A Method for Analyzing Tsaoguo Genetic Diversity Using Scot Molecular Markers

A technology of genetic diversity and molecular markers, applied in the field of DNA marker technology and application in molecular biology

Active Publication Date: 2021-07-06
HONGHE COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in Tsaoguo, the method of using SCoT molecular marker technology for germplasm resource identification and genetic diversity analysis has not been reported yet.

Method used

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  • A Method for Analyzing Tsaoguo Genetic Diversity Using Scot Molecular Markers
  • A Method for Analyzing Tsaoguo Genetic Diversity Using Scot Molecular Markers
  • A Method for Analyzing Tsaoguo Genetic Diversity Using Scot Molecular Markers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] The extraction of embodiment 1DNA

[0054] (1) Experimental materials

[0055] 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.

[0056] Table 1 Test material numbers and sampling points

[0057]

[0058] (2) Extraction of DNA

[0059] 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 reaction.

Embodiment 2

[0060] Embodiment 2PCR reaction system optimization and primer screening

[0061] (1) Using the orthogonal design method, for Mg 2+ , dNTPs and primer concentration, TaqDNA polymerase and the amount of template DNA and other 5 factors were screened, and the SCoT-labeled PCR reaction system suitable for Tsaoguo was obtained. The orthogonal design of SCoT-PCR is shown in Table 2. The selected SCoT-PCR primer was SCoT15, 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 0.5U, Mg 2+ 2.5mmol / L, dNTP 0.25mmol / L, primer 0.5μmol / L and template DNA 60ng comprehensive amplification effect is the best.

[0062] Table 2 SCoT-PCR reaction L16 (4 5 ) Orthogonal Experiment Design Table

[0063]

[0064] (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 50°C for 1 min, extension at 72°C ...

Embodiment 3

[0070] Application of Example 3 SCoT-PCR Reaction System in Analysis of Genetic Diversity of 91 Caoguo Varieties

[0071] (1) Cluster analysis

[0072] 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.

[0073] Based on SCoT markers, cluster analysis was performed on 91 Tsaoguo samples from 8 populations using NTSYSpc 2.10e software. Based on the 10 SCoT primers at the ge...

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Abstract

The invention discloses a method for analyzing Tsaoguo genetic diversity by using SCoT molecular markers, wherein the SCoT-PCR reaction system is as follows: each 25 μL reaction system contains 2+ 10×PCR buffer 3.0μL, Taq enzyme 0.5U, Mg 2+ 2.5mmol / L, dNTP 0.25mmol / L, primer 0.5μmol / L and template DNA 60ng; the above reaction mixture was amplified according to the following procedure: pre-denaturation at 95°C for 5min, denaturation at 95°C for 1min, annealing at 48‑52°C for 1min , extended at 72°C for 2min, 35 cycles, finally extended at 72°C for 10min, and stored at 4°C. The invention has great significance for the protection, breeding and utilization of near-threatened grass fruit resources, and further improves the economic income of the grass fruit planting farmers in mountainous areas.

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 Tsaoguo genetic diversity by using SCoT molecular markers. 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 temperature...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/156
Inventor 卢丙越马孟莉王田涛孟衡玲雷恩苏一兰李春燕刘艳红张虹张薇袁盛勇
Owner HONGHE COLLEGE
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