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1.
针对现有玉米单倍体核磁共振分选系统基于一个含油率阈值,无法对胚败育籽粒和单倍体籽粒正确分选的问题,分别对玉米生物诱导产生的二倍体、单倍体和胚败育3种不同籽粒类型的单粒质量和含油率进行分析,提出了利用籽粒含油率双阈值提高单倍体正确识别率的分选方法。该研究以2个普通玉米杂交种和3个自交系为母本,以高油型诱导系为父本,进行生物诱导产生的3种不同类型籽粒为研究对象,利用核磁共振分选系统分别对不同类型籽粒的单粒质量和含油率进行测定,结果表明：单粒质量整体表现为单倍体>二倍体>胚败育,除二倍体籽粒与胚败育籽粒间存在极显著差异外,其他籽粒类型间差异不显著;不同类型籽粒的单粒质量平均变异系数为16.62%,并且每个材料的3种籽粒类型间出现较大的重叠区域。而不同类型籽粒含油率整体表现为二倍体>单倍体>胚败育,变异性以二倍体最小,平均变异系数仅为12.52%,其次是单倍体,而胚败育籽粒最高（34.14%）,但其含油率最低且均≤2%;每个材料各自的3种类型籽粒间含油率呈现梯度分布,存在较明显的界限。由此可见,利用籽粒含油率能够区分玉米生物诱导的3种不同籽粒类型,而单粒质量则不能;通过设置二倍体籽粒的最小含油率为上限,胚败育籽粒的最大含油率为下限,利用含油率的双阈值可提高单倍体的正确识别率,为玉米生物诱导单倍体高效自动化分选提供依据。  相似文献   

2.
Several genetically modified (GM) cultivars are registered in Canada although they are not currently in commercial production. The GM cultivars can be distinguished from the non-GM and other GM cultivars by analyzing the DNA nucleotide sequence at the insertion site of the transgene corresponding to a single transformation event in the plant genome. Techniques based on modified polymerase chain reaction (PCR) strategies were used to generate sequence information from the plant genome flanking the insertion site of transgenic DNA for specific GM potato events. The plant genome sequence adjacent to the transgenic insertion was used to design PCR primers, which could be used in combination with a primer annealing to one of the nearby inserted genetic elements to amplify an event specific DNA fragment. The event specific PCR fragments generated were sequenced to confirm the specificity of the method.  相似文献   

3.
Because of the increasing use of maize hybrids with genetically modified (GM) stacked events, the established and commonly used bulk sample methods for PCR quantification of GM maize in non-GM maize are prone to overestimate the GM organism (GMO) content, compared to the actual weight/weight percentage of GM maize in the grain sample. As an alternative method, we designed and assessed a group testing strategy in which the GMO content is statistically evaluated based on qualitative analyses of multiple small pools, consisting of 20 maize kernels each. This approach enables the GMO content evaluation on a weight/weight basis, irrespective of the presence of stacked-event kernels. To enhance the method's user-friendliness in routine application, we devised an easy-to-use PCR-based qualitative analytical method comprising a sample preparation step in which 20 maize kernels are ground in a lysis buffer and a subsequent PCR assay in which the lysate is directly used as a DNA template. This method was validated in a multilaboratory collaborative trial.  相似文献   

4.
为建立一套适合玉米单倍体胚性愈伤培养和快速筛选体系,以单倍体诱导系MT-1为父本,18-599红为母本进行单倍体诱导,设置暗培养、全光照培养和光暗周期培养3种光照培养方式,均分别培养0、1、5、10、20、40 h后,观察幼胚形态和颜色。结果表明,自交系18-599红的单倍体和二倍体幼胚均能正常诱导形成胚性愈伤组织;不同光照培养方式对紫色(二倍体)愈伤率的检出效果依次为光培养>光暗周期培养>暗培养。通过光照筛选的方法可在早期鉴定愈伤组织,其二倍体愈伤的筛选率在培养20 h时可达68%,40 h时为71%,剔除了大部分非单倍体愈伤,综合分析确定光照强度为2 000 lx、20℃处理20 h为最适光照筛选处理。染色体压片技术获得的拟单倍体愈伤中有二倍体愈伤的检出,但经流式细胞仪检出获得的拟单倍体愈伤再经染色体压片检测,无二倍体愈伤,表明流式细胞仪检测单倍体愈伤的准确性高于染色体压片技术。通过光照初步筛选结合流式细胞仪的精确鉴定,迅速从3 000个单倍体愈伤中获得110个单倍体愈伤,单倍体愈伤率3.67%。本研究结果为以玉米单倍体愈伤为转基因受体,快速获得转基因植株提供了一定的技术支撑和理论参考。  相似文献   

