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Method for identifying tea aphids by utilizing mitochondrial molecular markers and application of method

A molecular marker, mitochondrial technology, applied in the field of molecular biology, can solve the problems of lack of good prevention methods, affecting the yield and quality of tea, and restriction of tea aphid, and achieves clear and easy-to-read bands, good application prospects, and polymorphism. high effect

Pending Publication Date: 2020-08-11
INST OF PLANT PROTECTION SICHUAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Tea aphids harm tea gardens in a large area in spring and autumn. The harmful behavior is to gather on the tea shoots and young leaves to suck the juice. When the stylet is inserted into the tea tree, the body secretes saliva, which destroys the normal physiological metabolism of the tea tree and eventually causes the buds and leaves to shrink. , growth stagnation, the dry tea produced has low aroma, light taste, and turbid soup color, which seriously affects the yield and quality of tea
[0003] Although the tea aphid is a serious hazard, the current prevention and control methods are mainly treated by chemical agents. The use of chemical agents can achieve certain effects, but long-term use is likely to cause chemical drug residues, and the tea aphid may also develop drug resistance. So far there is no good way to prevent
[0004] Biological prevention of pests and diseases is an effective and environmentally friendly way, but there have been few studies and reports on the genetic information of the tea aphid itself, which greatly limits how to use biological information to control the harm of the tea aphid
[0005] Due to the serious harm of tea aphids, which seriously affects the development of tea planting, it is of great significance to carry out the identification of tea aphids, the construction of fingerprints, the evaluation of genetic diversity and the study of phylogenetic relationships. However, the above work depends on the high polymorphism. Development of molecular markers with good reproducibility, stable labeling, and clear and easy-to-read band patterns

Method used

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  • Method for identifying tea aphids by utilizing mitochondrial molecular markers and application of method
  • Method for identifying tea aphids by utilizing mitochondrial molecular markers and application of method
  • Method for identifying tea aphids by utilizing mitochondrial molecular markers and application of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1 SSR primer design

[0043]The inventor tested the whole genome sequence of the citrus leaf miner in the early stage, and constructed a developmental tree, such as Figure 4 As shown, its species status was confirmed. For the SSR loci obtained from the whole genome sequencing determined in the previous stage, 5 pairs were randomly selected on each chromosome of the mitochondria, and primers were designed using the flanking sequences at both ends of the repeat sequence. The design principles were as follows:

[0044] ●PCR product length range is 100-350bp;

[0045] The annealing temperature Tm is 50-70°C, ensuring that the difference between the Tm values ​​of the two primers does not exceed 4°C;

[0046] ●GC% content is 40-65%;

[0047] ●The length of the primer is 18-28bp;

[0048] ●In order to ensure the specificity of the primers, the conserved flanking sequences used for primer design are at least 20-23 bases apart from the microsatellite sites.

[...

Embodiment 2

[0050] Example 2 Primer Screening

[0051] The SSR primers designed in the process of Example 1 are used for PCR amplification through the genome of the sample. In order to ensure the accuracy and reliability of subsequent data analysis, two conditions are required. First, use low cycle number amplification as much as possible; secondly, ensure that the cycle number of each sample amplification is uniform. Randomly select representative samples for pre-experiment to ensure that most of the samples can amplify products with appropriate concentration in the lowest number of cycles, making full preparations for the formal experiment of all samples. There is a total of 1 group of experiments to obtain high-quality PCR products. The statistics of PCR amplification results so far are as follows:

[0052] Glue number sample name Sequencing primers m(℃) cycle result Sample2 tea aphid cox1_Yachong 55 35 A

[0053] The primer sequences screened out a...

Embodiment 3

[0056] Embodiment 3 Utilizes the method for the identification of tea aphids by mitochondrial molecular markers

[0057] 1. Mitochondrial DNA extraction

[0058] Genomic DNA in the mitochondria of the test samples was extracted respectively:

[0059] DNA extraction kit: Brand: Tiangen Item No.: DP304-03;

[0060] Operation process of centrifugal adsorption column DNA extraction:

[0061] ●Add 200μl GA buffer to fresh tissue samples, after sample treatment before extraction, add 20μl proteinase K to lyse, and the lysis time is 1-3h;

[0062] After the tissue lysis is completed, add 200 μl GB buffer, mix thoroughly by inversion, let stand at 70°C for 10 minutes, and briefly centrifuge to remove water droplets on the inner wall of the tube;

[0063] ●Add 200μl of absolute ethanol, shake and mix well, you can see flocculent precipitate, briefly centrifuge to remove water droplets on the inner wall of the tube;

[0064] After the previous operation, all the solution and precipi...

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Abstract

The invention discloses a method for identifying tea aphids by utilizing mitochondrial molecular markers and an application of the method, and belongs to the technical field of molecular biology. Themolecular markers comprise the following primers: F: 5'-TGGAACAGGAACAGGATGAACT-3'; and R:5'-ACTCCTGTTAATCCTCCGATTGT-3'. The mitochondrial molecular marker has the advantages of good stability, high polymorphism and good repeatability; and the amplified strip is clear and easy to read, can be effectively applied to the fields of tea aphid identification, DNA fingerprint construction, genetic diversity evaluation, phylogenetic research and the like, provides a new tool for tea aphid molecular marker-assisted selection, and has a good application prospect.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a method for identifying tea aphids using mitochondrial molecular markers and an application thereof. Background technique [0002] The whole mitochondrial genome of the tea aphid was sequenced and analyzed. The tea aphid Toxoptera aurantii Boyer, also known as the tea aphid, belongs to the Hemiptera family Aphididae. It mainly feeds on the tender parts of tea trees and is one of the main pests of tea trees. Tea aphids are widely distributed in the four major tea regions of Jiangnan, Jiangbei, South China, and Southwest China, and are distributed in major tea-producing countries such as India, Kenya, and Japan abroad. Tea aphids harm tea gardens in a large area in spring and autumn. The harmful behavior is to gather on the tea shoots and young leaves to suck the juice. When the stylet is inserted into the tea tree, the body secretes saliva, which destroys the normal ph...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/6888C12Q1/686C12Q2565/125
Inventor 蒲德强刘虹伶刘超伍兴隆肖科军陈宇罗曦毛建辉
Owner INST OF PLANT PROTECTION SICHUAN ACAD OF AGRI SCI
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