[Comparison of DNA Extraction Methods for Processed Foods Containing Soybean or Maize].

Processed foods containing soybean or maize are subject to labeling regulations pertinent to genetically modified (GM) foods in Japan. To confirm the reliability of the labeling procedure of GM foods, the Japanese standard analytical methods (standard methods) using real-time PCR technique have been established. Although certain DNA extraction protocols are stipulated as standard in these methods, the use of other protocols confirmed to be equivalent to the existing ones was permitted. In this study, the equivalence testing of the techniques employed for DNA extraction from processed foods containing soybean or corn was conducted. In this study, the equivalence testing of the techniques employed for DNA extraction from processed foods containing soybean or maize was conducted. The silica membrane-based DNA extraction kits, GM quicker 4 and DNeasy Plant Maxi Kit (Maxi Kit), as an existing method were compared. GM quicker 4 was considered to be equivalent to or better than Maxi Kit.

Development and Validation of a New Robust Detection Method for Low-Content DNA Using ΔΔCq-Based Real-Time PCR with Optimized Standard Plasmids as a Control Sample.

Real-time polymerase chain reaction (PCR) is the gold standard for DNA detection in many fields, including food analysis. However, robust detection using a real-time PCR for low-content DNA samples remains challenging. In this study, we developed a robust real-time PCR method for low-content DNA using genetically modified (GM) maize at concentrations near the limit of detection (LOD) as a model. We evaluated the LOD of real-time PCR targeting two common GM maize sequences (P35S and TNOS) using GM maize event MON863 containing a copy of P35S and TNOS. The interlaboratory study revealed that the LOD differed among laboratories partly because DNA input amounts were variable depending on measurements of DNA concentrations. To minimize this variability for low-content DNA samples, we developed ΔΔCq-based real-time PCR. In this study, ΔCq and ΔΔCq are as follows: ΔCq = Cq (P35S or TNOS) - Cq (SSIIb; maize endogenous gene), ΔΔCq = ΔCq (analytical sample) - ΔCq (control sample at concentrations near the LOD). The presence of GM maize was determined based on ΔΔCq values. In addition, we used optimized standard plasmids containing SSIIb, P35S, and TNOS with ΔCq equal to the MON863 genomic DNA (gDNA) at concentrations near the LOD as a control sample. A validation study indicated that at least 0.2% MON863 gDNA could be robustly detected. Using several GM maize certified reference materials, we have demonstrated that this method was practical for detecting low-content GM crops and thus for validating GM food labeling. With appropriate standards, this method would be applicable in many fields, not just food.

Development and Interlaboratory Validation of a Novel Reproducible Qualitative Method for GM Soybeans Using Comparative Cq-Based Analysis for the Revised Non-GMO Labeling System in Japan.

Many countries have implemented the labeling system of genetically modified organisms (GMO). In Japan, the regulatory threshold for non-GMO labeling will be revised and restricted to undetectable by April 2023. The practical criterion for the revised system is based on the limit of detection (LOD). However, determining whether the commingling of GMO levels exceeds the LOD is challenging because GM contents close to the LOD are usually below the limit of quantification. In this study, we developed a qualitative method based on comparative Cq-based analysis targeting cauliflower mosaic virus 35S promoter and GM soybean MON89788 event-specific sequences that could be applicable to the revised non-GMO labeling. ΔCq values between the target and endogenous sequences were calculated, and the ΔΔCq value obtained was used as a criterion to determine analytical samples with GM contents exceeding the threshold. To improve the reproducibility of the method, we used a standard plasmid that yields equivalent and stable ΔCq values comparable with those obtained from LOD samples. The developed method was validated with an interlaboratory study. The new qualitative detection concept would be useful for ensuring robust and reproducible results among laboratories, particularly for detecting low-copy-number DNA samples.

Interlaboratory Evaluation of a Method for Quantification of Norovirus RNA as an Alternative Use for ISO 15216-1:2017 to Conduct Japan Baseline Survey of Oysters.

