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Molecular marking method for carrying out genetic relationship and clustering analysis on citrus fruit flies

A citrus fruit fly, kinship technology, applied in biochemical equipment and methods, microbial determination/inspection and other directions, can solve the problem of inability to accurately and quickly detect and identify citrus fruit fly, etc. High, optimized reaction conditions, stable effect of PCR amplification reaction

Inactive Publication Date: 2014-10-08
CITRUS RES INST SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional identification method of Bactrocera citrus has always been based on morphology, but this method cannot accurately and quickly detect and identify Bactrocera citrus in the larval stage

Method used

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  • Molecular marking method for carrying out genetic relationship and clustering analysis on citrus fruit flies
  • Molecular marking method for carrying out genetic relationship and clustering analysis on citrus fruit flies
  • Molecular marking method for carrying out genetic relationship and clustering analysis on citrus fruit flies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 extracts the DNA of Bactrocera citrus

[0023] 1 Gathering materials

[0024] The collected samples (adults and larvae) from 10 different geographical populations of Bactrocera citrus in my country were soaked in absolute ethanol and stored at -20°C. See Table 1 for information on the collection points of the population and the number of populations collected.

[0025] Table 1 Collection information of Bacteralis citrus samples tested

[0026]

[0027]

[0028] 2 Extraction of DNA from Bactrocera citrus

[0029] Get the citrus fruit fly worm bodies of 10 different geographical populations collected above, adopt the CTAB method to extract genomic DNA, and operate according to the following steps:

[0030] (1) Take 100 mg of the body of Bactrocera citrus, add liquid nitrogen and grind it into powder, and quickly transfer it into a 1.5 ml Eppendorf tube.

[0031] (2) Add 800 μl of CTAB extraction buffer to the centrifuge tube in step 1, mix well (CTAB i...

Embodiment 2

[0038] Embodiment 2RAPD analysis

[0039] Seven RAPD primers were used to perform PCR amplification on the DNA samples of 10 different geographic populations of B. citrus extracted in Example 1, and the RAPD random primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. The names and sequences of the seven RAPD primers are as follows: 1: ACGCCAGAGG, 2: GAGCGAGGCT, 3: CAGGGGTGGA, 4: GGTCTGGTTG, S75: GACGGATCAG, Y06: AAGGCTCACC, Y18: GCTGAGTCAG. The total volume of the PCR reaction system is 25uL, including 1×PCRBuffer2.0μL, 1.5mmol / μL MgCl 21.5 μL, 500 μmol / L dNTPs 1.5 μL, 0.5 μmoL / L primer 1.0 μL, 1U Taq enzyme 0.4 μL, 20ng / μL DNA template 1.0 μL, make up water to 25 μL. The amplification program was: 94°C pre-denaturation for 5 minutes; 94°C for 60 s, 38°C for 60 s, 72°C for 90 s, a total of 32 cycles; 72°C for 10 min.

[0040] The amplified product was electrophoresed on 1.5% agarose gel (1×TBE buffer, 4V / cm electrophoresis), stained with et...

Embodiment 3

[0041] Embodiment 3SRAP analysis

[0042] 10 pairs of SRAP primer combinations were used to perform PCR amplification on the DNA samples of B. citrus of 10 different geographical populations extracted in Example 1, and the SRAP primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. The total volume of the PCR reaction system is 25uL, including 1×PCR Buffer2.0μL, 1.5mmol / μL MgCl 2 1.5 μL, 500 μmol / L dNTPs 1.5 μL, 0.5 μmoL / L upstream and downstream primers 1.0 μL each, 1U Taq enzyme 0.4 μL, 20ng / μL DNA template 1.0 μL, water to 25 μL. Amplification program: 94°C pre-denaturation for 3 minutes; 94°C for 40s, 63°C for 40s, 72°C for 60s; cycle 15 times, each cycle annealing temperature decreased by 0.5°C; 94°C for 40s, 55.5°C for 40s, 72°C for 60s, cycle 25 times ; 72°C extension for 10 min. The 10 pairs of SRAP primer combinations are named: 3F-1R, 3F-2R, 9F-1R, 9F-3R, 9F-2R, 9F-11R, 4F-10R, 12F-11R, 10F-3R, 10F-10R ; Wherein, each primer seque...

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Abstract

The invention relates to a molecular marking method for carrying out genetic relationship and clustering analysis on citrus fruit flies and belongs to the technical field of molecular marking. The molecular marking method for carrying out genetic relationship and clustering analysis on the citrus fruit flies comprises the following steps: carrying out PCR (polymerase chain reaction) amplification on DNA of citrus fruit flies by utilizing seven RAPD (random amplification polymorphism DNA) primers and 10 pairs of SRAP (sequence-related amplified polymorphism) primer combinations, carrying out agarose gel electrophoresis on amplification products, taking pictures, recording results, and carrying out statistics on an amplification zone of a detected material; and analyzing with genetic analysis software to obtain genetic distance between individuals, and drawing a dendrogram of citrus fruit flies in different geographical populations for distinguishing different class groups. The molecular marking method for carrying out genetic relationship and clustering analysis on the citrus fruit flies has the advantages of stable reaction, clear amplified fragments and high polymorphism, a molecular reaction system for population genetic diversity of the citrus fruit flies is constructed, genetic diversity among populations can be shown, clustering analysis can be rapidly and accurately carried out on the citrus fruit flies, different populations can be distinguished, and the molecular marking method for carrying out genetic relationship and clustering analysis on the citrus fruit flies has an important significance on prevention and control of the citrus fruit flies.

Description

technical field [0001] The invention belongs to the technical field of molecular markers, and relates to a molecular marker method, in particular to a molecular marker method for performing kinship and cluster analysis on Bactrocera citrus. Background technique [0002] Citrus is the world's largest fruit, with an annual output of more than 100 million tons, accounting for about a quarter of the world's total fruit output. At present, the citrus industry has become one of the pillar industries of the rural economy in the vast areas of southern China, poor mountainous areas and the Three Gorges reservoir area. Citrus fruit fly Bactrocera (Tetradacus) minax (Enderlein) is commonly called as " citrus maggot ", has another name called citrus fruit fly, citrus fruit fly, and the victim fruit is called "maggot fruit" and "citrus maggot". Quarantine pests. The traditional identification method of Bactrocera citrus has always been based on morphology, but this method cannot detect...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6888C12Q2600/156C12Q2600/16
Inventor 刘浩强冉春李鸿筠丛林胡军华姚廷山
Owner CITRUS RES INST SOUTHWEST UNIV
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