Development of a certified genomic DNA reference material for detection and quantification of genetically modified rice KMD.

Qualitative and quantitative detection of genetically modified products is inseparable from the application of reference materials (RMs). In this study, a batch of genomic DNA (gDNA) certified reference materials (CRMs) was developed using genetically modified rice Kemingdao (KMD) homozygotes as the raw material. The gDNA CRMs in this batch showed good homogeneity; the minimum sample intake was determined to be 2 µL. The stability study showed that transportation by cold chain is preferable, no significant degradation trend was observed during a 12-month period when storing the gDNA CRMs at 4 °C and - 20 °C, and the number of freeze-thaw cycles cannot exceed 10. The property values of the copy number ratio of transgene and endogenous gene and the copy number concentration for gDNA CRMs were determined by a collaborative characterization of eight laboratories using the duplex KMD/PLD droplet digital PCR (ddPCR) assays. The uncertainty components of characterization, potential between-unit heterogeneity, and potential degradation during long-term storage were combined to estimate the expanded uncertainty of the certified value with a coverage factor k of 2.0. The certified value of copy number ratio for KMD gDNA CRM is 0.99 ± 0.05, and that of copy number concentration is (1.76 ± 0.10) × 105 copies/µL. Compared to the gDNA CRMs in availability, this batch of KMD gDNA CRMs is assigned accurate property values and can be directly used for qualitative and quantitative detection of GMOs as well as evaluation of the parameters of analytical methods with no need of further DNA concentration measurement. Graphical abstract.

Assessment of genetic purity of bulked-seed of rice CMS lines using capillary electrophoresis.

Genetically pure cytoplasmic male sterile line (A-line) is essential to generate pure hybrid seeds in order to harness the yield heterosis in rice. Conventionally, seed purity testing is carried by grow-out test, which has many limitations. Seed purity assessments based on molecular markers reduce the time required for analysis significantly. However, it is very tedious as at least 200-400 seeds/seedlings are needed to be analyzed individually. An assay based on bulked-seed and molecular markers will be an ideal system. Keeping these points in view, in the present study, a co-dominant mitochondrial marker was used to test the purity of bulked parental line (A-line) seed utilizing CE system in a genetic analyzer. The results indicate that this method is very simple, accurate, and can be used to test purity of large number of samples rapidly in a cost-effective way compared to grow-out test and conventional molecular marker analysis.

Towards metrologically traceable and comparable results in GM quantification.

The GM content in a food or feed product produced from or containing genetically modified organisms (GMO) has to be expressed in Europe in the form of a GM mass fraction. However, the most widely used quantification methods, based on PCR, are basically counting PCR-amplifiable DNA fragments in a sample extract. This paper outlines the requirements for obtaining comparable measurement results which are fit for regulatory decision-making. It introduces the concept of a reference measurement system which enables GMO analysis laboratories to relate their results to a universally accessible reference, thus establishing metrological traceability to a unique reference point. The conversion factors required for transforming data from one measurement unit into the other have to carry a minimum uncertainty and are anchored to specified certified reference materials. The establishment of such conversion factors and related calibration approaches to achieve comparable GM quantification results are sketched. Graphical abstract ᅟ.

Fast and inexpensive protocols for consistent extraction of high quality DNA and RNA from challenging plant and fungal samples for high-throughput SNP genotyping and sequencing applications.

Modern genotyping techniques, such as SNP analysis and genotyping by sequencing (GBS), are hampered by poor DNA quality and purity, particularly in challenging plant species, rich in secondary metabolites. We therefore investigated the utility of a pre-wash step using a buffered sorbitol solution, prior to DNA extraction using a high salt CTAB extraction protocol, in a high throughput or miniprep setting. This pre-wash appears to remove interfering metabolites, such as polyphenols and polysaccharides, from tissue macerates. We also investigated the adaptability of the sorbitol pre-wash for RNA extraction using a lithium chloride-based protocol. The method was successfully applied to a variety of tissues, including leaf, cambium and fruit of diverse plant species including annual crops, forest and fruit trees, herbarium leaf material and lyophilized fungal mycelium. We consistently obtained good yields of high purity DNA or RNA in all species tested. The protocol has been validated for thousands of DNA samples by generating high data quality in dense SNP arrays. DNA extracted from Eucalyptus spp. leaf and cambium as well as mycelium from Trichoderma spp. was readily digested with restriction enzymes and performed consistently in AFLP assays. Scaled-up DNA extractions were also suitable for long read sequencing. Successful RNA quality control and good RNA-Seq data for Eucalyptus and cashew confirms the effectiveness of the sorbitol buffer pre-wash for high quality RNA extraction.

