Development and collaborative validation of an event-specific quantitative real-time PCR method for detection of genetically modified CC-2 maize.

As one of the developed genetically modified (GM) maize varieties in China, CC-2 has demonstrated promising commercial prospects during demonstration planting. The establishment of detection methods is a technical prerequisite for effective supervision and regulation of CC-2 maize. In this study, we have developed an event-specific quantification method that targets the junction region between the exogenous gene and the 5' flanking genomic DNA (gDNA) of CC-2. The accuracy and precision of this method were evaluated across high, medium, and low levels of CC-2 maize content, revealing biases within ±25% and satisfactory precision data. Additionally, we determined the limits of quantification of the method to be 0.05% (equivalent to 20 copies) of the CC-2 maize. A collaborative trial further confirmed that our event-specific method for detecting CC-2 produces reliable, comparable, and reproducible results when applied to five different samples provided by various sources. Furthermore, we calculated the expanded uncertainty associated with determining the content level of CC-2 in these samples.

The CRISPR/Cas System: A Customizable Toolbox for Molecular Detection.

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) are promising molecular diagnostic tools for rapidly and precisely elucidating the structure and function of genomes due to their high specificity, programmability, and multi-system compatibility in nucleic acid recognition. Multiple parameters limit the ability of a CRISPR/Cas system to detect DNA or RNA. Consequently, it must be used in conjunction with other nucleic acid amplification techniques or signal detection techniques, and the reaction components and reaction conditions should be modified and optimized to maximize the detection performance of the CRISPR/Cas system against various targets. As the field continues to develop, CRISPR/Cas systems have the potential to become an ultra-sensitive, convenient, and accurate biosensing platform for the detection of specific target sequences. The design of a molecular detection platform employing the CRISPR/Cas system is asserted on three primary strategies: (1) Performance optimization of the CRISPR/Cas system; (2) enhancement of the detection signal and its interpretation; and (3) compatibility with multiple reaction systems. This article focuses on the molecular characteristics and application value of the CRISPR/Cas system and reviews recent research progress and development direction from the perspectives of principle, performance, and method development challenges to provide a theoretical foundation for the development and application of the CRISPR/CAS system in molecular detection technology.

Establishment and Validation of Reference Genes of Brassica napus L. for Digital PCR Detection of Genetically Modified Canola.

As an effective tool for genetically modified organism (GMO) quantification in complex matrices, digital PCR (dPCR) has been widely used for the quantification of genetically modified (GM) canola events; however, little is known about the quantification of GM canola events using endogenous reference gene (ERG) characteristics by dPCR. To calculate and quantify the content of GM canola using endogenous reference gene (ERG) characteristics, the suitability of several ERGs of canola, such as cruciferin A (CruA), acetyl-CoA carboxylase (BnAcc), phosphoenolpyruvate carboxylase (PEP), cruciferin storage (BnC1), oleoyl hydrolase (Fat(A)), and high-mobility-group protein I/Y (HMG-I/Y), was investigated by droplet dPCR. BnAcc and BnC1 were more specific and stable in copy number in the genome of Brassica napus L. than the other genes. By performing intra-laboratory validation of the suitability of ERG characteristics for the quantification of GM canola events, the ddPCR methods for BnAcc and BnC1 were comprehensively demonstrated in dPCR assays. The methods could provide technical support for GM labeling regulations.

Establishment and Application of Ligation Reaction-Based Method for Quantifying MicroR-156b.

Microribonucleic acids (miRNAs) play significant roles in the regulation of biological processes and in responses to biotic or abiotic environmental stresses. Therefore, it is necessary to quantitatively detect miRNAs to understand these complicated biological regulation mechanisms. This study established an ultrasensitive and highly specific method for the quantitative detection of miRNAs using simple operations on the ground of the ligation reaction of ribonucleotide-modified deoxyribonucleic acid (DNA) probes. This method avoids the complex design of conventional reverse transcription. In the developed assay, the target miRNA miR156b was able to directly hybridize the two ribonucleotide-modified DNA probes, and amplification with universal primers was achieved following the ligation reaction. As a result, the target miRNA could be sensitively measured even at a detection limit as low as 0.0001 amol, and differences of only a single base could be detected between miR156 family members. Moreover, the proposed quantitative method demonstrated satisfactory results for overexpression-based genetically modified (GM) soybean. Ligation-based quantitative polymerase chain reaction (PCR) therefore has potential in investigating the biological functions of miRNAs, as well as in supervising activities regarding GM products or organisms.

