A Cytosolic Triosephosphate Isomerase Is a Key Component in XA3/XA26-Mediated Resistance.

Bacterial blight caused by Xanthomonas oryzae pv oryzae (Xoo) causes severe damage to rice (Oryza sativa) production worldwide. The major disease resistance gene, Xa3/Xa26, confers broad-spectrum and durable resistance to Xoo at both seedling and adult stages. However, the molecular mechanism of the Xa3/Xa26-initiated defense pathway against Xoo is still largely unknown. Here, we show that a triosephosphate isomerase (TPI), OsTPI1.1, is a key component in XA3/XA26-mediated resistance to Xoo OsTPI1.1 is a glycolytic enzyme that catalyzes the reversible interconversion of dihydroxyacetone phosphate to glyceraldehyde-3-phosphate. Transcriptional suppression of OsTPI1.1 in plants harboring Xa3/Xa26 largely impaired the XA3/XA26-mediated resistance to Xoo, and constitutive overexpression of OsTPI1.1 in susceptible rice plants without Xa3/Xa26 only slightly decreased the susceptibility to Xoo Therefore, both XA3/XA26 and OsTPI1.1 are required in XA3/XA26-mediated resistance. We show that OsTPI1.1 participates in the resistance through its enzymatic activity, which was enhanced significantly by its binding with XA3/XA26. Reactive oxygen species (ROS), especially hydrogen peroxide, accumulated in the OsTPI1.1-overexpressing plants, and suppression of OsTPI1.1 decreased ROS accumulation. The changes in ROS are associated with the reduction of NADP+ to NADPH, which may act as a redox cofactor to scavenge ROS, leading to reduced resistance to Xoo These results suggest that OsTPI1.1 modulates ROS production as a resistance mechanism against Xoo.

Successful detection of foreign inserts in transgenic rice TT51-1 (BT63) by RNA-sequencing combined with PCR.

BACKGROUND: As event-specific sequence information for most unauthorised genetically modified organisms (GMOs) is currently still unavailable, detecting unauthorised GMOs remains challenging. Here, we used insect-resistant rice TT51-1 as an example to develop a novel approach via detecting GMOs by RNA-seq (sequencing) and PCR. RNA-seq of TT51-1 generated 4.8 million (M) 21-nt cDNA tags. Alignment to the Oryza sativa subsp. japonica reference genome revealed 24 098 unmapped tags. Foreign tags from the nopaline synthetic enzyme gene (NOS) terminator and insect-resistant genes were then identified by searching against the NCBI VecScreen and NT databases. RESULTS: To further isolate foreign DNA sequences, putative NOS terminator and insect-resistant gene tags were combined and used directly as primer pairs for long-range PCR, producing a 5016-bp fragment. The inserted DNA sequence of TT51-1 has been submitted to a database, and thus, similarity analysis using the database could identify a test sample. CONCLUSION: The novel approach has a great potential for application to the detection and identification of unauthorised GMOs in food and feed products. © 2016 Society of Chemical Industry.

Comparison of three common DNA concentration measurement methods.

Accurate measurement of DNA concentration is important for DNA-based biological applications. DNA concentration is usually determined by the ultraviolet (UV) absorption, fluorescence staining, and diphenylamine reaction methods. However, the best method for quality assurance of measurements is unknown. Here, we comprehensively compared these methods using different types of samples. We found that all three methods accurately determined the concentrations of high-purity DNA solutions. After digestion of DNA samples, concentration measurements revealed that the PicoGreen dye method was very sensitive to the degradation of DNA. The three methods displayed different anti-jamming ability when contaminants such as transfer RNA (tRNA), protein, and organic chemicals were included in DNA solutions. The diphenylamine reaction method gave the highest accuracy, with an average error of approximately 10% between measured and true values. The PicoGreen dye method was influenced by tRNA and protein, and the UV absorption method was susceptible to all kinds of impurities. Overall, the diphenylamine reaction method gave the most accurate results when DNA was mixed with contaminants, the PicoGreen dye method was most suitable for degraded DNA samples or DNA extracted from processed products, and the UV absorbance method was best for evaluating the impurities in DNA solutions.