In this study, we aimed to investigate the standard method used for quantification of norovirus in oysters in Japan for the provisional adaptation of the method as an alternative to ISO 15216-1:2017, to conduct a Japan baseline survey of norovirus in oysters. For this purpose, the method provided by the Japan Committee for Standardization of Virus Detection in Food was subjected to an interlaboratory study to determine the performance characteristics of the standard method used in Japan. As a result, the theoretical limit of quantification for norovirus GI and GII in oysters by the standard method used in Japan was expected to be 1.92 and 1.85 log10 copies/g, respectively. The repeatability standard deviations (Sr) were 0.26 and 0.30 log10 copies/g for GI and GII, respectively, and the reproducibility standard deviations (SR) were 0.47 and 0.44 log10 copies/g for GI and GII, respectively. Through the interlaboratory study, we specified several critical points to obtain scientifically reliable results by using the standard method used in Japan. Especially, necessity for application of using process control virus was the most crucial point that needed to be improved. In addition, there are many participating laboratories that could not handle dilution of standard and quantify or detect the viruses in the test samples. To ensure scientifically reliable test result, capacity building of laboratories and implementation of proficiency testing should be considered for future tasks in combination with an application of process control materials in the method. On the assumption that the problems revealed in this study will be solved, the standard method used in Japan would be suitable for use in Japan baseline survey of norovirus in oysters, which will contribute to the international action against norovirus in oysters, led by the EU.

[Evaluation of Conversion Factors for Genetically Modified Maize Quantification].

To quantify the amount of authorized GM maize or soybean, conversion factor (Cf) values are required for converting the copy number ratio of GM sequence to an endogenous sequence into weight-based GMO amounts. Cf values are available for the several latest real-time PCR instruments such as QuantStudio5, QuantStudio12K Flex, LightCycler 96, and LightCycler 480 for GM soybeans but not for GM maize. For the quantification of GM maize, we experimentally determined the Cf values targeting Cauliflower mosaic virus 35S promoter (P35S), GA21 construct specific, MIR604 event specific and MIR162 event specific sequences using the four real-time PCR instruments.

Development of a novel method for specific detection of genetically modified Atlantic salmon, AquAdvantage, using real-time polymerase chain reaction.

Genetically modified (GM) Atlantic salmon, AquAdvantage (AquAd), was the first GM animal approved officially for human consumption. Many countries monitor the use of this product under their GM regulations, but a pragmatic system for AquAd-specific detection is needed. Here, we developed a real-time polymerase chain reaction method with high sensitivity for detection of AquAd in foods. This method showed high specificity for the AquAd transgene and the detection limit was 12.5-25 targeted DNA copies per test reaction. An inter-laboratory study using the method developed demonstrated reproducibility at >0.1% (w/w) AquAd content.

Rapid DNA template preparation directly from a rice sample without purification for loop-mediated isothermal amplification (LAMP) of rice genes.

Rapid DNA template preparation directly from a single rice (Oryza sativa) grain or rice flour of its equivalent weight was developed for loop-mediated isothermal amplification (LAMP). LAMP efficiency using DNA extract obtained from consecutive addition of alkaline lysis reagent (25 mM NaOH, 0.2 mM EDTA) and neutralizing reagent (40 mM Tris-HCl [pH 5]) was comparable to that using an equivalent amount of purified DNA as template. The stability of the prepared DNA extract was confirmed for up to six-day storage at room temperature. Without using any special laboratory devices, the developed method enabled a rapid, simple, and low-cost DNA template preparation method for reliable LAMP testing to detect rice genes.

Data representing applicability of developed growth hormone 1 (GH1) gene detection method for detecting Atlantic salmon (Salmo salar) at high specificity to processed salmon commodities.