Event-specific quantitative polymerase chain reaction methods for detection of double-herbicide-resistant genetically modified corn MON 87419 based on the 3'-junction of the insertion site.

MON 87419 was one of the new transgenic corn events developed in US with the trait of herbicide resistance to both dicamba and glyphosate. To monitor unintended release of genetically modified organism in the future, as well as to meet GM-labeling requirements, it is requisite to develop a reliable method for the detection and quantification of MON 87419, an event-specific primer pair was designed to amplify the 3'-junction site between the endogenous genome sequence and the transferred DNA of GM event MON 87419, amplicons of desired size were produced by qualitative polymerase chain reaction (PCR) assay. For the validation of this quantitative method, the mixed samples containing 10%, 1%, and 0.1% MON 87419 ingredient were quantified. The precisions were expressed as relative standard deviations, deviated by 7.87%, 12.94%, and 19.98%, respectively. These results clearly demonstrate that the PCR methods we developed herein can be used for event-specific quantitative testing of the double-herbicide-resistant corn MON 87419.

Development and validation of a 48-target analytical method for high-throughput monitoring of genetically modified organisms.

The rapid increase in the number of genetically modified (GM) varieties has led to a demand for high-throughput methods to detect genetically modified organisms (GMOs). We describe a new dynamic array-based high throughput method to simultaneously detect 48 targets in 48 samples on a Fludigm system. The test targets included species-specific genes, common screening elements, most of the Chinese-approved GM events, and several unapproved events. The 48 TaqMan assays successfully amplified products from both single-event samples and complex samples with a GMO DNA amount of 0.05 ng, and displayed high specificity. To improve the sensitivity of detection, a preamplification step for 48 pooled targets was added to enrich the amount of template before performing dynamic chip assays. This dynamic chip-based method allowed the synchronous high-throughput detection of multiple targets in multiple samples. Thus, it represents an efficient, qualitative method for GMO multi-detection.

Development and application of loop-mediated isothermal amplification assays for rapid visual detection of cry2Ab and cry3A genes in genetically-modified crops.

The cry2Ab and cry3A genes are two of the most important insect-resistant exogenous genes and had been widely used in genetically-modified crops. To develop more effective alternatives for the quick identification of genetically-modified organisms (GMOs) containing these genes, a rapid and visual loop-mediated isothermal amplification (LAMP) method to detect the cry2Ab and cry3A genes is described in this study. The LAMP assay can be finished within 60 min at an isothermal condition of 63 °C. The derived LAMP products can be obtained by a real-time turbidimeter via monitoring the white turbidity or directly observed by the naked eye through adding SYBR Green I dye. The specificity of the LAMP assay was determined by analyzing thirteen insect-resistant genetically-modified (GM) crop events with different Bt genes. Furthermore, the sensitivity of the LAMP assay was evaluated by diluting the template genomic DNA. Results showed that the limit of detection of the established LAMP assays was approximately five copies of haploid genomic DNA, about five-fold greater than that of conventional PCR assays. All of the results indicated that this established rapid and visual LAMP assay was quick, accurate and cost effective, with high specificity and sensitivity. In addition, this method does not need specific expensive instruments or facilities, which can provide a simpler and quicker approach to detecting the cry2Ab and cry3A genes in GM crops, especially for on-site, large-scale test purposes in the field.

Dramatic genotypic difference in, and effect of genetic crossing on, tissue culture-induced mobility of retrotransposon Tos17 in rice.

