Testing Varietal Identity And Purity Of Crop By Molecular Bioloby Technologies

The identity and purity of variety have an signicant effects on the yield and quality, which are important evaluation index for seed quality. And the problem of varietal identity and purity can lead to dramatical yield declining, such events often happened in the past years, and which seriously affected the increase of the agricultural production. Genetically modified crops are obtained via the genetic transformation of crop genome. Although all countries hold a relatively cautious attitude to commercialization of the transgenic crops, but the acreage and varieties of genetically modified crops still are rapid increased. With the enaction of the relevant laws and regulations for the risk assessment and detection of genetically modified products, the detection and identification of genetically modified component have become increasingly important.Now there are a great many seed business entities in China, and market supervision technology and means are lagged. Fake and shoddy seeds could not be forbidden with absolutely, and infringement problems of sets licensing outstand issues, so the order of the seed market is chaotic, Which seriously infringe the legitimate rights and interests of farmers and legitimate enterprises."The view of the State Council on accelerating the development of modern crop seed industry" clearly pointed out that establishing the seed management system with advanced means and effective supervision, to crack down the behavior of infringement of licensing and counterfeit seed production and management, and feasible safeguarding the market order of fair competition. Molecular detection techniques provide effective measures to solve these problems mentioned above. It can break the bottlenecks of management technology. Therefore, It is an important and urgent task for current seed quality management to accelerate the development and application of molecular detection technology.Many studies have shown that the method is feasible, and that the technique of SSR molecular markers can be used to accurately appraise the varietal identity and purity. But most of these reports focused on the theoretical point of research. So in present work, we combined the practical supervision of seedand theoretical research together, to try to solve the existing problems in the practical application. The main results were as follows:1. Variety purity testing:screened out specific primers for23rice hybrids which are good sold variety on the market, checked the result of SSR molecular identification through field planting in growing season and field planting in Hainan province. By SSR molecular marker, use the primers screened to check226samples, which can stand for nearly4million kilograms "two-line" rice hybrids and contained23rice hybrids sold well on the market. The results showed:the identification results of SSR marker and the field planting in growing season were basically the same, the biggest difference was11.2%, the average gap was0.9%; the identification results of field planting in growing season and field planting in Hainan province were quite different, the biggest difference was44.0%, the average gap was3.6%.In addition, we selected20representative samples and identified per plant of the samples by SSR marker and the field planting in growing season. The result showed the goodness of fit of each individual plant with these two identification methods was99%. In conclusion, the purity of the two-line rice hybrids could be accurate and rapid identification with the primers screened, and the sterile lines can be distinguished out only with a pair of primers according the parents’ complementary types.In addition, through analyze the experimental data, at first to raise a viewpoint that the identification results of SSR marker were more accurate and reliable than field planting in Hainan province, when identify the purity of two-line rice hybrids. When purity testing, off-types could easy divided into female parent type and paternal type when parents as standard control, which conducive to analyze the sources of the off-types, being a good solution of the problem that difficult to distinguish off-types during the actual molecular detection.2. Varietal identity:when identify lower purity sample, electrophoretic analysis often appeared the bands that were not parents complementary bands, which brings difficulties to read tape, it often easy causes miscarriage of justice when identify the varietal identity. In this study, a good solution for the problems above was that use per plant to identify the varietal identity. Samples in the institutional capacity of national agricultural seed testing to verify the varietal identity of the SSR markers by DNA extraction of per plant, whose differences could not be detected about27%per plant checked in the24sites, whose differences were detected about73%per plant checked in the24sites. DNA extracted with a mixed sample, the amplification bands manifest as deep-undertone bands, deep band was the same with the check, shallow tape was inconsistent with the check, and this was not easy to accurately read the bands. By the method of extracting the DNA of per plant could be more accurately distinguish the differences of the electrophoretic bands, and could avoid the interference of the off-types.3.In making test of the varietal identity and purity, people always thought that the results of SSR Markers were more stringent than the results of field planting test, and the results were unfair to the enterprise when use SSR Marker to identify the varietal identity and purity. After making analysis on a large number of the results of SSR molecular marker test and the results of field planting test,this study showed that the results by6%polyacrylamide electrophoresis were closer to the results of field planting test,.The results in this study provided a strong scientific basis for chosen electrophoresis method, which was the setting standard use for varietal identity and purity. In this study,24pairs of primers which announced in agricultural industry standard NY/T1433-2007of China were used to identify11rice hybrid samples on behalf of the seed lots of77,680kg by SSR molecular marker test and the field planting inspection. The analysis by electrophoresing by6%non-denaturing polyacrylamide showed that:The result of SSR molecular marker test was86.6%,87.3%by field planting test, and the slightly difference of two methods was0.7%. The results of two identifications were all below the grade of96.0%that was the national quality standard, so the samples were unqualified. In addition, the average difference was0.9%between the results of3%agarose gel electrophoresis and the results of field planting test. The average difference was0.7%between the results of6%non-denaturing polyacrylamide gel electrophoresis and field planting test. It clearly showed that the results of6%non-denaturing polyacrylamide gel electrophoresis much more close to the results of field planting test. Use24pairs of primers which published on agricultural industry standard NY/T1433-2007of the People’s Republic of China to identify the varietal identity of448samples with standard samples which provided by the Ministry of Agriculture, and37samples was unqualified tested with6%non-denaturing polyacrylamide electrophoresis. Comparing the results of SSR molecular marker test and the results of field planting test show that13samples with no standard sample were not made SSR detection,6samples with no field control samples could not be carried out field test, and the remaining429samples SSR identification results consistentted with the field test results. This shows that the results of the SSR molecular marker identification of varietal identity and purity by6%non-denaturing polyacrylamide gel electrophoresis were accurate and reliable.The international community still was no uniform methods and standards to identify genetically modified component. With the development of identification technology for genetically modified crops, the technology of PCR has become one of the most important identification methods. But regular PCR only can amplify a gene one time, due to the limitations of multiplex PCR technology, therefore, the technology combined the individual multiplex PCR and regular electrophoresis could not meet the current requirement of transgenic crops test. And the number of gene amplification was very limited at one time. Gene chip was a basic chip which had a number of oligonucleotide probes, each point containing complementary linked with the sequence-specific probe and the target gene. Therefore, the gene chips could be combined with multiplex PCR technology, this could be use to detect many target genes simultaneously. This study applied the hybridization between the amplified product of multiple asymmetric PCR and the gene chip which fixed target exogenous gene-specific probes, in order to achieve the purpose to detect more target genes at one time. In this work, by collecting the commercial information on existing transgenic corn, designed11pairs of primers and probes by using the gene sequence information that had published, and optimized the conditions of asymmetric PCR amplification and hybridization, Finally the technology of bio-chip was used to accurate and rapid detect10genetically modified corn varieties sold well in the market.