5.
Toward the development of reliable qualitative and quantitative Polymerase Chain Reaction (PCR) detection methods of transgenic tomatoes, one tomato (Lycopersicon esculentum) species specific gene, LAT52, was selected and validated as suitable for using as an endogenous reference gene in transgenic tomato PCR detection. Both qualitative and quantitative PCR methods were assayed with 16 different tomato varieties, and identical amplified products or fluorescent signals were obtained with all of them. No amplified products and fluorescent signals were observed when DNA samples from 20 different plants such as soybean, maize, rapeseed, rice, and Arabidopsis thaliana were used as templates. These results demonstrated that the amplified LAT52 DNA sequence was specific for tomato. Furthermore, results of Southern blot showed that the LAT52 gene was a single-copy gene in the different tested tomato cultivars. In qualitative and quantitative PCR analysis, the detection sensitivities were 0.05 and 0.005 ng of tomato genomic DNA, respectively. In addition, two real-time assays employing this gene as an endogenous reference gene were established, one for the quantification of processed food samples derived from nontransgenic tomatoes that contained degraded target DNA and the other for the quantification of the junction region of CaMV35s promoter and the anti-sense ethylene-forming enzyme (EFE) gene in transgenic tomato Huafan No. 1 samples. All of these results indicated that the LAT52 gene could be successfully used as a tomato endogenous reference gene in practical qualitative and quantitative detection of transgenic tomatoes, even for some processed foods derived from transgenic and nontransgenic tomatoes.  相似文献   

6.
The applicability of quantifying genetically modified (GM) maize and soy to processed foods was investigated using heat treatment processing models. The detection methods were based on real-time quantitative polymerase chain reaction (PCR) analysis. Ground seeds of insect resistant GM maize (MON810) and glyphosate tolerant Roundup Ready (RR) soy were dissolved in water and were heat treated by autoclaving for various time intervals. The calculated copy numbers of the recombinant and taxon specific deoxyribonucleic acid (DNA) sequences in the extracted DNA solution were found to decrease with time. This decrease was influenced by the PCR-amplified size. The conversion factor (Cf), which is the ratio of the recombinant DNA sequence to the taxon specific DNA sequence and is used as a constant number for calculating GM% at each event, tended to be stable when the sizes of PCR products of two DNA sequences were nearly equal. The results suggested that the size of the PCR product plays a key role in the quantification of GM organisms in processed foods. It is believed that the Cf of the endosperm (3n) is influenced by whether the GM originated from a paternal or maternal source. The embryos and endosperms were separated from the F1 generation seeds of five GM maize events, and their Cf values were measured. Both paternal and maternal GM events were identified. In these, the endosperm Cf was lower than that of the embryo, and the embryo Cf was lower than that of the endosperm. These results demonstrate the difficulties encountered in the determination of GM% in maize grains (F2 generation) and in processed foods from maize and soy.  相似文献   

