Amplification primers, screening methods and identification methods for identification of olive varieties based on SNP sites

A technology for amplification primers and species identification, which is applied in biochemical equipment and methods, microbiological determination/inspection, recombinant DNA technology, etc., can solve the problems of various products, chaotic peak shapes, and expensive SSR detection, etc., to achieve expansion High efficiency, clear peak shape, low cost detection effect

Active Publication Date: 2021-10-29
INST OF FORESTRY CHINESE ACAD OF FORESTRY +1
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, in practical applications, SSR markers are not particularly ideal, mainly in the following aspects: (1) The repeat region causes an increase in the error rate of amplification, and the amplification is more or less of one or several repeat units, resulting in a variety of products, The peak shape is chaotic, which affects the interpretation; (2) For the rare alleles with large differences in the length of the main alleles in the population, different researchers have different subjective judgments, which may easily cause identification bias; (3) SSR detection is expensive, Its product detection is mainly detected by a first-generation sequencer, which needs to be labeled with a fluorescent dye (such as HEX) at one end, and this kind of primer is not resistant to storage

Method used

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  • Amplification primers, screening methods and identification methods for identification of olive varieties based on SNP sites
  • Amplification primers, screening methods and identification methods for identification of olive varieties based on SNP sites
  • Amplification primers, screening methods and identification methods for identification of olive varieties based on SNP sites

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Effect test

Embodiment 1

[0047] In 2016, the Forestry Research Institute of the Chinese Academy of Forestry collected an olive variety resource A from the Wudu olive planting base in Gansu. There is still a difference. In order to identify whether the variety resource A is 'Dou Guo', the following processing analysis was carried out:

[0048] 1. Collect the leaf materials of olive variety A and the known variety 'Douguo' respectively, and extract DNA;

[0049] 2. Use 8 pairs of olive amplification primers in Table 1 of the present invention to amplify and sequence the two DNAs respectively;

[0050] PCR amplification uses 30uL reaction system: 20ng DNA, 10×buffer (Promega, USA) buffer 3uL, 2.4mM dNTPs 2.4uL, 10uM primer 2.4uL, forward primer and reverse primer 1.2uL each, TaqDNA polymerase (5U / uL, Takara, Shiga, Japan) 0.15uL. ABI 96U Thermo cycler PCR instrument was used for PCR amplification.

[0051] The reaction procedure of PCR amplification is:

[0052]

[0053] 3. Use ContigExpress soft...

Embodiment 2

[0057] In 2016, the Forestry Research Institute of the Chinese Academy of Forestry collected an olive variety resource B from the olive planting base in Mianyang, Sichuan. Since there are no important varieties such as fruits, it is not clear what variety it is. To identify this variety resource B, the following treatment analyzes were performed:

[0058] 1. Collect the leaf material of olive variety B and extract DNA;

[0059] 2. Use 8 pairs of olive amplification primers in Table 1 of the present invention to amplify and sequence the leaf DNA of this variety resource respectively;

[0060] PCR amplification uses 30uL reaction system: 20ng DNA, 10×buffer (Promega, USA) buffer 3uL, 2.4mM dNTPs 2.4uL, 10uM primer 2.4uL, forward primer and reverse primer 1.2uL each, TaqDNA polymerase (5U / uL, Takara, Shiga, Japan) 0.15uL. ABI 96U Thermo cycler PCR instrument was used for PCR amplification.

[0061] The reaction procedure of PCR amplification is:

[0062]

[0063] 3. Use Co...

Embodiment 3

[0068] In 2017, the Subtropical Forestry Research Institute of the Chinese Academy of Forestry collected an olive variety resource C from the Lishui olive planting base in Zhejiang. The resource of this variety was introduced from Sichuan, and its phenotypic traits are similar to the existing olive variety 'Leixing', but there are still differences in traits such as leaf texture. In order to identify whether the variety resource C is 'Leixing', the following processing analysis was carried out:

[0069] 1. Collect the leaf material of olive variety C and extract DNA;

[0070] 2. Use 8 pairs of olive amplification primers in Table 1 of the present invention to amplify and sequence the leaf DNA of this variety resource respectively;

[0071] PCR amplification uses 30uL reaction system: 20ng DNA, 10×buffer (Promega, USA) buffer 3uL, 2.4mM dNTPs 2.4uL, 10uM primer 2.4uL, forward primer and reverse primer 1.2uL each, TaqDNA polymerase (5U / uL, Takara, Shiga, Japan) 0.15uL. ABI 96...

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Abstract

The invention discloses an amplification primer, a screening method and an identification method for identifying olive varieties based on SNP sites, and eight pairs of olive amplification primers. The present invention selects highly reliable and reproducible single-copy fragments through restriction endonuclease digestion, cloning and sequencing, and genome comparison analysis of the leaf DNA of olive varieties, and designs amplification primers for single-copy fragments; and then selects single-copy Fragment-designed amplification primers were used to amplify the leaf DNA of olive varieties by PCR, and 8 pairs of single-copy nuclear gene markers with high amplification efficiency and rich variation were screened out. The amplification products of 8 pairs of olives were relatively pure, with clear peak shapes and no Fluorescent primers are needed, and the detection price is low; 8 pairs of single-copy nuclear gene markers in olive can be used for identification of olive varieties.

Description

technical field [0001] The present invention relates to the field of biotechnology. Specifically, the invention relates to an amplification primer, a screening method and an identification method for identification of olive varieties based on SNP loci. Background technique [0002] Olive (Olea europaea L.) is the second largest woody oil-bearing plant in the world after oil palm. It is an evergreen tree of Oleaceae Oleaceae. It is mainly used for fresh fruit processing olive oil. Olive oil is rich in monounsaturated fatty acids and antioxidants Oxidizing substances have important nutritional value and are known as "liquid gold". [0003] Olives are widely distributed in the Mediterranean region, and the planting area of ​​olives in the world has exceeded 8.8 million hm2. Since the first introduction of olives from Albania in the 1960s, my country has collected and introduced a large number of foreign olive varieties, and there are nearly 200 registered varieties. The freq...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/6811C12N15/11
CPCC12Q1/6811C12Q1/6895C12Q2600/13C12Q2521/301C12Q2535/122C12Q2537/165
Inventor 邵文豪王兆山张建国
Owner INST OF FORESTRY CHINESE ACAD OF FORESTRY
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