Fine mapping of a dominant thermo-sensitive genic male sterility gene (BntsMs) in rapeseed (Brassica napus) with AFLP- and Brassica rapa-derived PCR markers.

KEY MESSAGE: A new thermo-sensitive dominant genic male sterility (TSDGMS) line of Brassica napus was found and mapped in this paper. Our result will greatly accelerate the map-based cloning of the BntsMs gene. TE5A is a thermo-sensitive dominant genic male sterility line originating from spontaneous mutation of the inbred line TE5 in Brassica napus and provides a promising system for the development of hybrid cultivars. Genetic analysis has revealed that the BntsMs mutant is controlled by a single, dominant gene. Here, we describe the fine mapping of BntsMs using amplified fragment length polymorphism (AFLP) and intron polymorphism (IP) methodologies. We screened 1,024 primer combinations and then identified five AFLP markers linked to the BntsMs gene, two of which were successfully converted into sequence-characterised amplified region (SCAR) markers. The linkage of the markers was identified by analysing a large BC2 population of 700 recessive-fertility individuals. Two SCAR markers were found in the flanking region of the BntsMs gene at distance of 3.5 and 4.8 cm. Based on sequence information from the previously screened AFLP markers and on genome organisation comparisons of the A genome of Brassica rapa and Arabidopsis, seven IP markers linked to the BntsMs gene were developed. By analysing the 700 recessive-fertility individuals, two IP markers, IP004 and IP470, were localised to the flanking region of the BntsMs gene at a distance of 0.3 and 0.2 cm, respectively. A comparison of the B. rapa and Arabidopsis genomes revealed 27 genes of B. rapa in the flanking region of these two IP markers. It is likely that the molecular markers developed from these investigations will greatly accelerate the positional cloning of the BntsMs gene.

Correction of the lack of commutability between plasmid DNA and genomic DNA for quantification of genetically modified organisms using pBSTopas as a model.

Plasmid calibrators are increasingly applied for polymerase chain reaction (PCR) analysis of genetically modified organisms (GMOs). To evaluate the commutability between plasmid DNA (pDNA) and genomic DNA (gDNA) as calibrators, a plasmid molecule, pBSTopas, was constructed, harboring a Topas 19/2 event-specific sequence and a partial sequence of the rapeseed reference gene CruA. Assays of the pDNA showed similar limits of detection (five copies for Topas 19/2 and CruA) and quantification (40 copies for Topas 19/2 and 20 for CruA) as those for the gDNA. Comparisons of plasmid and genomic standard curves indicated that the slopes, intercepts, and PCR efficiency for pBSTopas were significantly different from CRM Topas 19/2 gDNA for quantitative analysis of GMOs. Three correction methods were used to calibrate the quantitative analysis of control samples using pDNA as calibrators: model a, or coefficient value a (Cva); model b, or coefficient value b (Cvb); and the novel model c or coefficient formula (Cf). Cva and Cvb gave similar estimated values for the control samples, and the quantitative bias of the low concentration sample exceeded the acceptable range within ±25% in two of the four repeats. Using Cfs to normalize the Ct values of test samples, the estimated values were very close to the reference values (bias -13.27 to 13.05%). In the validation of control samples, model c was more appropriate than Cva or Cvb. The application of Cf allowed pBSTopas to substitute for Topas 19/2 gDNA as a calibrator to accurately quantify the GMO.

Structural basis for hormone recognition and distinctive Gq protein coupling by the kisspeptin receptor.