An optimized protocol for DNA extraction in plants with a high content of secondary metabolites, based on leaves of Mimosa tenuiflora (Willd.) Poir. (Leguminosae).

Some species are characterized by a high content of tannins, alkaloids, and phenols in their leaves. These secondary metabolites are released during DNA extraction and might hinder molecular studies based on PCR (polymerase chain reaction). To provide an efficient method to extract DNA, Mimosa tenuiflora, an important leguminous plant from Brazilian semiarid region used in popular medicine and as a source of fuelwood or forage, was used. Eight procedures previously reported for plants were tested and adapted from leaf tissues of M. tenuiflora stored at -20°C. The optimized procedure in this study encompassed the utilization of phenol during deproteinization, increased concentrations of cetyltrimethylammonium bromide and sodium chloride, and a shorter period and lower temperature of incubation concerning other methods. The extracted DNA did not present degradation, and amplification via PCR was successful using ISSR, trnL, ITS, and ETS primers. Besides M. tenuiflora, this procedure was also tested and proved to be efficient in genetic studies of other plant species.

Selection of suitable endogenous reference genes for relative copy number detection in sugarcane.

Transgene copy number has a great impact on the expression level and stability of exogenous gene in transgenic plants. Proper selection of endogenous reference genes is necessary for detection of genetic components in genetically modification (GM) crops by quantitative real-time PCR (qPCR) or by qualitative PCR approach, especially in sugarcane with polyploid and aneuploid genomic structure. qPCR technique has been widely accepted as an accurate, time-saving method on determination of copy numbers in transgenic plants and on detection of genetically modified plants to meet the regulatory and legislative requirement. In this study, to find a suitable endogenous reference gene and its real-time PCR assay for sugarcane (Saccharum spp. hybrids) DNA content quantification, we evaluated a set of potential "single copy" genes including P4H, APRT, ENOL, CYC, TST and PRR, through qualitative PCR and absolute quantitative PCR. Based on copy number comparisons among different sugarcane genotypes, including five S. officinarum, one S. spontaneum and two S. spp. hybrids, these endogenous genes fell into three groups: ENOL-3--high copy number group, TST-1 and PRR-1--medium copy number group, P4H-1, APRT-2 and CYC-2--low copy number group. Among these tested genes, P4H, APRT and CYC were the most stable, while ENOL and TST were the least stable across different sugarcane genotypes. Therefore, three primer pairs of P4H-3, APRT-2 and CYC-2 were then selected as the suitable reference gene primer pairs for sugarcane. The test of multi-target reference genes revealed that the APRT gene was a specific amplicon, suggesting this gene is the most suitable to be used as an endogenous reference target for sugarcane DNA content quantification. These results should be helpful for establishing accurate and reliable qualitative and quantitative PCR analysis of GM sugarcane.

C-value reassessment of plant standards: an image cytometry approach.

Image cytometry (ICM) has been used to measure DNA 2C-values by evaluating the optical density of Feulgen-stained nuclei. This optical measurement is carried out using three basic tools: microscopy, digital video camera, and image analysis software. Because ICM has been applied to plants, some authors have remarked that studies should be performed before this technique can be accepted as an accurate method for determination of plant genome size. Based on this, the 2C-value of eight plants, which are widely used as standards in DNA quantifications, was reassessed in a cascade-like manner, from A. thaliana through R. sativus, S. lycopersicum, Glycine max, Z. mays, P. sativum, V. faba, to A. cepa. The mean 2C-values of all plants were statistically compared to the values reported by other authors using flow cytometry and/or ICM. These analyses demonstrated that ICM is an accurate and reliable method for 2C-value measurement, representing an attractive alternative to flow cytometry. Statistical comparison of the results also indicated Glycine max 'Polanka' as the most adequate primary standard. However, distinct authors have been advised that 2C DNA content of the reference standard should be close to that of the sample. As three further approaches also revisited the 2C-value of these eight plants, we have thus proposed a mean 2C-value for each eight species.

Nuclear genome size: are we getting closer?