KEY MESSAGE : We show for the first time that intraspecific crossing may impact mobility of the prominent endogenous retrotransposon Tos17 under tissue culture conditions in rice. Tos17, an endogenous copia retrotransposon of rice, is transpositionally active in tissue culture. To study whether there exists fundamental genotypic difference in the tissue culture-induced mobility of Tos17, and if so, whether the difference is under genetic and/or epigenetic control, we conducted this investigation. We show that dramatic difference in tissue culture-induced Tos17 mobility exists among different rice pure-line cultivars sharing the same maternal parent: of the three lines studied that harbor Tos17, two showed mobilization of Tos17, which accrued in proportion to subculture duration, while the third line showed total quiescence (immobility) of the element and the fourth line did not contain the element. In reciprocal F1 hybrids between Tos17-mobile and -immobile (or absence) parental lines, immobility was dominant over mobility. In reciprocal F1 hybrids between both Tos17-mobile parental lines, an additive or synergistic effect on mobility of the element was noticed. In both types of reciprocal F1 hybrids, clear difference in the extent of Tos17 mobility was noted between crossing directions. Given that all lines share the same maternal parent, this observation indicates the existence of epigenetic parent-of-origin effect. We conclude that the tissue culture-induced mobility of Tos17 in rice is under complex genetic and epigenetic control, which can be either enhanced or repressed by intraspecific genetic crossing.

Spaceflight-induced genetic and epigenetic changes in the rice (Oryza sativa L.) genome are independent of each other.

An array of studies have reported that the spaceflight environment is mutagenic and may induce phenotypic and genetic changes in diverse organisms. We reported recently that in at least some plant species (e.g., rice) the spaceflight environment can be particularly potent in generating heritable epigenetic changes in the form of altered cytosine methylation patterns and activation of transposable elements. To further study the issue of spaceflight-induced genomic instability, and in particular to test whether the incurred genetic and epigenetic changes are connected or independent of each other, we performed the present study. We subjected seeds of the standard laboratory rice (Oryza sativa L.) cultivar Nipponbare to a spaceflight in the spaceship Long March 2 for 18 days. We then investigated the genetic and DNA methylation stabilities of 11 randomly selected plants germinated from the spaceflown seeds by using two kinds of DNA markers, amplified fragment length polymorphism (AFLP) and methylation sensitive amplified polymorphism (MSAP). For AFLP, by using 15 primer combinations, we assessed 460 genomic loci and found that the frequencies of genetic changes across the 11 plants ranged from 0.7% to 6.7% with an average frequency of 3.5%. For MSAP, by using 14 primer combinations, we assessed 467 loci and detected the occurrence of four major types of cytosine methylation alterations at the CCGG sites, namely CG or CNG hypomethylation and CG or CNG hypermethylation. Collectively, the frequencies of the two kinds of hypermethylation, CG (1.95%) and CNG (1.44%), are about two times higher than those of the two kinds of hypomethylation, CG (0.76%) and CNG (0.80%), though different plants showed variable frequencies for each type of alteration. Further analysis suggested that both the genetic and cytosine methylation changes manifested apparent mutational bias towards specific genomic regions, but the two kinds of instabilities are independent of each other based on correlation analysis.

The spaceflight environment can induce transpositional activation of multiple endogenous transposable elements in a genotype-dependent manner in rice.

Spaceflight represents a unique environmental condition whereby dysregulated gene expression and genomic instability can be provoked. However, detailed molecular characterization of the nature of genetic changes induced by spaceflight is yet to be documented in a higher eukaryote. Transposable elements (TEs) are ubiquitous and have played a significant role in genome evolution. Mounting evidence indicates that TEs constitute the genomic fraction that is susceptible and responsive to environmental perturbations, and hence, most likely manifesting genetic instabilities in times of stress. A predominant means for TEs to cause genetic instability is via their transpositional activation. Here we show that spaceflight has induced transposition of several endogenous TEs in rice, which belong to distinct classes including the miniature inverted terminal repeat TEs (MITEs) and long-terminal repeat (LTR) retrotransposons. Of three rice lines studied, transposition of TEs were detected in the plants germinated from space-flown dry seeds of two lines (RZ1 and RZ35), which are genetically homogeneous and stabilized recombinant inbred lines (RILs) derived from a pure-line rice cultivar, Matsumae. In contrast, the TEs remained immobile in plants derived from space-flown seeds of Matsumae itself, indicating a genotype-dependent manner of TE transposition under the spaceflight environment. Further examination showed that at least in some cases transposition of TEs was associated with cytosine demethylation within the elements. Moreover, the spaceflight-induced TE activity was heritable to organismal progenies. Thus, our results implicate that the spaceflight environment represents a potent mutagenic environment that can cause genetic instabilities by eliciting transposition of otherwise totally quiescent endogenous TEs in a higher eukaryote.