Kisspeptin signaling through its G protein-coupled receptor, KISS1R, plays an indispensable role in regulating reproduction via the hypothalamic-pituitary-gonadal axis. Dysregulation of this pathway underlies severe disorders like infertility and precocious puberty. Here, we present cryo-EM structures of KISS1R bound to the endogenous agonist kisspeptin-10 and a synthetic analog TAK-448. These structures reveal pivotal interactions between peptide ligands and KISS1R extracellular loops for receptor activation. Both peptides exhibit a conserved binding mode, unveiling their common activation mechanism. Intriguingly, KISS1R displays a distinct 40° angular deviation in its intracellular TM6 region compared to other Gq-coupled receptors, enabling distinct interactions with Gq. This study reveals the molecular intricacies governing ligand binding and activation of KISS1R, while highlighting its exceptional ability to couple with Gq. Our findings pave the way for structure-guided design of therapeutics targeting this physiologically indispensable receptor.

A pair of orthologs of a leucine-rich repeat receptor kinase-like disease resistance gene family regulates rice response to raised temperature.

BACKGROUND: Rice Xa3/Xa26 disease-resistance gene encodes a leucine-rich repeat (LRR) receptor kinase-type protein against Xanthomonas oryzae pv. oryzae (Xoo) and belongs to a multigene family. However, the functions of most genes in this family are unknown. RESULTS: Here we report that two orthologs of this family, the NRKe from rice variety Nipponbare and 9RKe from variety 93-11 at the RKe locus, have similar functions although they encode different proteins. This pair of orthologs could not mediate resistance to Xoo, but they were transcriptionally induced by raised temperature. Transcriptional activation of NRKe or 9RKe resulted in the formation of temperature-sensitive lesion mimics, which were spots of dead cells associated with accumulation of superoxides, in different organs of the transgenic plants. These plants were more sensitive to high temperature shock than wild-type controls. Transgenic plants carrying a chimeric protein consisting of the LRR domain of NRKe and the kinase domain of Xa3/Xa26 developed the same lesion mimics as the NRKe-transgenic plants, whereas transgenic plants carrying another chimeric protein consisting of the LRR domain of Xa3/Xa26 and the kinase domain of NRKe were free of lesion mimic. All the transgenic plants carrying a chimeric protein were susceptible to Xoo. CONCLUSION: These results suggest that the RKe locus is involved in rice response to raised temperature. The LRR domain of RKe protein appears to be important to sense increased temperature. The RKe-involved temperature-related pathway and Xa3/Xa26-mediated disease-resistance pathway may partially overlap.

Whole-genome sequence of Phellinus gilvus (mulberry Sanghuang) reveals its unique medicinal values.

Phellinus gilvus (Schwein.) Pat, a species of 'Sanghuang', has been well-documented for various medicinal uses, but the genome information and active constituents are largely unknown. Here, we sequenced the whole-genome of P. gilvus, identified phenylpropanoids as its key anti-cancer components, and deduced their biosynthesis pathways. A 41.11-Mb genome sequence was assembled and the heatmap created with high-throughput chromosome conformation capture techniques data suggested all bins could be clearly divided into 11 pseudochromosomes. Cellular experiments showed that P. gilvus fruiting body was more effective to inhibit hepatocellular carcinoma cells than mycelia. High resolution electrospray ionization mass spectroscopy (HR-ESI-MS) analysis revealed P. gilvus fruiting body was rich in phenylpropanoids, and several unique phenylpropanoids in Phellinus spp. exhibited potent anti-carcinogenesis activity. Based on genomic, HR-ESI-MS information and differentially expressed genes in transcriptome analysis, we deduced the biosynthesis pathway of four major phenylpropanoids in P. gilvus. Transcriptome analysis revealed the deduced genes expressions were synergistically changed with the production of phenylpropanoids. The optimal candidate genes of phenylpropanoids' synthesis pathway were screened by molecular docking analysis. Overall, our results provided a high-quality genomic data of P. gilvus and inferred biosynthesis pathways of four phenylpropanoids with potent anti-carcinogenesis activities. These will be a valuable resource for further genetic improvement and effective use of the P. gilvus.