7.
For implementation of the issued regulations and labeling policies for genetically modified organism (GMO) supervision, the polymerase chain reaction (PCR) method has been widely used due to its high specificity and sensitivity. In particular, use of the event-specific PCR method based on the flanking sequence of transgenes has become the primary trend. In this study, both qualitative and quantitative PCR methods were established on the basis of the 5' flanking sequence of transgenic soybean A2704-12 and the 3' flanking sequence of transgenic soybean A5547-127, respectively. In qualitative PCR assays, the limits of detection (LODs) were 10 copies of haploid soybean genomic DNA for both A2704-12 and A5547-127. In quantitative real-time PCR assays, the LODs were 5 copies of haploid soybean genomic DNA for both A2704-12 and A5547-127, and the limits of quantification (LOQs) were 10 copies for both. Low bias and acceptable SD and RSD values were also achieved in quantification of four blind samples using the developed real-time PCR assays. In addition, the developed PCR assays for the two transgenic soybean events were used for routine analysis of soybean samples imported to Shanghai in a 6 month period from October 2010 to March 2011. A total of 27 lots of soybean from the United States and Argentina were analyzed: 8 lots from the Unites States were found to have the GM soybean A2704-12 event, and the GM contents were <1.5% in all eight analyzed lots. On the contrary, no GM soybean A5547-127 content was found in any of the eight lots. These results demonstrated that the established event-specific qualitative and quantitative PCR methods could be used effectively in routine identification and quantification of GM soybeans A2704-12 and A5547-127 and their derived products.  相似文献   

8.
Real-time polymerase chain reaction is currently being used for the identification and quantification of plant and animal species as well as microorganisms in food or feed samples based on the amplification of specific sequences of low copy genes. We report here the development of a new real-time PCR method for the detection and quantification of the pea (Pisum sativum) based on the amplification of a specific region of the legS gene. The specificity was evaluated in a wide range of plant species (51 varieties of Pisum sp., and 32 other plant species and varieties taxonomically related or nonrelated). The method allows the detection and quantification of as low as 21.6 pg of DNA, which corresponds to 5 haploid genome copies. The system has been shown to be sensitive, reproducible and 100% specific for the rapid detection and quantification of pea DNA in processed food and feed samples, being therefore suitable for high-throughput analysis.  相似文献   

9.
Four real-time polymerase chain reaction systems aiming at the specific detection and quantification of maize DNA are described. They have been developed in four independent laboratories targeting different maize sequences, i.e., alcohol dehydrogenase (Adh1), high mobility group protein (hmga), invertase A (ivr1), and zein, respectively. They were all fully specific, showing a very similar quantification accuracy along a number of distantly related maize cultivars and being either single or low copy number genes. They were highly sensitive and exhibited limits of quantification below 100 maize genomic copies. In consequence, they are considered suitable for use as maize specific endogenous reference genes in DNA analyses, including GMO quantitative tests.  相似文献   

10.
The fate of DNA during steeping, wet-milling, and subsequent processing of maize was examined using a sensitive polymerase chain reaction (PCR-based) detection system. The system used specific amplification of maize DNA sequences by primers generated toward plant nuclear- and chloroplast-encoded genes. The PCR method facilitated analysis of DNA content in food products, which is an important issue in use of genetically modified organisms. In a conventional laboratory wet-milling countercurrent steep system, DNA was detected in maize kernels throughout the process but was not found in steepwater. After kernels were wet-milled, DNA was detected in the starch, germ, coarse fiber, and wet gluten fractions but not in the fine fiber fraction. When dried by heating at 135°C for 2 hr, DNA was degraded to undetectable levels in the wet-milled gluten fraction and hydrated kernels. DNA was not detected in feed pellets, starch, dextrose, sorbitol, or high-fructose maize syrup made from industrial wet-milled samples. Although DNA could be detected in laboratory wet-milled fractions, some degree of degradation occurred after extended exposure to steepwater. Countercurrent steepwater samples from the later stages of the steeping process were able to degrade DNA. The level of DNA degradation appeared to correspond to the presence of sulfur dioxide and may represent a physiochemical rather than an enzyme-mediated process. Our results indicate that some steps in the steeping and wet-milling process can degrade maize genomic and plastid DNA.  相似文献   