Spaceflight induces both transient and heritable alterations in DNA methylation and gene expression in rice (Oryza sativa L.).

Spaceflight represents a complex environmental condition in which several interacting factors such as cosmic radiation, microgravity and space magnetic fields are involved, which may provoke stress responses and jeopardize genome integrity. Given the inherent property of epigenetic modifications to respond to intrinsic as well as external perturbations, it is conceivable that epigenetic markers like DNA methylation may undergo alterations in response to spaceflight. We report here that extensive alteration in both DNA methylation and gene expression occurred in rice plants subjected to a spaceflight, as revealed by a set of characterized sequences including 6 transposable elements (TEs) and 11 cellular genes. We found that several features characterize the alterations: (1) All detected alterations are hypermethylation events; (2) whereas alteration in both CG and CNG methylation occurred in the TEs, only alteration in CNG methylation occurred in the cellular genes; (3) alteration in expression includes both up- and down-regulations, which did not show a general correlation with alteration in methylation; (4) altered methylation patterns in both TEs and cellular genes are heritable to progenies at variable frequencies; however, stochastic reversion to wild-type patterns and further de novo changes in progenies are also apparent; and (5) the altered expression states in both TEs and cellular genes are also heritable to selfed progenies but with markedly lower transmission frequencies than altered DNA methylation states. Furthermore, we found that a set of genes encoding for the various putative DNA methyltransferases, 5-methylcytosine DNA glycosylases, the SWI/SNF chromatin remodeller (DDM1) and siRNA-related proteins are extremely sensitive to perturbation by spaceflight, which might be an underlying cause for the altered methylation patterns in the space-flown plants. We discuss implications of spaceflight-induced epigenetic variations with regard to health safety issues of spaceship crews and potentiality of spaceflight as a means for mutagenesis in crop breeding.

In planta mobilization of mPing and its putative autonomous element Pong in rice by hydrostatic pressurization.

The miniature Ping (mPing) is a recently discovered endogenous miniature inverted repeat transposable element (MITE) in rice, which can be mobilized by tissue culture or irradiation. It is reported here that mPing, together with one of its putative transposase-encoding partners, Pong, was efficiently mobilized in somatic cells of intact rice plants of two distinct cultivars derived from germinating seeds subjected to high hydrostatic pressure, whereas the other autonomous element of mPing, Ping, remained static in the plants studied. mPing excision was detected in several plants of both cultivars in the treated generation (P0), which were selected based on their novel phenotypes. Southern blot analysis and transposon-display assay on selfed progenies (P1 generation) of two selected P0 plants, one from each of the cultivars, revealed polymorphic banding patterns consistent with mobilization of mPing and Pong. Various mPing excisions and de novo insertions, as detected by element-bracketing, locus-specific PCR assays, occurred in the different P1 plants of both cultivars. Pong excision at one locus for each cultivar was also detected by using a Pong internal primer together with locus-specific flanking primers in the P1 plants. In contrast to the pressurized plants, immobility of both mPing and Pong in control plants, and the absence of within-cultivar heterozygosity at the analysed loci were verified by Southern blotting and/or locus-assay. Sequencing at 18 mPing empty donor sites isolated from the pressurized plants indicated properties characteristic of the element excision. Sequence-based mapping of 10 identified mPing de novo insertions from P1 progenies of pressurized plants indicated that all were in unique or low-copy regions, conforming with the targeting propensity of mPing. No evidence for further mPing activity was detected in the P2 plants tested. In spite of the high activity of mPing and Pong in the pressurized plants, amplified fragment length polymorphism (AFLP) analysis denoted their general genomic stability, and several potentially active retrotransposons also remained largely immobile. Further investigation showed that the same hydrostatic pressure treatments also caused mobilization of mPing in the standard laboratory cultivar for japonica rice, Nipponbare. Thus, a simple and robust approach for in planta MITE-mobilization in rice has been established by using high hydrostatic pressure treatment, which may be useful as an alternative for gene-tagging in this important crop plant.

Alterations in DNA methylation and genome structure in two rice mutant lines induced by high pressure.