Breeding response of transcript profiling in developing seeds of Brassica napus.

BACKGROUND: The upgrading of rapeseed cultivars has resulted in a substantial improvement in yield and quality in China over the past 30 years. With the selective pressure against fatty acid composition and oil content, high erucic acid- and low oil-content cultivars have been replaced by low erucic acid- and high oil-content cultivars. The high erucic acid cultivar Zhongyou 821 and its descendent, low erucic acid cultivar Zhongshuang 9, are representatives of two generations of the most outstanding Chinese rapeseed cultivars (B. napus) developed the past 2 decades. This paper compares the transcriptional profiles of Zhongshuang 9 and Zhongyou 821 for 32 genes that are principally involved in lipid biosynthesis during seed development in order to elucidate how the transcriptional profiles of these genes responded to quality improvement over the past 20 years. RESULTS: Comparison of the cultivar Zhongyou 821 with its descendent, Zhongshuang 9, shows that the transcriptional levels of seven of the 32 genes were upregulated by 30% to 109%, including FAD3, ACCase, FAE1, GKTP, Caleosin, GAPDH, and PEPC. Of the 32 genes, 10 (KAS3, beta-CT, BcRK6, P450, FatA, Oleosin, FAD6, FatB, alpha-CT and SUC1) were downregulated by at least 20% and most by 50%. The Napin gene alone accounted for over 75% of total transcription from all 32 genes assessed in both cultivars. Most of the genes showed significant correlation with fatty acid accumulation, but the correlation in ZS9 was significantly different from that in ZY821. Higher KCR2 activity is associated with higher C16:0, C18:0, and C18:2 in both cultivars, lower C22:1 and total fatty acid content in ZY821, and lower 18:1 in ZS9. CONCLUSION: This paper illustrates the response of the transcription levels of 32 genes to breeding in developing rapeseed seeds. Both cultivars showed similar transcription profiles, with the Napin gene predominantly transcribed. Selective pressure for zero erucic acid, low glucosinolate, high oleic acid and high oil content, as well as high yield, resulted in higher FAD3, ACCase, FAE1, GKTP, Caleosin, GAPDH, and PEPC expression levels and lower KAS3, beta-CT, BcRK6, P450, FatA, Oleosin, FAD6, FatB, alpha-CT and SUC1 expression levels. It also resulted in altered relationships between these genes during storage accumulation in seed development.

Multiple gene loci affecting genetic background-controlled disease resistance conferred by R gene Xa3/Xa26 in rice.

The function of bacterial-blight resistance gene Xa3/Xa26 in rice is influenced by genetic background; the Oryza sativa L. ssp. japonica background can increase Xa3/Xa26 expression, resulting in an enhanced resistance. To identify whether Xa3/Xa26 transcript level is the only factor contributing to genetic background-controlled resistance, we screened an F(2) population that was developed from a cross between Oryza sativa L. ssp. indica and japonica rice lines and was segregating for Xa3/Xa26, and compared the expression profiles of a pair of indica and japonica rice lines that both carried Xa3/Xa26. Eight quantitative trait loci (QTLs), in addition to Xa3/Xa26, were identified as contributing to the bacterial resistance of this population. Four of the eight QTLs were contributed to the japonica line. The resistance of this population was also affected by epistatic effects. Some F(2) individuals showed significantly increased Xa3/Xa26 transcripts, but the increased transcripts did not completely correlate with the reduced disease in this population. The analysis of the expression profile of Xa3/Xa26-mediated resistance using a microarray containing approximate 7,990 rice genes identified 44 differentially expressed genes. Thirty-five genes were rapidly activated in the japonica background, but not in the indica background, during disease resistance. These results suggest that multiple factors, including the one resulting in increased Xa3/Xa26 expression, may contribute to the enhanced resistance in the japonica background. These factors can cause a variation in gene expression profile that differs from that in the indica background during disease resistance.