This article is referred to the research article entitled "Development of a novel method for specific detection of genetically modified Atlantic salmon, AquAdvantage, using real-time polymerase chain reaction" by Soga et al. (2020). Applicability of the developed growth hormone 1 (GH1) and 18S ribosomal DNA (18S rDNA) detection methods using real-time polymerase chain reaction (PCR) for detecting Atlantic salmon (Salmo salar) to processed food commodities was examined. DNAs extracted and purified from 24 commodities labelled to include salmon as an ingredient were used as template. Yield and purity of DNAs obtained and Cq values from real-time PCR analyses were provided.

Novel Bioprinting Application for the Production of Reference Material Containing a Defined Copy Number of Target DNA.

Nucleic acid amplification methods, such as polymerase chain reaction (PCR), are extensively used in many applications to detect target DNA because of their high sensitivity, good reproducibility, and wide dynamic range of quantification. However, analytical quality control when detecting low copy number target DNA is often missing because of a lack of appropriate reference materials. Recent advances in analytical sciences require a method to accurately quantify DNA at the single molecule level. Herein, we have developed a novel method to produce reference material containing a defined copy number of target DNA (referred to as "cell number-based DNA reference material"). In this method, a suspension of cells carrying a single target DNA sequence was ejected by an inkjet head, and the number of cells in each droplet was counted using highly sensitive cameras. The resulting solutions contained a defined copy number of target DNA and could be used as reference materials. The use of the newly developed reference material was compared with that of diluted solutions of target DNA to evaluate the performance of qualitative real-time PCR in terms of the limit of detection (LOD). Our results demonstrated that cell number-based DNA reference material provides more accurate information regarding performance quality. The reference material produced by this method is a promising tool to evaluate assay performance.

[Detection of DNA in Highly Processed Foods].

Highly processed foods, including soy sauce, cornflakes, starch sugar, beet sugar and vegetable oil, are not currently subject to genetically modified (GM) food labeling, because DNA could not be detected in these food products. Here we re-examined the method of DNA extraction from starch syrup, beet sugar and vegetable oil using commercially available DNA extraction kits. We found that DNA was not stably detected by PCR targeting a species-specific endogenous plant gene. The reason for this may have been that the DNA yield was below the detection limit, because PCR inhibition was not observed.

A set of tetra-nucleotide core motif SSR markers for efficient identification of potato (Solanum tuberosum) cultivars.

Simple sequence repeat (SSR) is a popular tool for individual fingerprinting. The long-core motif (e.g. tetra-, penta-, and hexa-nucleotide) simple sequence repeats (SSRs) are preferred because they make it easier to separate and distinguish neighbor alleles. In the present study, a new set of 8 tetra-nucleotide SSRs in potato (Solanum tuberosum) is reported. By using these 8 markers, 72 out of 76 cultivars obtained from Japan and the United States were clearly discriminated, while two pairs, both of which arose from natural variation, showed identical profiles. The combined probability of identity between two random cultivars for the set of 8 SSR markers was estimated to be 1.10 × 10-8, confirming the usefulness of the proposed SSR markers for fingerprinting analyses of potato.

Allocation of absorbed light energy in PSII to thermal dissipations in the presence or absence of PsbS subunits of rice.

The thermal dissipation (TD) of absorbed light energy in PSII is considered to be an important photoprotection process in photosynthesis. A major portion of TD has been visualized through the analysis of Chl fluorescence as energy quenching (qE) which depends on the presence of the PsbS subunit. Although the physiological importance of qE-associated TD (qE-TD) has been widely accepted, it is not yet clear how much of the absorbed light energy is dissipated through a qE-associated mechanism. In this study, the fates of absorbed light energy in PSII with regard to different TD processes, including qE-TD, were quantitatively estimated by the typical energy allocation models using transgenic rice in which psbS genes were silenced by RNA interference (RNAi). The silencing of psbS genes resulted in a decrease in the light-inducible portion of TD, whereas the allocation of energy to electron transport did not change over a wide range of light intensities. The allocation models indicate that the energy allocated to qE-TD under saturating light is 30-50%. We also showed that a large portion of absorbed light energy is thermally dissipated in manners that are independent of qE. The nature of such dissipations is discussed.

