| Use molecular markers to identify the authenticity and purity of crop varieties indoors.It has the characteristics of short identification cycle,free from seasonal and climatic conditions,and provinces of industry and land.It is used in new variety approval,germplasm resource protection,market supervision and management,etc.It has been widely used and has become an effective technical means for assisting rice field planting identification.The optimization of DNA extraction materials is a key step in the identification system of variety authenticity molecular markers.In view of the advantages of leaf tissues such as tender and fragile,traditional DNA extraction generally uses leaves as materials,but to obtain leaves,the seeds must be germinated to further grow into seedlings for sampling,which takes a long time.This thesis studies the feasibility of extracting DNA with a kit method using seeds as materials,with a view to using seeds to extract DNA for rapid and efficient identification of the authenticity of varieties.On this basis,the industry standards of the Ministry of Agriculture(NY/1433-2007)The recommended 24 pairs of SSR primers are used to construct the authenticity fingerprints of77 rice varieties approved in Jiangxi Province in recent years,aiming to provide a way to quickly identify and distinguish rice seed varieties for Jiangxi Province seed enterprises and municipal and county seed quality inspection agencies in the future.According to the basis,use the molecular characteristics of the varieties in the map to assist the DUS test to strengthen the protection of variety resources and provide technical support.The main results of the research are as follows:1.The feasibility of extracting rice genomic DNA using a kit method from seeds was studied.The concentration and purity of DNA extracted from seeds,grains and leaves were compared and analyzed.The results showed that the overall concentration and purity of DNA extracted from the three materials were higher.Although there is a small amount of RNA residue,there are fewer contaminants such as carbohydrates and peptides.Two pairs of SSR primers RM267 and RM274 were used to perform PCR amplification on the DNA extracted from the three materials by agarose gel electrophoresis.The results showed that the amplification results of seed and leaf materials showed clear and bright band patterns,but the amplification results of grain materials were partially missing.The experimental study found that using the seed as the material and using the kit method can extract high-quality genomic DNA,which meets the requirements of PCR amplification in the authenticity identification of varieties.2.Using 24 pairs of SSR primers,the fingerprint maps of 77 rice varieties approved in Jiangxi Province in recent years were constructed.A total of 103 alleles were detected in24 pairs of primers,with an average of 4.29 alleles detected per primer,and the highest detection of 7 alleles;a total of 155 band types were amplified,of which 17 varieties were labeled A specific band pattern is shown on the primer.The results showed that the 24 pairs of SSR primers used in the experiment showed abundant polymorphism in 77 rice varieties,but some rice varieties also showed a low degree of heterogeneity,reflecting that there may be a homogeneity problem in the region at the emerging stage.3.The ntsys cluster analysis was performed on 77 rice varieties,and the results showed that the genetic similarity coefficient was between 0.62 and 1.00.The genetic relationship among most rice varieties was close.Among them,Yongyou 6715 and Yongyou 6763 were three-line hybrids of indica and japonica.Rice,Jinyou 207 is an indica three-line hybrid rice and can be clearly distinguished from the other two-line rice varieties on the cluster tree.For some varieties to be clearly identified,the 48 pairs of primers in the "NY/T 1433-2014 Rice Variety Identification Technical Regulations SSR Marking Method" can be used to improve the identification effect or refer to the field trait performance to distinguish. |