Gene transferability from transgenic Brassica napus L. to various subspecies and varieties of Brassica rapa.

Gene transferability from transgenic rapeseed to various subspecies and varieties of Brassica rapa was assessed in this study. Artificial crossability was studied in 118 cultivars of 7 B. rapa subspecies and varieties with the transgenic rapeseed GT73 (Brassica napus) as the pollen donor. On average 5.7 seeds were obtained per pollination, with a range from 0.05 to 19.4. The heading type of B. rapa L. showed significantly higher crossability than non-heading types of B. rapa. The spontaneous outcrossing rate between B. rapa (female) and the transgenic rapeseed Ms8 x Rf3 (B. napus) (male) ranged from 0.039 to 0.406%, with an average of 0.19%. The fertilization process and the development of the hybrid seeds as shown by fluorescent staining techniques indicated that the number of adhered pollens on the stigma was reduced by 80%, the number of pollen tubes in the style was reduced by 2/3 and the fertilization time was delayed by over 20 h when pollinated with the transgenic rapeseed Ms8 x Rf3 in comparison with the bud self-pollination of B. rapa as control. About 10-70% of the interspecific hybrid embryos were aborted in the course of development. Some seeds looked cracked in mature pods, which showed germination abilities lower than 10%. The spontaneous outcrossing rates were much lower than the artificial crossability, and their survival fitness of the interspecific hybrid was very low, indicating that it should be possible to keep the adventitious presence of the off-plants under the allowed threshold, if proper measures are taken.

The expression pattern of a rice disease resistance gene xa3/xa26 is differentially regulated by the genetic backgrounds and developmental stages that influence its function.

Genetic background and developmental stage influence the function of some disease resistance (R) genes. The molecular mechanisms of these modifications remain elusive. Our results show that the two factors are associated with the expression of the R gene in rice Xa3 (also known as Xa26)-mediated resistance to Xanthomonas oryzae pv. oryzae (Xoo), which in turn influences the expression of defense-responsive genes. The background of japonica rice, one of the two major subspecies of Asian cultivated rice, facilitates the function of Xa3 more than the background of indica rice, another rice subspecies. Xa3 expression gradually increases from early seedling stage to adult stage. Japonica plants carrying Xa3 regulated by the native promoter showed an enlarged resistance spectrum (i.e., resistance to more Xoo races), an increased resistance level (i.e., further reduced lesion length), and whole-growth-stage resistance compared to the indica rice; this enhanced resistance was associated with an increased expression of Xa3 throughout the growth stages in the japonica plants, which resulted in enhanced expression of defense-responsive genes. Overexpressing Xa3 with a constitutive strong promoter further enhanced rice resistance due to further increased Xa3 transcripts in both indica and japonica backgrounds, whereas regulating Xa3 with a pathogen-induced weak promoter impaired rice resistance.

OsWRKY13 mediates rice disease resistance by regulating defense-related genes in salicylate- and jasmonate-dependent signaling.

Although 109 WRKY genes have been identified in the rice genome, the functions of most are unknown. Here, we show that OsWRKY13 plays a pivotal role in rice disease resistance. Overexpression of OsWRKY13 can enhance rice resistance to bacterial blight and fungal blast, two of the most devastating diseases of rice worldwide, at both the seedling and adult stages, and shows no influence on the fertility. This overexpression was accompanied by the activation of salicylic acid (SA) synthesis-related genes and SA-responsive genes and the suppression of jasmonic acid (JA) synthesis-related genes and JA-responsive genes. OsWRKY13 bound to the promoters of its own and at least three other genes in SA- and JA-dependent signaling pathways. Its DNA-binding activity was influenced by pathogen infection. These results suggest that OsWRKY13, as an activator of the SA-dependent pathway and a suppressor of JA-dependent pathways, mediates rice resistance by directly or indirectly regulating the expression of a subset of genes acting both upstream and downstream of SA and JA. Furthermore, OsWRKY13 will provide a transgenic tool for engineering wider-spectrum and whole-growth-stage resistance rice in breeding programs.

