 11 HS loci in the callus population. The fine mapping of S₁ and S₁₉ loci using CSSLs revealed precise distances of markers from the positions of HS loci exhibiting excessive TRD.

Conclusions

We demonstrated that AC to generate callus populations derived from immature microspores is a useful methodology for genetic study. The callus population facilitated detection of TRD at multiple HS loci and dramatically shortened the process for mapping hybrid sterility genes.

     

Quantitative loop-mediated isothermal amplification method for the detection of <Emphasis Type="Italic">Vibrio nigripulchritudo</Emphasis> in shrimp

Vibrio nigripulchritudo is considered one of the major pathogens threatening shrimp aquaculture. In this study, we developed a novel and highly specific quantitative loop-mediated isothermal amplification (Q-LAMP) assay. A set of four specific primers were designed targeting the V. nigripulchritudo intergenic spacer region. The reaction time and temperature were optimized for 60 min at 63°C. Quantitative analysis was then performed by measuring the turbidity of the reaction solution using a real-time turbidimeter, allowing for quantification of the initial DNA concentration with a sensitivity of 10² copy numbers equivalent to 2.3 colony forming units/ml or 0.3 fg/μl. The LAMP assay was able to specifically detect two representative strains of V. nigripulchritudo, whereas other Vibrio and non-Vibrio species were not amplified. A standard curve was generated for V. nigripulchritudo by plotting the threshold time (T _t) versus the log of bacterial number. A high correlation coefficient (R ² = 0.9749) was observed for the Q-LAMP reaction. In conclusion, Q-LAMP assay is a sensitive, rapid, and simple tool that can be used for the detection and quantification of V. nigripulchritudo in shrimp, thereby facilitating surveillance of vibriosis infection.     

Comparison of morphological and DNA-based techniques for stomach content analyses in juvenile chum salmon <Emphasis Type="Italic">Oncorhynchus keta</Emphasis>: a case study on diet richness of juvenile fishes

To identify the stomach contents of marine organisms, morphological observations are commonly conducted. However, the results obtained by this traditional method are frequently biased, as it is difficult to detect partially digested, soft-bodied organisms. To resolve this, recent studies have used DNA-based (DNA barcoding) analyses to examine the diet breadth of marine organisms. Here, we compared the prey richness of juvenile fishes using morphological observations and DNA-based analyses, with a focus on juvenile chum salmon Oncorhynchus keta. A higher number of prey taxa were successfully identified using DNA-based analyses than morphological observations. However, we also noticed several shortcomings of the DNA-based analyses, as reported in other diet-analysis studies that used molecular techniques. For example, the degree of digestion among prey taxa might have resulted in differential sensitivity to DNA detection. Additionally, several prey taxa could not be precisely identified, as the sequence data for some of the targeted organismal groups are unavailable in public gene databases. Remarkably, it is also possible that DNA-based analyses detected secondary prey, and therefore, the richness of prey taxa was likely overestimated. Thus, dietary analyses of juvenile fishes need to be carefully conducted, considering the respective advantages and disadvantages of DNA-based and morphological techniques.     

Effects of acid and alkaline pretreatment on the discoloration rates of dark muscle and myoglobin extract of skinned tilapia fillet during iced storage

Discoloration profiles of dark muscle of skinned tilapia fillets were examined during iced storage after pretreatment with lactic acid (LA) or sodium carbonate (SC). During the subsequent storage, the a* values decreased gradually, and changed more rapidly when the pH was lower than 6.3. The fillet pretreated with 10% (v/v) LA exhibited the highest metmyoglobin formation ratio (MetMb%), followed by the fillet pretreated with 5% (v/v) LA, the control fillet, and the fillet pretreated with 10% (w/v) SC. The sample pretreated with 10% LA showed a marked decrease in the a* value. Discoloration of the control was not observed until the ninth day of iced storage, and no discoloration was observed up to the 11th day for the fillet pretreated with 10% SC. These fillet discoloration profiles were subsequently verified using the myoglobin (Mb) fraction prepared from the dark muscle. MetMb% of the Mb fraction gradually increased during storage, and this increase accelerated at pH values of <6.3. Discoloration of the Mb fraction also showed a similar tendency, and no significant discoloration was observed at pH values of >6.5. These results suggest that pH greatly affects the discoloration rate of the dark muscle of skinned fillet, and the critical pH for the accelerated autooxidation of tilapia Mb is in the range 6.3–6.5.     

