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Method for identifying Shida tea varieties through SSR fingerprint spectrum

A technology of fingerprinting and persimmon tea, applied in the field of molecular biology, can solve problems such as difficult to distinguish, and achieve high accuracy, high polymorphism, and good stability

Pending Publication Date: 2018-11-20
ANHUI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In recent years, the demand for my country's famous and high-quality tea has been increasing and new varieties of tea trees have been successfully cultivated. Only Shida Cha has cultivated Shida Tea No. 1, Shida Tea No. 2, Shida Tea No. 6, and Shida Tea Yellow Variety, etc. There are many new varieties and famous green teas made from different persimmon teas. The quality and price of green tea are also different. It is difficult for consumers to distinguish the true from the fake and the difference in raw materials from the appearance and shape with the naked eye.

Method used

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  • Method for identifying Shida tea varieties through SSR fingerprint spectrum
  • Method for identifying Shida tea varieties through SSR fingerprint spectrum
  • Method for identifying Shida tea varieties through SSR fingerprint spectrum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The extraction of embodiment 1 tea fresh leaf total DNA

[0029] (1) The selected tea tree varieties: No. 1 Shidacha; No. 2 Shidacha; No. 6 Shidacha; Huangzhong Shidacha. Provided by the Industry-University-Research Base of Guohe Modern Agriculture Demonstration Zone of Anhui Agricultural University

[0030] (2) Using the selected tea tree species as material, the CTAB method was used to extract the total DNA, and the specific operation process was:

[0031] ① Take a 2ml centrifuge tube, add 0.1g of ground dry tea powder, add 900mL of CTAB extract (preheated to 65°C in advance), add 20mL of β-mercaptoethanol, bathe in water at 65°C for 20min, shake gently up and down several times every 10min , then add 10mL RNAase and place in a 65°C water bath for 15min, shake up and down several times every 5min.

[0032] ② Centrifuge at 13000r / min for 10min, take 800mL of supernatant, add 400mL Tris to balance phenol, add an equal volume (800mL) of chloroform: isoamyl alcohol to 2...

Embodiment 2

[0036] The selection of embodiment 2SSR site and the preliminary screening of primer

[0037] Among the 100,000 sequences containing SSR sites, the SSR sites with hexanucleotides as repeating units were more and more polymorphic. Therefore, 4,000 sequences containing SSR sites were selected according to the following principles, and 450 pairs of primers (synthesized by General Biology) were successfully designed. The principles of SSR primer screening are as follows:

[0038] ①The length of the primer is 18-22bp, and the target fragment is between 250bp-380bp.

[0039] ②The GC content is 40%-60%, avoid three or four consecutive bases in the primer sequence;

[0040] ③The annealing temperature should be between 45°C and 55°C, preferably around 50°C, and the difference between the Tm values ​​of the upstream and downstream primers should not be greater than 4°C;

[0041] ④ Avoid more than 3 consecutive bases at the 3' end of the primer, and try to avoid primer dimers and hairp...

Embodiment 3

[0042] Embodiment 3PCR amplification and Fragment Analyzer TM Automatic capillary electrophoresis system primary screening

[0043]The total reaction system is 20μL, including 2μL of 50ng / μL DNA solution, 0.5μL of 10μM upstream and downstream primers, 2μL of 10×EasyTaq Buffer, 1units of EasyTaq DNA Polymerase, 2μL of 2.5mM dNTP, ddH 2 O 12.8 μL. (Reagents are from Transgeng Biological Company)

[0044] The PCR reaction program was: pre-denaturation at 94°C for 5 min, 30 cycles (denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s), extension at 72°C for 10 min, and storage at 4°C. The annealing temperature was determined according to each primer. PCR amplification and capillary electrophoresis primary screening. Specifically, 2 microliters of the PCR product was taken and passed through the Fragment AnalyzerTM automatic capillary electrophoresis system for primary screening, and the primary screening of primers was performed by comparing with...

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Abstract

The invention discloses a method for identifying Shida tea varieties through an SSR fingerprint spectrum. Specific SSR locus in tea tree whole genome and conserved sequences on the two sides are as shown in SEQ ID NO: 1 to SEQ ID NO: 3. The method comprises the following steps: taking four Shida tea good varieties comprising Shida tea I, Shida tea II, Shida tea VI and Shida tea yellow variety, selecting 450 SSR primer pairs in the tea tree whole genome to perform polymorphic screening, ensuing that identification flow mainly comprises tea tree total DNA extraction, primer design, PCR amplification and allele polymorphic statistics, and finally determining 1 primer pair as core primer pairs for specie identification. The method effectively separates four Shida tea varieties from one another, is beneficial to protection and promotion of the Shida tea varieties, and meanwhile provides a rapid and accurate identification method for various tea which are asserted to take fresh leaves of Shida tea as raw materials on the market.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a method for identifying persimmon tea varieties by using SSR fingerprints. Background technique [0002] Persimmon tea, a provincial-level tea tree variety in Anhui Province. The sexual propagation line is a unique tea tree variety in the Taiping Houkui tea producing area in Huangshan District. It is a shrub type, large-leaved, and medium-bud species. It is the best variety suitable for making Taiping Houkui tea among the top ten traditional teas, and it is also one of the necessary conditions for the development of Taiping Houkui tea production. The plant of persimmon tea is moderate, the tree posture is half open, the branches are thin, the internodes are short, the leaves grow obliquely, the leaves are thick, the leaves are oval like lung lobes, the leaves are dark green, shiny, the leaves are raised, and the leaves are thick and thick. Soft texture. The buds and l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6858
CPCC12Q1/6858C12Q1/6895C12Q2600/13C12Q2531/113C12Q2565/125
Inventor 韦朝领安焱林刘升锐郭凌霄密孝增程道杰
Owner ANHUI AGRICULTURAL UNIVERSITY
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