11.
  【目的】  石灰性土壤高pH和高重碳酸盐含量严重影响土壤中有效铁含量,导致作物缺铁黄化、减产,铁高效玉米品种的推广应用是实现石灰性土壤玉米高产稳产的重要途径。 本研究探讨不同铁效率玉米品种适应低铁胁迫的根系特征与铁积累差异,旨在为铁高效玉米品种的推广应用提供科学依据。  【方法】  试验以铁高效玉米品种正红2号 (ZH2)、正大619 (ZD619) 和铁低效玉米品种川单418 (CD418)、先玉508 (XY508) 为材料,设置极低铁处理 (Fe0,Fe浓度为0 μmol/L)、低铁处理 (Fe10,Fe浓度为10 μmol/L) 和正常供铁 (Fe100,Fe浓度为100 μmol/L) 3个处理,通过砂培试验,研究不同铁效率玉米品种适应低铁胁迫的根系形态特征、干物质重、铁积累及铁吸收利用差异。  【结果】  低铁胁迫下,玉米幼苗的根干重、单株干重、铁积累量、根系相对铁吸收效率均显著降低,而根冠比与铁素生理效率均显著升高,且随胁迫程度的增加变幅加大;总根长、根表面积、根体积和根直径则表现出明显的品种差异,与正常铁处理 (Fe100)相比,低铁处理下铁低效品种的总根长、根表面积和根体积显著降低,根直径显著增加,而铁高效品种的总根长和根表面积差异不显著,根体积显著增加,根直径在极低铁处理(Fe0)下显著降低,低铁处理 (Fe10)下差异不显著;铁高效品种总根长、根表面积、根体积、根干重、单株干物重、铁积累量和根系铁吸收效率的降幅及根冠比的增幅均明显低于铁低效品种,而铁生理效率的增幅高于铁低效品种。相关性分析结果表明,玉米幼苗铁积累量与总根长、根表面积、根体积和根干重均呈显著正相关,而与根冠比呈负相关,其中与总根长 (R2 = 0.8546) 和根表面积 (R2 = 0.8983) 相关性最强。  【结论】  与铁低效玉米品种相比,铁高效玉米品种低铁胁迫下具有较优的总根长、根表面积及较高的根系铁吸收效率与铁生理效率,促进了其对铁的高效吸收与利用,提高了其对低铁环境的适应能力。  相似文献   

12.
Sorghum is a critical source of food in the semiarid regions of sub-Saharan Africa and India and a potential source of dietary phytochemicals including carotenoids. The objective of this study was to determine the carotenoid profiles of sorghum cultivars, selected on the basis of their yellow-endosperm kernels, at various developmental stages. Following extraction from sorghum flours, carotenoids were separated by high-performance liquid chromatography (HPLC) with diode array detection. Total carotenoid content in fully matured yellow-endosperm sorghum kernels (0.112-0.315 mg/kg) was significantly lower (p < 0.05) than that in yellow maize (1.152 mg/kg) at physiological maturity. Variation in total carotenoids and within individual carotenoid species was observed in fully mature sorghum cultivars. For developing kernels, large increases in carotenoid content occurred between 10 and 30 days after half bloom (DAHB), resulting in a peak accumulation between 6.06 and 28.53 microg of total carotenoids per thousand kernels (TK). A significant (p < 0.05) decline was noted from 30 to 50 DAHB, resulting in a final carotenoid content of 2.62-15.02 microg/TK total carotenoids. (all-E)-Zeaxanthin was the most abundant carotenoid, ranging from 2.22 to 13.29 microg/TK at 30 DAHB. (all-E)-Beta-carotene was present in modest amounts (0.15-3.83 microg/TK). These data suggest the presence of genetic variation among sorghum cultivars for carotenoid accumulation in developing and mature kernels.  相似文献   