Cultivar identification of Korean rice based on DNA markers for blast resistance.

Cultivar identification of seven Korean domestic rice using DNA markers related to blast resistance was conducted. By PCR analyses using six markers, which we developed in a previous study, and one newly-developed marker for pib, it became possible to differentiate the seven cultivars from each other. This result should contribute not only to cultivar identification but also, to molecular breeding of blast resistance for Korean rice.

Grain qualities and their genetic derivation of 7 new rice for Africa (NERICA) varieties.

NERICA are interspecific rice varieties from crossing between the high-yielding Asian rice ( Oryza sativa spp. Japonica) with locally adapted African rice ( Oryza glaberrima). In this study, we analyzed grain qualities of 7 NERICA varieties (NERICA 1 to 7) and genetic derivation of quality-related genes. Quality analyses of NERICA grains showed that 7 NERICA varieties were clearly classified into two groups based on the difference of amylose content, and the difference influenced the pasting and physical properties of grains. Genetic analysis of the gene encoding granule-bound starch synthase I (GBSSI), which is known as a key enzyme on amylose synthesis in rice grain, revealed that varieties with higher amylose content ( approximately 29%) have the gene derived from O. glaberrima parent, and group 2 with lower amylose content ( approximately 22%) have the gene from O. sativa parent. These results indicated that the difference in amylose content as well as grain properties among 7 NERICA varieties is mainly determined by the genetic derivation of GBSSI. Further genetic analysis of starch synthesis-related genes suggested that the genetic derivation of SSIIa also influences the chain length of amylopectin in 7 NERICA varieties.

Differential use of two cyclic electron flows around photosystem I for driving CO2-concentration mechanism in C4 photosynthesis.

Whereas linear electron flow (LEF) in photosynthesis produces both ATP and NADPH, the cyclic electron flow (CEF) around photosystem I has been shown to produce only ATP. Two alternative routes have been shown for CEF; NAD(P)H dehydrogenase (NDH)- and ferredoxin:plastoquinone oxidoreductase (FQR)-dependent flows, but their physiological relevance has not been elucidated in detail. Meanwhile, because C(4) photosynthesis requires more ATP than does C(3) photosynthesis to concentrate CO(2), it has not been clear how the extra ATP is produced. In this study, to elucidate whether CEF contributes to the additional ATP needed in C(4) photosynthesis, we estimated the amounts of PGR5, which participates in FQR-dependent flow, and NDH-H, a subunit of NDH, in four C(4) species. Although the expression profiles of PGR5 did not correlate well with the additional ATP requirement, NDH was greatly expressed in mesophyll cells in the NAD-malic enzyme (ME) species, and in bundle-sheath cells in NADP-ME species, where there is a strong need for ATP in the respective cells. Our results indicate that CEF via NDH plays a central role in driving the CO(2)-concentrating mechanism in C(4) photosynthesis.

Ribosomal RNA processing and an RNase R family member in chloroplasts of Arabidopsis.

An Arabidopsis mutant rnr1 , which has a defect in the basic genetic system in chloroplasts, was isolated using the screening of the high chlorophyll fluorescence phenotype. Whereas chlorophyll fluorescence and immunoblot studies showed the mutant had reduced activities of photosystems I and II, molecular characterization of the mutant suggested that a T-DNA insertion impaired the expression of a gene encoding a RNase R family member with a targeting signal to chloroplasts. Since RNase R family members have a 3'-5' exoribonuclease activity, we examined the RNA profile in chloroplasts. In rnr1 the intercistronic cleavage between 23S and 4.5S rRNA was impaired, and a significant reduction in rRNA in chloroplasts was found, suggesting that RNR1 functions in the maturation of chloroplast rRNA. The present results suggest that defects in the genetic system in chloroplasts cause high chlorophyll fluorescence, pale green leaf, and marked reduction in the growth rate, whereas the levels of some chloroplast RNA were higher in rnr1 than in the wild-type.