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Primer for genuine ligusticum wallichii cpSSR molecular identification and kit and application thereof

A molecular identification and kit technology, applied in the field of genetic engineering, can solve problems such as irregular drug use, and achieve high polymorphism, good repeatability, and accurate results

Pending Publication Date: 2022-01-14
四川省农业科学院经济作物育种栽培研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The counterfeit products of Chuanxiong from different origins are mixed as authentic Chuanxiong medicinal materials locally, and the problem of non-standard use of "Chuanxiong" with the same name and different species also occurs in various places

Method used

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  • Primer for genuine ligusticum wallichii cpSSR molecular identification and kit and application thereof
  • Primer for genuine ligusticum wallichii cpSSR molecular identification and kit and application thereof
  • Primer for genuine ligusticum wallichii cpSSR molecular identification and kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Design screening of primers

[0022] 1, CTAB method to extract genomic DNA

[0023] (1) Each material (see Table 1) Take 3 pieces of young leaves in 2 ml EP tube, simultaneously add 5mm glass beads, quickly put into liquid nitrogen to quickly freeze, then put liquid nitrogen pre-cooling Scientz-48 Type High General Tissue Grinder (Ningbo Xinzhi Co., Ltd.) grinds with 50Hz and 60S program.

[0024] (2) After the grinding is completed, 870 L CTAB liquid (containing 0.4% β-mercaptoethanol) is added, and the shake is mixed, and the water bath preheated to 65 ° C is taken after 50min, the water bath, every 10 minutes will be sampled every 10 minutes. Tube takes out a shake.

[0025] (3) After the water bath is completed, take the template and drop to room temperature. Chloroform: isopentyl alcohol (24: 1) mixture 870L, which was placed on a shaker for 15 min, 10,000 rpm, 25 ° C for 10 min.

[0026] (4) Using a tip tab to absorb the supernatant to 2 MLP tube with 1 ml o...

Embodiment 2

[0038] Example 2 Primer specificity and polymorphism detection

[0039] 1, specific verification

[0040] For 14 pairs of Chuanxiong CPSSR primers with polymorphism, it is found that 2 pairs of primers can be completely distinguished by Chuanxiong and 5 mixed goods materials, which can be used as specific primers identified by Chuanxiong and mixed counter, CY13 can be district , Gansu Chuanxiong, Liaoyota and 4 materials ( figure 1 ), CY18 can distinguish between Fuxion and Yunnan Chuanxiong (2 materials ( figure 2 . In the figure, CX: L.chuanxiong, GS: L.Chuanxiong Cv.gansu, LGB: L. Jeholensen, GB: 藁本 (L. Sinense), FX: Fuxion (L. SINENSE CV.FUXIONG), YN: Yunnan Chuanxiong (L.chuanxiong Cv.yunnan).

[0041] 2, polymorphism detection

[0042]PCR amplification and detection using this 14 pair of primers, and the results showed that 14 pairs of primers were expanded in the test material, 32 multi-state strips, The percentage of polymorphism is 97.0%. Analysis of the genetic diversity...

Embodiment 3

[0046] Example 3 Chuanxiong and its hybrid product cluster analysis

[0047] The similar coefficient matrix of the test sample is established by NTSYS2.10E using the marker information generated by the 14 CPSR primers. The genetic similarity factor of Chuanxiong and the mixed counter is between 0.364 to 0.970, and the average genetic similar coefficient is 0.664. The results of CPSSR cluster analysis of Chuanxiong and mixed goods image 3 . At the genetic similarity coefficient, 6 materials are gathered into 3, Chuanxiong, Gansu Chuanxiong and Liao Hao are a class, and the 藁本 is a class, Fuxim and Yunnan Chuanxiong gather together.

[0048] The 21-to-CPSSR primer is tested in Chuanxiong and its mixed officers in 21 chloroplast genomic data designs, and 14-to-CPSR primers can be expanded in the test materials. Sex is 0.4716, the polymorphic information content is 0.3954, wherein the two pairs of primer combinations can directly distinguish between 6 Test materials. Cluster analysis ...

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Abstract

The invention discloses a primer for genuine ligusticum wallichii cpSSR molecular identification and a kit and application thereof. The specific sequences of the primers are as shown in SEQ ID NO. 1-28. Szechuan lovage rhizome and adulterants thereof are detected by 21 pairs of cpSSR primers designed according to data of chloroplast genomes of 7 ligusticum, 14 pairs of cpSSR primers can amplify polymorphic bands in test materials, the gene diversity is 0.4716, the polymorphic information content is 0.3954, and 6 test materials can be directly distinguished by combining two pairs of primers. The designed primer sequence can be used for researches such as genetic map construction, variety identification and the like of ligusticum wallichii.

Description

Technical field [0001] The present invention belongs to the field of genetic engineering, and more particularly to primers identified by Tao Kagawa CPSSR molecules and their kits and applications. Background technique [0002] Chuanxiong is a dry root stem in the umbrella, and the dry roots of the "Shennong Herb", which is listed as top, sex, temperature, with a blood circulation, hurricane pain relief. Chuanxion is a kawaki plant, the total cultivation area is stable for more than 90,000 mu, and the annual output is more than 20,000 tons, which is an important component of herbody. Chuanxion is part of the martial arts, and this is one of the most difficult groups in the umbrella department. It often has the same name of the same name or the same number of abnormalities, leading to the circulation of Chuanxiong in the market unknown, more introduce Chuanxiong And near-territorial species, the phenomenon of Tao Chuanxion, and the source of 农 购 源In terms of cultivated plants, the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6858C12N15/11
CPCC12Q1/6895C12Q1/6858C12Q2600/13C12Q2600/156C12Q2531/113C12Q2565/125
Inventor 陶珊张超廖旭红彭芳毛常清吴宇李旭东李生翠赵豆豆
Owner 四川省农业科学院经济作物育种栽培研究所