Correct information on genome size is important in many areas of research. For a long time, scientists have been struggling to understand the reason for the huge variation in eukaryotic genome size and its biological significance. More recently, the knowledge on genome size has become important to structure genome sequencing projects as their scale and cost depend on genome size. Despite the fact that the first estimates of genome size in eukaryotes were made more than 50 years ago, we are still not quite sure about the exact genome size in practically all animal and plant species. Moreover, different estimates continue to be published for the same species. These discrepancies compromise data comparison and interpretation and point to methodological problems, which include standardization. This article assesses the current state of DNA reference standards for flow cytometry and the issues related to their calibration.

Evaluation of modified PCR quantitation of genetically modified maize and soybean using reference molecules: interlaboratory study.

Real-time polymerase chain reaction (PCR)-based quantitative methods were previously developed and validated for genetically modified (GM) maize or soy. In this study, the quantification step of the validated methods was modified, and an interlaboratory study was conducted. The modification included the introduction of the PCR system SSIIb 3 instead of SSIIb 1 for the detection of the taxon-specific sequence of maize, as well as the adoption of colE1 as a carrier included in a reference plasmid solution as a replacement for salmon testis. The interlaboratory study was conducted with the ABI PRISM 7700 and consisted of 2 separate stages: (1) the measurement of conversion factor (Cf) value, which is the ratio of recombinant DNA (r-DNA) sequence to taxon-specific sequence in each genuine GM seed, and (2) the quantification of blind samples. Additionally, Cf values of other instruments, such as the ABI PRISM 7900 and the ABI PRISM 7000, were measured in a multilaboratory trial. After outlier laboratories were eliminated, the repeatability and reproducibility for 5.0% samples were <15.8 and 20.6%, respectively. The quantitation limits of these methods were 0.5% for Bt11, T25, and MON810, and 0.1% for GA21, Event176, and RR soy. The quantitation limits, trueness, and precision of the current modified methods were equivalent to those of the previous methods. Therefore, it was concluded that the modified methods would be a suitable replacement for the validated methods.

DNA internal standard for the quantitative determination of hallucinogenic plants in plant mixtures.

Here, we show a new, simple, and rapid SYBR Green-based Real-Time PCR assay for the quantification of hallucinogenic plants in plant mixtures. As a test plant, Salvia divinorum Epling & Játiva-M., a perennial herb belonging to the Lamiaceae family able to induce hallucinations, changes in perception, or other psychologically induced changes with similar potency as LSD, was used. The method was tested on seven mixtures 100/0%, 80/20%, 60/40%, 40/60%, 20/80%, 10/90%, 0/100% (w/w) S. divinorum versus a non-hallucinogenic plant, Salvia officinalis. Total DNA was extracted from samples and quantified by Real-Time PCR. Arabidopsis thaliana genomic DNA was added, as internal standard, at the beginning of each extraction. A new formula for the interpretation of Real-Time PCR data, based on the relative quantification of DNA extracted from mixture versus a reference DNA extracted from a known amount of pure S. divinorum, was developed. The results of this work show an almost perfect correspondence between Real-Time PCR-calculated weight and the weight estimated by an analytical weighted method, proving the effectiveness of this method for the quantitative analysis of a given species in a plant mixture.

Selection of international molecular standards for DNA fingerprinting of Theobroma cacao.

A collaborative international program was initiated to identify and describe the genetic diversity of living germplasm collections of Theobroma cacao genotypes that are maintained in several international collections scattered throughout tropical cacao growing countries of the world. Simple sequence repeat (SSR) DNA analysis was identified as the most appropriate molecular tool for DNA fingerprinting these collections during an international forum representing academic, government and industry scientists in the cacao community. Twenty-five SSR primers, which had been previously described, were evaluated as potential candidates to define an efficient, standardized, molecular fingerprinting protocol for T. cacao accessions. These primers have been evaluated for reliability, widespread distribution across the cacao genome, number of alleles produced by the SSR primers in cacao and their ability to discriminate between cacao accessions. Approximately 690 cacao accessions were used to evaluate the utility of these SSR primers as international molecular standards, and a small number of test samples of T. cacao were sent to two other independent laboratories for verification. DNA fragments were selectively amplified by PCR, using the SSR primers labeled with fluorescent dyes, and separated by capillary electrophoresis. Based on this study, the 15 SSR primers that had the highest reproducibility and consistency within a common genotype, while allowing the differentiation of separate divergent genotypes, were selected as international molecular standards for DNA fingerprinting of T. cacao.