Improvement of multiple agronomic traits by a disease resistance gene via cell wall reinforcement.

The major disease resistance gene Xa4 confers race-specific durable resistance against Xanthomonas oryzae pv. oryzae, which causes the most damaging bacterial disease in rice worldwide. Although Xa4 has been one of the most widely exploited resistance genes in rice production worldwide, its molecular nature remains unknown. Here we show that Xa4, encoding a cell wall-associated kinase, improves multiple traits of agronomic importance without compromising grain yield by strengthening the cell wall via promoting cellulose synthesis and suppressing cell wall loosening. Strengthening of the cell wall by Xa4 enhances resistance to bacterial infection, and also increases mechanical strength of the culm with slightly reduced plant height, which may improve lodging resistance of the rice plant. The simultaneous improvement of multiple agronomic traits conferred by Xa4 may account for its widespread and lasting utilization in rice breeding programmes globally.

BraLTP1, a lipid transfer protein gene involved in epicuticular wax deposition, cell proliferation and flower development in Brassica napus.

Plant non-specific lipid transfer proteins (nsLTPs) constitute large multigene families that possess complex physiological functions, many of which remain unclear. This study isolated and characterized the function of a lipid transfer protein gene, BraLTP1 from Brassica rapa, in the important oilseed crops Brassica napus. BraLTP1 encodes a predicted secretory protein, in the little known VI Class of nsLTP families. Overexpression of BnaLTP1 in B. napus caused abnormal green coloration and reduced wax deposition on leaves and detailed wax analysis revealed 17-80% reduction in various major wax components, which resulted in significant water-loss relative to wild type. BnaLTP1 overexpressing leaves exhibited morphological disfiguration and abaxially curled leaf edges, and leaf cross-sections revealed cell overproliferation that was correlated to increased cytokinin levels (tZ, tZR, iP, and iPR) in leaves and high expression of the cytokinin biosynthsis gene IPT3. BnaLTP1-overexpressing plants also displayed morphological disfiguration of flowers, with early-onset and elongated carpel development and outwardly curled stamen. This was consistent with altered expression of a a number of ABC model genes related to flower development. Together, these results suggest that BraLTP1 is a new nsLTP gene involved in wax production or deposition, with additional direct or indirect effects on cell division and flower development.

Collaborative validation of an event-specific quantitative real-time PCR method for genetically modified rice event TT51-1 detection.

In this study, a collaborative trial of validating a real-time PCR method for the TT51-1 rice event was organized, including six participating laboratories. In this validation, serially diluted solutions from homogeneous genomic DNA of the TT51-1 event were used to construct standard curves of the TT51-1 event and phospholipase D (PLD) assays. The PCR efficiency was 95%, and the R(2) coefficient was 0.99 for the TT51-1 system. The mean quantitative values for blind samples containing 0.1%, 0.5% 1%, 5%, and 10% (w/w) TT51-1 corresponded to 0.1%, 0.51%, 1.06%, 4.83%, and 9.62%, respectively, with a bias (%) ranging from -3.77% to 5.87%. The repeatability and reproducibility were all below 25% across the entire dynamic range. Furthermore, the measurement uncertainties of the quantitative results were estimated to be 0.10%, 0.20%, 0.40%, 1.76%, and 3.52% (w/w) for the tested samples. Both the LOD and LOQ were calculated to be 0.22%. This collaborative trial demonstrated that the TT51-1 method produces reliable, comparable, and reproducible results for a given sample set and can be adopted as a detection standard for testing laboratories.

A novel approach for the construction of plant amiRNA expression vectors.