Effects of light quality on conidiophore formation of the melon powdery mildew pathogen <Emphasis Type="Italic">Podosphaera xanthii</Emphasis>

The lengths of conidiophores in fungal colonies of the melon powdery mildew pathogen Podosphaera xanthii Pollacci KMP-6 N cultured under greenhouse (natural) conditions differed markedly from those cultured in a growth chamber. We hypothesized that light wavelength was responsible for the differences in conidiophore length. In this study, we examined the effects of light-emitting diode (LED) irradiation (purple, blue, green, orange, and red light) and white light on colony development and conidiophore formation in KMP-6 N using a stereomicroscope and a high-fidelity digital microscope. Colonies on leaves were flat under greenhouse conditions and under red LED light irradiation but were stacked under growth chamber conditions and under purple, blue, green, and orange LED light irradiation. In addition, KMP-6 N formed catenated conidia comprising six conidia per conidiophore under greenhouse conditions and red light but more than seven conidia per conidiophore under growth chamber conditions and purple, blue, green, and orange light. Furthermore, almost none of the conidia on top of the conidiophores grown under blue light were fully constricted. Therefore, these fungi could not scatter their conidia and spread infection. This is the first report of the effects of LED lights on conidiophore formation in the melon powdery mildew fungus P. xanthii. The results provide insight into the mechanisms underlying the responses of conidiophores to light of specific wavelengths and conidial scatter from conidiophores of melon powdery mildew fungi.     

Response of the leaf photosynthetic rate to available nitrogen in erect panicle-type rice (Oryza sativa L.) cultivar,Shennong265

Increasing the yield of rice per unit area is important because of the demand from the growing human population in Asia. A group of varieties called erect panicle-type rice (EP) achieves very high yields under conditions of high nitrogen availability. Little is known, however, regarding the leaf photosynthetic capacity of EP, which may be one of the physiological causes of high yield. We analyzed the factors contributing to leaf photosynthetic rate (P_n) and leaf mesophyll anatomy of Nipponbare, Takanari, and Shennong265 (a EP type rice cultivar) varieties subjected to different nitrogen treatments. In the field experiment, P_n of Shennong265 was 33.8 μmol m^?2 s^?1 in the high-N treatment, and was higher than that of the other two cultivars because of its high leaf nitrogen content (LNC) and a large number of mesophyll cells between the small vascular bundles per unit length. In Takanari, the relatively high value of P_n (31.5 μmol m^?2 s^?1) was caused by the high stomatal conductance (g_s; .72 mol m^?2 s^?1) in the high-N treatment. In the pot experiment, the ratio of P_n/C_i to LNC, which may reflect mesophyll conductance (g_m), was 20–30% higher in Nipponbare than in Takanari or Shennong265 in the high N availability treatment. The photosynthetic performance of Shennong265 might be improved by introducing the greater ratio of P_n/C_i to LNC found in Nipponbare and greater stomatal conductance found in Takanari.     

