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Method for analyzing genetic diversity of Amomum tsao-ko by using inter simple sequence repeat (ISSR) reaction system

A technology of genetic diversity and reaction system, applied in the field of molecular biology DNA labeling technology and application, to achieve the effects of high polymorphism, clear amplified fragments, and speeding up identification

Active Publication Date: 2017-09-15
HONGHE COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there have been reports on the use of ISSR markers to analyze the genetic diversity of Amomum amomum from different origins in Zingiberaceae plants, but in Tsaoguo, the method of using ISSR molecular marker technology for germplasm resource identification and genetic diversity analysis has not been reported yet.

Method used

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  • Method for analyzing genetic diversity of Amomum tsao-ko by using inter simple sequence repeat (ISSR) reaction system
  • Method for analyzing genetic diversity of Amomum tsao-ko by using inter simple sequence repeat (ISSR) reaction system
  • Method for analyzing genetic diversity of Amomum tsao-ko by using inter simple sequence repeat (ISSR) reaction system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] The extraction of embodiment 1DNA

[0056] (1) Experimental materials

[0057] The 91 Caoguo populations used in this experiment were collected from 8 Caoguo populations in Yunnan Province, including 13 in Jinping County, 12 in Yuanyang County, 11 in Luchun County, 9 in Pingbian County, 13 in Lancang County, and Baoshan County. There are 11 copies in the city, 12 copies in Lianghe County, and 10 copies in Yunxian County. See Table 1 for details.

[0058] Table 1 Test material numbers and sampling points

[0059]

[0060]

[0061] (2) Extraction of DNA

[0062] Young leaves were collected at the same time as the germplasm resource investigation for genome-wide DNA extraction. DNA was extracted by 2*CTAB method, and the extracted DNA samples were dissolved in TE buffer and stored at -20°C for later use. Before amplification, dilute the DNA sample with double distilled water to a working solution of 20ng / μl, which is used as a template for PCR amplification react...

Embodiment 2

[0063] Embodiment 2 ISSR-PCR reaction system of the present invention and screening of primers

[0064] (1) Using the orthogonal design method, for Mg 2+ , dNTPs and primer concentration, TaqDNA polymerase and the amount of template DNA were screened to obtain an ISSR-labeled PCR reaction system suitable for Caoguo. The orthogonal design of ISSR-PCR is shown in Table 2. The selected ISSR-PCR primer was UBC888, and the experiment was repeated 3 times. After score comparison, 10×PCR buffer (without Mg) in the 25 μl reaction system 2+ )3.0μL, Taq enzyme 1.5U, Mg 2+ 1.5mmol / L, dNTP 0.25mmol / L, primer 0.3μmol / L (each) and template DNA 50ng comprehensive amplification effect is the best.

[0065] Table 2 ISSR-PCR reaction L16 (4 5 ) Orthogonal Experiment Design Table

[0066]

[0067]

[0068] (2) The PCR amplification reaction program of the above reaction system is: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 1 min, annealing at 52°C for 1 min, extensi...

Embodiment 3I

[0075] Example 3 Application of ISSR-PCR Reaction System in Analysis of Genetic Diversity of 91 Caoguo Varieties

[0076] (1) Cluster analysis and principal coordinate analysis

[0077] A clear and reproducible band within the range of 100 to 2000 bp on the electrophoretic map was recorded as 1, and no band at the same position was recorded as 0, thereby generating the original matrix of 0 and 1. The total number of bands and the number of polymorphic bands amplified by each primer were counted. The SimQual program in NTSYS-pc (2.10e) software was used to calculate the similarity coefficient matrix, and the SHAN in the Clustering program was used to perform UPGMA (unweighted pair-group method with arithmetic means) clustering; the Tree plot module was used to generate a clustering diagram and a molecular evolutionary tree was constructed. According to the calculated similarity coefficient matrix, the Decenter data conversion is carried out, and then the principal coordinate a...

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Abstract

The invention belongs to the field of molecular biology DNA marker technique and application. The invention discloses a method for analyzing genetic diversity of Amomum tsao-ko by using inter simple sequence repeat (ISSR) reaction system. The ISSR reaction system comprises (per 25 muL) Mg<2+>-free 10*PCR buffer 3.0 muL, Taq DNA polymerase 1.5 U, Mg<2+> 1.5 mmol / L, dNTP 0.25 mmol / L, primers 0.3 mumol / , and template DNA 50 ng. The reaction mixture is amplified by the following procedures: pre-degeneration at 95 DEG C for 5 min, degeneration at 95 DEG for 1 min, annealing at 50-58 DEG C for 1 min, extension at 72 DEG C for 1 min, after 35 cycles, extension at 72 DEG C for 10 min, and storage at 4 DEG C. The method of the invention provides technical guide and theoretical support for Amomum tsao-ko resource identification, genetic diversity analysis and other scientific researches.

Description

technical field [0001] The invention belongs to the field of DNA marker technology and application of molecular biology, and in particular relates to a method for analyzing hereditary diversity of Tsaoguo by using an ISSR reaction system. Background technique [0002] Tsaoko (Amomum tsaoko Crevost et Lemaire) is a perennial herb in the genus Cardamom of the ginger family. It is not only an important raw material for food processing and light industry in my country, but also a traditional Chinese medicinal material. It has the functions of eliminating phlegm, warming the body, smoothing qi, and antimalarial. It is also a seasoning food for the public, and it is in great demand in the international and domestic markets. Tsaoguo has high requirements on the growth environment conditions. It generally grows in the northern tropics and mid- and south subtropical mid-low mountainous areas with an altitude of 800-1500m. The annual rainfall is 1200-1600mm, and the annual average temp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11G06F19/24
CPCC12Q1/686C12Q1/6895C12Q2600/156G16B40/00C12Q2565/125
Inventor 卢丙越马孟莉雷恩王田涛孟衡玲袁盛勇刘艳红苏一兰李春燕张薇张虹
Owner HONGHE COLLEGE
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