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InDel markers for identifying purple tea tree varieties, and combination and application of InDel markers

A technology for marking and tea trees, which is applied in the fields of molecular biology and plant genetics and breeding, can solve problems such as difficult to control spread, impure varieties, and varieties with low market access threshold, and achieve high accuracy, good stability, and high sensitivity. Effect

Active Publication Date: 2020-01-10
ANHUI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current tea tree seedling cultivation technology is simple, the market access threshold is low, and the varieties are various and messy. There are phenomena of false ones and impure varieties.
Famous teas made from different purple tea leaves are not the same in quality and price. It is difficult for consumers to distinguish the authenticity from the appearance with the naked eye.
In addition, since tea trees can be rapidly propagated using the "short ear cutting" technology, once excellent varieties enter the market, it is difficult to control their spread

Method used

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  • InDel markers for identifying purple tea tree varieties, and combination and application of InDel markers
  • InDel markers for identifying purple tea tree varieties, and combination and application of InDel markers
  • InDel markers for identifying purple tea tree varieties, and combination and application of InDel markers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all in accordance with conventional experimental conditions, such as Sambrook et al. Molecular Cloning Experiment Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or in accordance with the conditions suggested by the manufacturer's instructions. The extraction of embodiment 1 tea fresh leaf total DNA

[0051] 1. Experimental materials and reagents

[0052] Tea tree varieties: Ziyan, Zixian, Zihong and Zijuan. Provided by the Tea Tree Germplasm Resource Garden of Wanzhong Experimental Station of Anhui Agricultural University (Hefei, Anhui, 31°49′21″N, 117°13′18″E).

[0053] For DNA extraction, kit EZgeneTM CP Plant Miniprep Kit was used, catalog number GD2621-02.

[0054] 2. Experimental method

[0055] The selected tea tree species were used as materials, and...

Embodiment 2

[0060] ⑤ Use a nucleic acid quantifier to measure the DNA content, and electrophoresis with 1.2% agarose. Electrophoresis results see figure 1 . The selection of embodiment 2InDel site and the preliminary screening of primer

[0061] According to the following principles, 30 pairs of primers (synthesized by General Biosystems (Anhui) Co., Ltd.) were successfully designed, and the principles of InDel primer screening were as follows:

[0062] ①The length of the primer is 18-22bp, and the target fragment is between 250bp-380bp;

[0063] ②The GC content is 40%-60%, avoid three or four consecutive bases in the primer sequence;

[0064] ③The annealing temperature is 50°C-60°C, preferably around 58°C, and the difference between the Tm values ​​of the upstream and downstream primers is not more than 4°C;

[0065] ④ Avoid more than 3 consecutive bases at the 3′ end of the primer, and try to avoid primer dimers and hairpin structures.

Embodiment 3

[0066] Example 3 Primer Screening

[0067] PCR Amplification and Fragment Analyzer TM Automatic capillary electrophoresis system for primary screening, details are as follows:

[0068] The total reaction system is 10 μL, including 1 μL of S0ng / μL DNA solution, 0.5 μL of 10 μM upstream and downstream primers, 5 μL of 2×TaqPlus Master Mix (Dye), ddH 2 O 3 μL. After centrifugation, add 20 μL Mineral oil to seal. Among them, Taq PlusMaster Mix comes from Beijing Kangwei Century Biotechnology Co., Ltd., product number CW2849M.

[0069] The PCR reaction program was: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 45 s, 35 cycles; extension at 72°C for 5 min, and storage at 4°C.

[0070] PCR amplification and capillary electrophoresis primary screening, specifically, pipetting 2 μL of the PCR product through the Fragment Analyzer TM The automatic capillary electrophoresis system performs the primary screening, ...

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Abstract

The invention provides InDel markers for identifying purple tea tree varieties, and a combination and application of the InDel markers. The InDel markers include the InDel marker 1 and the InDel marker 2. InDel fingerprints are utilized for identifying four purple tea tree varieties including the ziyan variety, the zixian variety, the zihong variety and the zijuan variety, and 30 pairs of InDel primers are selected from whole tea tree genomes to be subjected to polymorphic screening. An identification process mainly includes the steps of: extracting total DNA of tea trees, designing the primers, conducting PCR amplification, and carrying out allele polymorphism statistics; and finally, two pairs of InDel primers (SEQ ID NO:5-8) are determined to serve as core primers for variety identification. By adopting the InDel markers, the four purple tea tree varieties can be effectively and completely distinguished, and therefore, not only can protection and popularization of the purple tea tree varieties be promoted, but also a simple, rapid, accurate and efficient identification method is provided for the four purple tea varieties which are easy to confuse on the market.

Description

technical field [0001] The invention belongs to the fields of molecular biology and plant genetic breeding, and in particular relates to an InDel marker for identifying purple tea tree varieties and its combination and application. Background technique [0002] Purple tea is a rare variety of tea. Lu Yu, a tea sage, once said: "Tea, the purple one is the best." The reason why purple tea appears purple is mainly because of its rich anthocyanin content. Studies have shown that anthocyanins, as a powerful antioxidant, can not only protect the human body from free radical damage, but also enhance blood vessel elasticity, improve circulatory system and skin smoothness, inhibit inflammation and allergies, Improves joint flexibility. The antioxidant capacity of anthocyanins is 50 times higher than that of vitamin E and 20 times higher than that of vitamin C. At the same time, anthocyanins have good solubility in hot water and can be effectively ingested by drinking tea. Theref...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/686C12N15/11
CPCC12Q1/6895C12Q1/686C12Q2600/156C12Q2565/125
Inventor 韦朝领郭锐刘升锐安焱林夏小波
Owner ANHUI AGRICULTURAL UNIVERSITY
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