DNA Fingerprinting Inspection Technique For Citrus Nursery Tree Purity And Genuineness

Citrus is the largest production and consumption fruit in the world, the annual output exceeds one billion tons and the annual international trade is greater than 23 billions. At present, citrus cultivated area in china are more than 2 million ha, annual output surpass 2,400 tons, ranking first in the world. Citrus nursery tree is one of the key factors in achieving sustainable development of citrus industry. However, the quality of citrus nursery trees is still a serious problem, and incidents of impure varietals and false varietals in citrus nursery tree occur occasionally, which seriously damaged the planters' economic benefit. Therefore, it is of great significance to establish a fast and accurate identification technology for varietals purity and genuineness in citrus nursery tree. It is helpful to reduce impure varietals and false varietals, regulate citrus nursery stocks, protect the benefits of growers and resolve the di spute of vari etal s.In this paper, by means of perfecting the DNA fingerprint database, establishing a reasonable sampling plans and rapid DNA extraction techniques, a DNA fingerprinting technique has been developed for varietals purity and genuineness in citrus nursery tree The main results were as follows:1. Supplement of Citrus varieties fingerprint database selecting 30 citrus varieties, in which their DNA fingerprints were not constructed in our laboratory, were selected to establish their DNA fingerprints through SSR analysis. DNA characteristic fingerprints of 30 varieties were converted into the standard model fingerprint through automated fingerprint identification software Gel 2.0, added to the standard DNA fingerprinting database. 2. The study on DNA rapid extraction methodBy comparing the results of DNA quality and PCR amplification with different conditions of lysis time, extraction time and number, centrifugal force etc, a rapid DNA extraction technique, which can extract high quality and Integrity DNA meeting SSR analysis in one hour, was established based on the CTAB method.3. Study on sampling planThe sampling plan affects the accuracy of test results of purity and authenticity in citrus nursery tree. According to purity demand in production industry and sampling investigation in citrus nursery tree, the quality accepts limits value (AQL) was determined as 1 and then sampling plant contain 13 ranks range from 10 to 500,000 was established according to the national standards GB/T2828.1-2003. The plan overcomes shortcomings that sample test based on full inspection or percentage sampling inspection, and further perfect the technology to assay varietals purity and genuineness of nursery tree in citrus based on DNA fingerprinting.4. The application example of seedling purity and genuineness testAccording to the sampling plan, the DNA of 200 samples from 10000 plants (Amakusa or Nanko) were extracted with rapid extraction method for SSR analysis, the results showed that all of citrus varietals had clear and stable bands. Compared with standard fingerprints, these samples were consistent with the standard fingerprint.It showed that the DNA fingerprinting technique could be effectively applied to inspect varietals purity and genuineness in citrus nursery trees.