13.
One tomato ( Lycopersicon esculentum) gene, LAT52, has been proved to be a suitable endogenous reference gene for genetically modified (GM) tomato detection in a previous study. Herein are reported the results of a collaborative ring trial for international validation of the LAT52 gene as endogenous reference gene and its analytical systems; 14 GMO detection laboratories from 8 countries were invited, and results were finally received from 13. These data confirmed the species specificity by testing 10 plant genomic DNAs, less allelic variation and stable single copy number of the LAT52 gene, among 12 different tomato cultivars. Furthermore, the limit of detection of LAT52 qualitative PCR was proved to be 0.1%, which corresponded to 11 copies of haploid tomato genomic DNA, and the limit of quantification for the quantitative PCR system was about 10 copies of haploid tomato genomic DNA with acceptable PCR efficiency and linearity. Additionally, the bias between the test and true values of 8 blind samples ranged from 1.94 to 10.64%. All of these validated results indicated that the LAT52 gene is suitable for use as an endogenous reference gene for the identification and quantification of GM tomato and its derivates.  相似文献   

14.
All transgenic cultivars of potatoes registered in Canada and the United States have been modified to express a synthetic cry3A gene as a means of conferring resistance against the Colorado potato beetle, an important economic pest of potatoes. A PCR method was developed to amplify a 499 bp region of the synthetic cry3A gene. Using this method, synthetic cry3A could be detected in six different transgenic cultivars. Positive results could be confirmed with PvuII restriction digestion of the PCR-generated amplicon, which resulted in two fragments that were 283 and 216 bp in size. Of the 52 tuber extracts tested with this method, no false positive or false negative results were obtained, suggesting the method could be used with a high degree of accuracy. The absolute limit of detection was the number of cry3A copies present in one or perhaps two haploid copies of the potato genome. The practical limit of detection in tubers on a fresh weight basis was 0.02% for the NL 10-SUP and 0.01% for the remaining cultivars. Synthetic cry3A could also be detected in processed food products such as potato chips, shoestring potatoes, and frozen French fries. The method was suitable for screening potato tuber lots and some processed foods for the presence of synthetic cry3A.  相似文献   

15.
Chemical composition (moisture, total lipids, protein, and apparent amylose) and some physical features (1,000 kernel weight, hardness, and anatomical composition) were determined in 71 accessions representing races of maize from Latin America. Their microstructural characteristics (size and compaction of endosperm cell bodies, pericarp thickness, horny‐floury endosperm ratio, and morphology and size of starch granules) were also evaluated using environmental scanning electron microscopy (ESEM). Compaction was the most important microstructural feature of the maize kernels, representing kernel hardness. Highly compact kernels tended to be hard, with high protein, pericarp, and hard‐endosperm content and high pericarp thickness, but with low moisture, amylose content, and kernel weight and size. The opposite was observed in the least compact kernels. Highly compact kernels tended to have small, polygonal starch granules (<10 μm), while the least compact kernels contained large, spherical granules (>10 μm). These results suggest that microstructure is responsible for the physical features of maize kernels and that microstructure is related to chemical composition.  相似文献   

16.
Because of the genetically modified organisms (GMOs) labeling policies issued in many countries and areas, polymerase chain reaction (PCR) methods were developed for the execution of GMO labeling policies, such as screening, gene specific, construct specific, and event specific PCR detection methods, which have become a mainstay of GMOs detection. The event specific PCR detection method is the primary trend in GMOs detection because of its high specificity based on the flanking sequence of the exogenous integrant. This genetically modified maize, MON863, contains a Cry3Bb1 coding sequence that produces a protein with enhanced insecticidal activity against the coleopteran pest, corn rootworm. In this study, the 5'-integration junction sequence between the host plant DNA and the integrated gene construct of the genetically modified maize MON863 was revealed by means of thermal asymmetric interlaced-PCR, and the specific PCR primers and TaqMan probe were designed based upon the revealed 5'-integration junction sequence; the conventional qualitative PCR and quantitative TaqMan real-time PCR detection methods employing these primers and probes were successfully developed. In conventional qualitative PCR assay, the limit of detection (LOD) was 0.1% for MON863 in 100 ng of maize genomic DNA for one reaction. In the quantitative TaqMan real-time PCR assay, the LOD and the limit of quantification were eight and 80 haploid genome copies, respectively. In addition, three mixed maize samples with known MON863 contents were detected using the established real-time PCR systems, and the ideal results indicated that the established event specific real-time PCR detection systems were reliable, sensitive, and accurate.  相似文献   

