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Method for identifying camellia oleifera cultivar and special primer and kit thereof

A kit and variety technology, applied in the direction of DNA / RNA fragments, recombinant DNA technology, etc., can solve the problems of allelic site length polymorphism among varieties, and achieve reliable detection results, good practicability, and simple operation

Inactive Publication Date: 2012-01-18
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] There are differences in the number of SSR repeats at the same site among different varieties of Camellia oleifera, resulting in the length polymorphism of allelic sites among varieties

Method used

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  • Method for identifying camellia oleifera cultivar and special primer and kit thereof
  • Method for identifying camellia oleifera cultivar and special primer and kit thereof
  • Method for identifying camellia oleifera cultivar and special primer and kit thereof

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Experimental program
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Effect test

Embodiment 1

[0024] The microsatellite primers were developed based on a large number of Camellia oleifera genome sequences obtained by next-generation high-throughput sequencing. First, high-throughput sequencing was used to complete the "Shotgun" sequence determination of 348 million bases in the Camellia oleifera genome. The number of chromosomes in the diploid genome of Camellia oleifera is 2n=30, and the genome size is about 3800Mb. However, polyploidy of Camellia oleifera is common in cultivated varieties. In order to improve the efficiency of sequencing and assembly, diploid Camellia oleifera is preferably used for DNA sequencing. Zhejiang safflower camellia is diploid, and the sequencing individual selected in this patent is a Zhejiang safflower camellia collected from Taining, Fujian.

[0025]In order to meet the quality requirements of DNA sequencing, the sequencing DNA template was extracted with Qiagen DNeasy Plant Mini Kit, the concentration was detected by a micro-nucleic aci...

Embodiment 2

[0033] The 10 pairs of primers screened in Example 1 were used to detect the genotypes of 128 Camellia oleifera varieties collected from Guangxi, Hubei, Hunan, Jiangxi, Anhui, Fujian and other places. The specific method is as follows:

[0034] DNA was extracted from tea leaves according to the method of Murry and Thomson (1990). Then, using this as a template, 10 pairs of primers obtained by screening were used for PCR amplification on an ABI-9700 thermal cycler. The total reaction system of PCR is 15uL, and the reaction system includes: template DNA 25ng, upstream and downstream primers 20ng each (FAM label), 200uM dNTP, 0.5U Taq polymerase, 1.5uL 10×buffer (containing 100uMTris-HCl, pH 8.3, 500mM KCl, 20mM MgCl 2 and 10.0g / L BSA), add sterilized deionized water to supplement the reaction volume to 15uL. The PCR reaction conditions were: pre-denaturation at 94°C for 2 min; then denaturation at 94°C for 30 s, annealing at 50°C for 30 s, and extension at 72°C for 1 min, for...

Embodiment 3

[0046] Using the primers screened in Example 1, a batch of samples sent by the Southern Seed Inspection Center of the State Forestry Administration that may be Cenruan No. 3 or may be counterfeit Cenruan No. 3 were tested, and a total of 9 samples were sent for inspection. During the experiment, the standard Cenruan No. 3 was used as the control. In order to verify the repeatability of PCR, two replicates were set up as the control. The 11 samples including the control were optionally amplified by PCR with primers NJFUC-243 and NJFUC-57. The genotypes amplified by these two primers in Cenruan No. 3 had a low frequency in the 128 varieties listed in Table 2.

[0047] In this experiment, the amplification reaction system and amplification conditions were the same as in Example 1. After the amplification, the amplified products were detected by electrophoresis using 8% polyacrylamide gel. The specific operation steps are as follows: prepare 8% polyacrylamide gel; after polymeri...

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Abstract

The invention discloses a method for identifying camellia oleifera cultivars and a special primer and a kit thereof; according to the invention, DNA of a camellia oleifera sample to be identified and DNA of a standard control sample are used as templates; PCR amplification is performed with the leading of a same primer pair to obtain a product; the product is treated by electrophoresis; genotype comparison of the electrophoresis results is performed; if the genotypes are different, the cultivars are different; otherwise similarity probability calculation is performed; if the detection requirements are not met, a primer is selected again for PCR identification till the identification probability requirements are met; compared with present camellia oleifera identification methods, the invention comprises the following outstanding advantages: 10 pairs of special microsatellite primers for camellia oleiferaare used to identify the genetic authenticity of camellia oleifera cultivars; the identification method has simple and rapid operations, and the identification accuracy is high. In addition, a corresponding detection kit developed by the method of the invention can be used to identify the genetic authenticity of camellia oleifera cultivars, and has the advantages of simple application method, speediness, and sensitivity; the detection results are reliable, stable, and accurate; and the kit provides reliable technical means for the identification of genetic authenticity of camellia oleifera cultivars. The invention plays an important role in the supervision and examination of the application of camellia oleifera improved varieties, has wide application prospects, has very good practicality, and can provide good economic benefit and social benefit.

Description

technical field [0001] The invention relates to an identification method of camellia oleifera varieties, in particular to a gene-based method for identification of camellia oleifera varieties and its special primers and method. Background technique [0002] Camellia oleifera is one of the world's four major woody oil tree species, and it is also the most important woody oil economic crop in my country. It has a long lifespan and has the characteristics of one-time planting and many years of benefit. The stable harvest period can be as long as more than 80 years. However, once improper or inferior varieties are misused for afforestation, the impact will be far-reaching. In recent years, the Chinese government has invested heavily in the development of Camellia oleifera industry. According to the national development plan, the national Camellia oleifera planting scale will reach about 60 million mu by 2015. During the vigorous development of the camellia oleifera industry, w...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 尹佟明施季森史洁管宏伟戴晓港陈金慧
Owner NANJING FORESTRY UNIV
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