By using high-pressure treatment, two mutant lines were obtained from a genetically stable japonica rice cultivar Bijing38. Genomic DNA of the mutant lines, together with the original line (Bijing38), was either undigested or digested by Hpa IIMsp I, and then subjected to molecular analysis using two markers, ISSR and RAPD. Results indicated that changes in the PCR amplification profiles of both markers are apparent in the two mutant lines compared with the original rice cultivar, suggesting that there had been both sequence changes and DNA methylation modifications in the mutant lines. Southern blot analysis using diverse sequences, including two cellular genes (S2 and S3), a set of retrotransposons (Osr7, Osr36, Tos19 and more), and a MITE transposon family (mPing and Pong), confirmed the results, and indicated that changes in DNA methylation pattern, genomic structure, and possible activation of some transposons indeed occurred in the mutant lines. Moreover, these changes are stably maintained through selfed generations and in different organs. Thus, our results indicate that it is possible to obtain stable mutants in rice by high pressure treatments, and the molecular basis of the mutants may include both genetic and epigenetic changes. Therefore, high hydrostatic pressure seems a promising approach for plant mutagenesis.

Isolation and characterization of a set of disease resistance-gene analogs (RGAs) from wild rice, Zizania latifolia Griseb. I. Introgression, copy number lability, sequence change, and DNA methylation alteration in several rice-Zizania introgression lines.

Eight resistance-gene analogs (RGAs) were isolated from wild rice, Zizania latifolia Griseb., by degenerate primers designed according to conserved motifs at or around the nucleotide-binding site (NBS) of known NBS-containing plant resistance genes. The 8 RGAs were classified into 6 distinct groups based on their deduced amino acid sequence similarity of 60% or greater. Gel-blot hybridization of each of the RGAs to 4 rice - Z. latifolia intro gression lines indicated an array of changes at either introgressed Zizania RGAs or, more likely, their rice homologs. The changes included dramatic increase in copy number, modification at the primary DNA sequence, and alteration in DNA methylation patterns.

Heritable alteration in DNA methylation pattern occurred specifically at mobile elements in rice plants following hydrostatic pressurization.

Intrinsic DNA methylation pattern is an integral component of the epigenetic network in many eukaryotes. Exploring the extent to which DNA methylation patterns can be altered under a specific condition is important for elucidating the biological functions of this epigenetic modification. This is of added significance in plants wherein the newly acquired methylation patterns can be inherited through organismal generations. We report here that DNA methylation patterns of mobile elements but not of cellular genes were specifically altered in rice plants following hydrostatic pressurization. This was evidenced by methylation-sensitive gel-blot analysis, which showed that 10 out of 10 studied low-copy transposons and retrotransposons manifested methylation alteration in at least one of the 8 randomly chosen pressure-treated plants, whereas none of the 16 studied low-copy cellular genes showed any change. Both gel-blotting and genome-wide fingerprinting indicated that the methylation alteration in mobile elements was not accompanied by a general genetic instability. Progeny analysis indicated retention of the altered methylation patterns in most progeny plants, underscoring early occurrence of the alterations, and their faithful epigenetic inheritance.

Mobilization of the active MITE transposons mPing and Pong in rice by introgression from wild rice (Zizania latifolia Griseb.).

Hybridization between different species plays an important role in plant genome evolution, as well as is a widely used approach for crop improvement. McClintock has predicted that plant wide hybridization constitutes a "genomic shock" whereby cryptic transposable elements may be activated. However, direct experimental evidence showing a causal relationship between plant wide hybridization and transposon mobilization has not yet been reported. The miniature-Ping (mPing) is a recently isolated active miniature inverted-repeat transposable element transposon from rice, which is mobilized by tissue culture and gamma-ray irradiation. We show herein that mPing, together with its putative transposase-encoding partner, Pong, is mobilized in three homologous recombinant inbred lines (RILs), derived from hybridization between rice (cultivar Matsumae) and wild rice (Zizania latifolia Griseb.), harboring introgressed genomic DNA from wild rice. In contrast, both elements remain immobile in two lines sharing the same parentage to the RILs but possessing no introgressed DNA. Thus, we have presented direct evidence that is consistent with McClintock's insight by demonstrating a causal link between wide hybridization and transposon mobilization in rice. In addition, we report an atypical behavior of mPing/Pong mobilization in these lines, i.e., the exclusive absence of footprints after excision.