17.
Polymerase chain reaction (PCR) methods are very useful techniques for the detection and quantification of genetically modified organisms (GMOs) in food samples. These methods rely on the amplification of transgenic sequences and quantification of the transgenic DNA by comparison to an amplified reference gene. Reported here is the development of specific primers for the rapeseed (Brassica napus) BnACCg8 gene and PCR cycling conditions suitable for the use of this sequence as an endogenous reference gene in both qualitative and quantitative PCR assays. Both methods were assayed with 20 different rapeseed varieties, and identical amplification products were obtained with all of them. No amplification products were observed when DNA samples from other Brassica species, Arabidopsis thaliana, maize, and soybean were used as templates, which demonstrates that this system is specific for rapeseed. In real-time quantitative PCR analysis, the detection limit was as low as 1.25 pg of DNA, which indicates that this method is suitable for use in processed food samples which contain very low copies of target DNA.  相似文献   

18.
An international CCQM-P60 pilot study involving eight national metrological institutes was organized to investigate if the quantification of genetically modified (GM) corn powder by real-time PCR was affected by the DNA extraction method applied. Four commonly used extraction methods were compared for the extraction of DNA from a GM Bt176 corn powder. The CTAB-based method yielded the highest DNA template quantity and quality. A difference in the 260 nm/230 nm absorbance ratio was observed among the different extraction methods. Real-time amplification of sequences specific for endogenous genes zein and hmg as well as transgenic sequences within the cryIA(b) gene and a fragment covering the junction between the transformed DNA and the plant genome were used to determine the GM percentage. The detection of the transgenic gene was affected by the quantity and quality of template used for the PCR reaction. The Bt176 percentages measured on diluted or purified templates were statistically different depending on the extraction method applied.  相似文献   

19.
Yellow and white maize kernels, masas, tortillas, and nejayote solids were analyzed in terms of lutein, zeaxanthin, cryptoxanthin, β‐carotene, and lipophilic antioxidant (AOX) capacity. The germplasm analyzed included two normal yellow maize, two high‐carotenoid genotypes, and one white for comparison purposes. In general, the yellow maize required 34% more lime‐cooking time compared with the white counterpart. Lime‐cooking significantly changed the extractability of carotenoids in masa and tortillas. No carotenoids were detected in the steepwater or nejayote. The lipophilic AOX activity increased 280‐fold from kernel to masa, but only 70% was retained in the baked tortillas. When masa was baked into tortillas, less than 10% of the carotenoids were retained because of the high temperatures used during baking. Interestingly, tortillas made with the maize kernels with the highest carotenoid content did not have the highest amount of these phytochemicals. Therefore, maize varieties should be evaluated based on the carotenoid content in finished food products instead of the amounts originally found in raw kernels.  相似文献   

20.
Mitochondrial DNA RFLP in genus Oryza and cultivated rice   总被引：1，自引：0，他引：1  
Summary Ninety-three accessions representing 23 species from the genus Oryza were surveyed for restriction fragment length polymorphism (RFLP) in mitochondrial (mt) DNA by probing total DNA with 15 known mt sequences cloned in plasmids from higher plants, and five mt genomic cosmid clones from maize. Very low levels of intra-specific and even intra-cytologically-defined nuclear genome mt DNA RFLP were found. High between-genome differentiation appeared, suggesting phylogenetic relationships consistent with data from previous nuclear and chloroplast (cp) DNA studies. Parallel inheritance of cp and mt DNA was found. There was one major exception: the mt DNA of the allotetraploid CD genome is apparently equally related to two putative diploid progenitors, which is suggestive of an interspecific recombination.RRLP in mt DNA was also probed in 82 cultivars, with four plasmid probes. Some bands not seen in the wild species appeared in O. sativa, with intra-specific polymorphism relatively higher than in the wild species. The pattern of variation paralleled that at the cp DNA level between the indica and japonica subspecies.  相似文献   

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