Artificial microRNA (amiRNA) technology has been applied in Arabidopsis thaliana and other plants to efficiently silence target genes of interest. Here we described a novel approach to construct plant amiRNA expression vectors with seamless enzyme-free cloning (SEFC) and mating-assisted genetically integrated cloning (MAGIC). Two pairs of primers were designed when the loop of amiRNA precursor was longer than 60 bp while three oligonucleotides were used to amplify the linearized vector containing the amiRNA precursor whose loop was smaller than 60 bp. The PCR products were transformed into Escherichia coli to generate the donor plasmid containing the amiRNA expression cassette through homologous recombination in vivo. The amiRNA expression cassette was then transferred to the recipient plasmid via MAGIC and an amiRNA expression plasmid was created. More than 200 amiRNA expression vectors were generated with this approach, three of which have been transformed into A. thaliana and successfully silence the target genes. Given its low-cost and simplicity, this novel approach of plant amiRNA expression vectors construction will benefit the study of individual gene function and establishment of plant amiRNA libraries.

Characterization of the transgenic rice event TT51-1 and construction of a reference plasmid.

Transgenic rice TT51-1 (BT63) is an insect resistant strain that was granted for safety certificate in China in 2009. This study characterizes the transgenic event TT51-1 using a GenomeWalker strategy. The organization of the transgenes indicated that the transgenes on two plasmids, pFHBT1 and pGL2RC7, had been integrated at the same locus. The sequence of the event TT51-1 spanned 8725 bp, including a truncated Cry1Ab/Ac cassette, an intact Cry1Ab/Ac cassette, two Amp gene segments, and an Hph gene segment. The 5' and 3' plant flanking sequences were isolated and used to locate the transgenes to chromosome 10 in TT51-1. The isolated TT51-1 fragment and a fragment of the rice PLD gene were integrated into a plasmid vector, to create plasmid pK-TT51 as a calibrator for detecting rice containing TT51-1. Analysis of unknown samples indicated that the reference plasmid was a reliable alternative to TT51-1 genomic DNA.

Comparison of five endogenous reference genes for specific PCR detection and quantification of Brassica napus.

Five previously reported Brassica napus endogenous reference genes, including acetyl-CoA carboxylase gene (BnACCg8), phosphoenolpyruvate carboxylase (PEP), oleoyl hydrolase gene (FatA), high-mobility-group protein I/Y gene (HMG-I/Y) and cruciferin A gene (CruA), were analyzed for their PCR specificity between B. napus and other species and the quantification stability among different B. napus cultivars. PCR and sequencing results indicated that none of these systems was species-specific as required by the genetically modified organism labeling policy. When these genes were employed in real-time PCR, BnACCg8 and HMG-I/Y systems showed relatively greater heterogeneity among 10 different cultivars. The sequencing results showed that the single nucleotide polymorphism in the primer binding sites was the potential source of the instability in the HMG-I/Y system. The bias of BnACCg8 was thought to be associated with the inconsistent copy number of this gene.

The gene MT3-B can differentiate palm oil from other oil samples.

The practice of blending cheap palm oil with more expensive oils is currently rampant owing to the increased global price of oil and the price gap between types of oils. This adulteration poses a serious threat to the trade of edible oil and negatively affects consumers. The aim of this study was to identify the presence of palm oil as an additive in more expensive oils using a PCR-based technique. A taxon-specific gene, MT3-B, was found by searching the GenBank database. MT3-B showed high oil palm (Elaeis guineensis Jacq.) specificity, low intraspecies variability, and a low copy number. On the basis of the MT3-B sequence, conventional and real-time PCR assays were established to detect palm oil contamination by amplifying an amplicon of 109 bp. The lowest copy number that the conventional PCR method could detect was five haploid copies; the limit of detection (LOD) for the real-time PCR assay was estimated to be five haploid copies. Experimental results demonstrated that the PCR-based methods were specific, sensitive, and reliable and could successfully detect the palm oil component of mixed oil samples.