An improved method for bright-field imaging of arbuscular mycorrhizal fungi in plant roots

Easy observation methods to assess the colonization levels of arbuscular mycorrhizal (AM) fungi in plant roots are crucial for studying the biology of AM symbiosis and for considering agricultural use. Many AM studies employ Trypan Blue (TB) coupled with lactic acid to stain AM fungal structures as bright-field images; however, TB staining can be difficult to use owing to its noxiousness and high viscosity. Here, we report the development of an easy method for visualizing AM fungal structures as bright-field images using 3,3′-diaminobenzidine (DAB). Wheat germ agglutinin (WGA)-conjugated horseradish peroxidase (HRP), which specifically targets N-acetylglucosamine polymers and detects AM fungal cell walls, penetrated the cortical layers of 10% potassium hydroxide (KOH)-treated soybean roots and stained AM fungal mycelia in the presence of DAB and hydrogen peroxide (H₂O₂). Comparison between DAB and TB staining of soybean (Glycine max L.) roots suggested that the intactness of root systems and image contrast using DAB staining were superior. Background signals in stele observed by DAB staining were negligible as compared with those observed by WGA-fluorescein isothiocyanate staining. DAB staining, which combines the advantages of TB (easy bright-field imaging) and WGA-fluorophore (specific and high-quality) staining, provides a robust imaging method for macro- and micro-level analyses of AM roots and is applicable to at least six crops: soybean, onion (Alium cepa L.), potato (Solanum tuberosum L.), maize (Zea mays L.), rice (Oryza sativa L.), and sunflower (Helianthus annuus L.).     

Psycho-educational Horseback Riding to Facilitate Communication Ability of Children with Pervasive Developmental Disorders

In this study, we applied a novel psycho-educational horseback riding (PEHR) program to the treatment of four Japanese children with pervasive developmental disorders (PDD) in order to facilitate the acquisition of verbal and nonverbal communication skills. The behavioral changes in each child were evaluated using a psychological and behavioral scale. The scale for evaluating the effect of Human-Equips-Interaction on Mental activity (HEIM scale) was designed to assess the behavioral improvement of children based on the following 10 items: Human relationships, Imitation, Emotional expression, Sudden physical movement, Fixative behavior, Adaptation to change, Visual response, Fear or nervousness, and Verbal and nonverbal communication. After taking part in the PEHR program for several months, all subjects showed remarkably improved HEIM scores and marked improvements were observed in eye contact with others (instructors, side walkers, and leaders) in the riding area. A statistical difference was found in items 1, 2, 3, 6, 7, 8, and 9. However, no statistical difference was found in items 4, 5, and 10. As the program progressed, the children showed enhanced verbal and nonverbal communication skills, and became more expressive in their emotional and empathetic interaction with their parents. These observations suggest that the normal functioning of pleasurable emotions and empathy may facilitate further improvements in joint attention, imitation and empathy, and may result in successful verbal expression by PDD children. Therefore, horseback riding can play a very important role in the psycho-educational support required for the communication ability of PDD children.     

Bait method to detect Pythium species that grow at high temperatures in hydroponic solutions

Pythium helicoides, P. aphanidermatum and P. myriotylum are important pathogens that cause root rot of several crops in hydroponic culture and in ebb-and-flow irrigation systems. These species belong to a group of Pythium species that can grow at temperatures higher than 40°C. We developed a method for baiting these high-temperature Pythium species and evaluated its practicality to monitor their presence in nutrient solutions. Seeds of cucumber, tomato, radish, hemp, perilla and millet and leaves of bent grass and rose were tested as baits in hydroponic systems. Hemp, perilla and radish seeds and bent grass and rose leaves were more effective than the other baits for Pythium zoospores, and bent grass leaves were the most effective. In a sensitivity test, bent grass leaf traps (BLTs) detected three Pythium species after only a 1 day exposure to suspensions of 40 zoospores per liter of water, and the frequency of detection increased with zoospore density and with baiting period. A temperature of 38°C was optimum for the selective reisolation of the high-temperature Pythium species from the BLTs. The BLT was also tested with inoculated and noninoculated miniature roses that shared a recirculating nutrient solution. The pathogen was detected in the nutrient solution 23 days before the disease spread to the noninoculated roses. In addition, P. helicoides was detected 30 days before the disease was evident in a commercial greenhouse. The baiting method described here will be useful for monitoring high-temperature Pythium species in recirculating hydroponic culture systems.