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A method for analyzing the genetic diversity of Tsaoguo germplasm resources based on srap markers

A technology of genetic diversity and fruit, applied in the field of genetic diversity analysis of grass and fruit germplasm resources based on SRAP markers, which can solve the problem of large experimental error, limitation of identification of grass and fruit resources and genetic diversity analysis, and morphological characters susceptible to the environment. Condition effects and other problems, to achieve the effect of clear bands, accelerated identification speed, and stable and reliable results

Active Publication Date: 2021-02-23
HONGHE COLLEGE
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In view of this, the present invention still adopts the traditional morphological identification method for the identification of Tsaoguo at present, mainly divides according to the shape, size, color and luster of the fruit, and the morphological traits are easily affected by environmental conditions, and the experimental error is relatively large, which limits people. For the identification of Tsaoguo resources and the analysis of genetic diversity, a relatively scientific, reasonable and effective method for analyzing the genetic diversity of Tsaoko germplasm resources based on SRAP markers is provided, which can provide good technical guidance for subsequent scientific research and theoretical support

Method used

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  • A method for analyzing the genetic diversity of Tsaoguo germplasm resources based on srap markers
  • A method for analyzing the genetic diversity of Tsaoguo germplasm resources based on srap markers
  • A method for analyzing the genetic diversity of Tsaoguo germplasm resources based on srap markers

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Experimental program
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Effect test

Embodiment 1

[0045] Example 1 Method for analyzing genetic diversity of Tsaoguo germplasm resources based on SRAP markers

[0046] (1) Tsaoguo genomic DNA was extracted by 2*CTAB method for future use.

[0047] (2) Caoguo SRAP-PCR reaction system optimization, the optimized reaction system is characterized in that: 25 μ L reaction system includes: 10 × PCR buffer (without Mg 2+ )3.0μL, Taq enzyme 1U, Mg 2+ 1.5mmol / L, dNTP 0.25mmol / L, primer 0.2μmol / L and template DNA 30ng.

[0048] (3) Improvement of the PCR amplification reaction program, the improved program: pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 1 minute, annealing at 35°C for 1 minute, extension at 72°C for 1 minute, 35 cycles, final extension at 72°C for 10 minutes, and storage at 4°C .

[0049] (4) Screening of SRAP polymorphic primers. From 153 pairs of SRAP primers, 12 pairs of stable, clear and reproducible polymorphic primers were screened out for PCR amplification reaction of different populations o...

Embodiment 2

[0051] The extraction of embodiment 2DNA

[0052] (1) Experimental materials

[0053] The 96 Tsaoguo used in this experiment were collected from 8 Tsaoguo producing areas in Yunnan Province, including 13 from Jinping County, 12 from Yuanyang County, 11 from Luchun County, 10 from Pingbian County, 14 from Pu’er Lancang County, Baoshan There are 11 copies in the city, 13 copies in Dehong Lianghe County, and 12 copies in Lincang Yun County. See Table 1 and Table 2 for details.

[0054] Table 1 Test material number (1-48) and sampling points

[0055]

[0056]

[0057] Table 2 Test material number (49-96) and sampling points

[0058]

[0059]

[0060] (2) Extraction of DNA

[0061] 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 sampl...

Embodiment 3

[0062] Embodiment 3PCR reaction system optimization and primer screening

[0063] Using the orthogonal design method, the Mg 2+ , dNTPs and primer concentration, TaqDNA polymerase and the amount of template DNA were screened, and the SRAP-labeled PCR reaction system suitable for Tsaoguo was obtained. The orthogonal design of SRAP-PCR is shown in Table 3. After three repeated tests of primers me1em15, me4em8, me7em1, and me8em14, 10×PCR buffer (excluding Mg 2+ )3.0μL, Taq enzyme 1U, Mg 2+ 1.5mmol / L, dNTP 0.25mmol / L, primer 0.2μmol / L and template DNA 30ng comprehensive amplification effect is the best.

[0064] Table 3 SRAP-PCR reaction L16 (4 5 ) Orthogonal Experiment Design Table

[0065]

[0066]

[0067] The PCR amplification reaction program of the above reaction system is an improved program: pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 1 minute, annealing at 35°C for 1 minute, extension at 72°C for 1 minute, 35 cycles, and finally extension ...

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Abstract

The invention discloses a SRAP marker-based Amomum tsao-ko germplasm resource genetic diversity analysis method. A SRAP-labeled primer pair is selected from me1-em11, me1-em12, me1-em15, me2-em11, me2-em12, me5-em5, me5-em6, me6-em2, me6-em5, me6-em14, me9-em6 or me9-em11. The SRAP-PCR reaction system is characterized in that each 25 microliters of the reaction system contains 3.0 microliters of a 10*PCR buffer without Mg<2+>, 1U of Taq enzyme, 1.0 mmol / L of Mg<2+>, 0.25 mmol L of dNTP, 0.2 micromoles per liter of primers and 30ng of a template DNA. The method provides good technical guidance and theoretical support for subsequent scientific research.

Description

technical field [0001] The invention belongs to the field of molecular biology DNA marker technology and application, in particular to a method for analyzing the genetic diversity of Tsaoko germplasm resources based on SRAP 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 temperatur...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/6858C12N15/11G16B40/00G16B30/00
CPCC12Q1/6858C12Q1/6895C12Q2600/156G16B30/00G16B40/00C12Q2531/113C12Q2565/125
Inventor 卢丙越马孟莉孟衡玲王田涛雷恩李春燕张虹张薇苏一兰刘艳红袁盛勇
Owner HONGHE COLLEGE
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