DNA barcode primer pair for amplification of liriodendron chinense(Hemsl.)Sarg. and identification method of liriodendron chinense(Hemsl.)Sarg. and germplasm source of liriodendron chinense(Hemsl.)Sarg.

A technology of barcode primers and amplification primers, which is applied in biochemical equipment and methods, recombinant DNA technology, DNA/RNA fragments, etc., can solve the problems of insufficient identification dimension and large variation of morphological markers in Liriodendron tulipifera, and achieve High accuracy and simple operation

Active Publication Date: 2019-09-17
INST OF BIOLOGICAL RESOURCES JIANGXI ACAD OF SCI
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AI Technical Summary

Problems solved by technology

[0004] Traditional classification and identification mostly rely on differences in phenotypic characteristics, but morphological markers vary greatly, and usually require complete plant tissue parts; although the emergence of SSR molecular markers in recent years has solved the problem of Chinese tulip tree, North American tulip tree, and North American to a certain extent. Part of the identification of Liriodendron tulipifera and hybrid Liriodendron tulipifera, but the identification dimension within the species of Liriodendron tulipifera is not enough, and multiple pairs of primers are needed in combination

Method used

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  • DNA barcode primer pair for amplification of liriodendron chinense(Hemsl.)Sarg. and identification method of liriodendron chinense(Hemsl.)Sarg. and germplasm source of liriodendron chinense(Hemsl.)Sarg.
  • DNA barcode primer pair for amplification of liriodendron chinense(Hemsl.)Sarg. and identification method of liriodendron chinense(Hemsl.)Sarg. and germplasm source of liriodendron chinense(Hemsl.)Sarg.
  • DNA barcode primer pair for amplification of liriodendron chinense(Hemsl.)Sarg. and identification method of liriodendron chinense(Hemsl.)Sarg. and germplasm source of liriodendron chinense(Hemsl.)Sarg.

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Liriodendron tulipifera and Liriodendron tulipifera were collected covering the entire distribution area of ​​Liriodendron, a total of 120 samples (sample number 1-120). See Table 1 for sample sources and other relevant information.

[0044] These samples were identified using the DNA barcode primer pair and method of the present invention.

[0045] details as follows:

[0046] 1) Using the improved CTAB method to extract the whole genome DNA of young leaves of the sample to be tested.

[0047] 2) The obtained whole genome DNA is used as a template, and PCR amplification is performed using the DNA barcode primer pair to obtain an amplified product. The PCR amplification system is calculated in 20 μL, including 10 μL of 2×Taq PCR Master Mix, 2.0 μL of 20-40 ng / L whole genome DNA, 0.6 μL of 10 μmol / L upstream amplification primer, 0.6 μL of 10 μmol / L downstream amplification primer, ddH 2 O 6.8 μL; PCR amplification program includes: 95°C pre-denaturation for 4 minutes...

Embodiment 2

[0052] 153 individuals (sample numbers 13-164) from 43 provenances in the entire distribution area of ​​Liriodendron were collected, covering the largest distribution area of ​​Liriodendron tulipifera. See Table 1 for the source of samples and other related information. Young leaves of all individuals were collected and dried with silica gel for preservation.

[0053] 1) The modified CTAB method was used to extract the total DNA of Liriodendron tulipifera.

[0054] 2) The obtained whole genome DNA is used as a template, and PCR amplification is performed using the DNA barcode primer pair to obtain an amplified product. The PCR amplification system is calculated in 20 μL, including 10 μL of 2×Taq PCR Master Mix, 2.0 μL of 20-40 ng / L whole genome DNA, 0.6 μL of 10 μmol / L upstream amplification primer, 0.6 μL of 10 μmol / L downstream amplification primer, ddH 2 O 6.8 μL; PCR amplification program includes: 95°C pre-denaturation for 4 minutes; 95°C denaturation for 40 s, 59°C anne...

Embodiment 3

[0059] Sample source and operating steps are the same as in Example 2.

[0060] According to the results of sequencing and sequence comparison, it was found that starting from 375bp, samples No. 13 to 26 had 8 A base repeats, which was consistent with the standard sequence SEQ ID No.6 of the tropical provenance of Liriodendron tulipifera of the present invention, and was identified as a tropical Provenance, the remaining samples are all 9 A base repeats starting from 375bp. For the sequence alignment of samples numbered 24-26 and 100-102 with DNA barcode sequences (SEQ ID No.5 and SEQ ID No.6) in the 320-395bp region, see Figure 4 According to the source of the control samples, it was found that samples 13-26 all came from tropical regions, and the rest of the samples all came from subtropical regions. Therefore, the identification accuracy rate of the barcode of the present invention is 100%.

[0061] It can be known from the above examples that the method for identifying ...

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Abstract

The invention provides a DNA barcode primer pair for amplification of liriodendron chinense(Hemsl.)Sarg. and an identification method of the liriodendron chinense(Hemsl.)Sarg. and a germplasm source of the liriodendron chinense(Hemsl.)Sarg., and belongs to the technical field of plant molecular identification. The DNA barcode primer pair for amplification of the liriodendron chinense(Hemsl.)Sarg. comprises an upstream amplification primer and a downstream amplification primer. The sequence of the upstream amplification primer is shown in SEQ ID No.1, and the sequence of the downstream amplification primer is shown in SEQ ID No.2. The identification method of the liriodendron chinense(Hemsl.)Sarg. comprises the following steps of 1, extracting a whole genome DNA of a to-be-detected sample; 2, taking the whole genome DNA as a template, and utilizing the DNA barcode primer pair for PCR amplification to obtain an amplification product; 3, sequencing the amplification product to obtain specific sequence information of the amplification product, comparing the specific sequence information with DNA barcode standard sequences of the liriodendron chinense(Hemsl.)Sarg., and then determining tree species and the regional germplasm source of the to-be-detected sample. The method is easy to operate and high in accuracy.

Description

technical field [0001] The invention belongs to the technical field of plant molecular identification, and in particular relates to a pair of primers for amplifying the DNA barcode of Liriodendron tulipifera and an identification method for Liriodendron tulipifera and its provenance. Background technique [0002] Liriodendron chinense (Hemsl.) Sarg. belongs to the genus Liriodendron L. of Magnoliaceae, and is a perennial deciduous tree species. Liriodendron tulipiflora has beautiful flowers and leaves, high ornamental value; fast-growing, straight wood, and fine material, it is also a good timber tree species and a potential energy plant. The genus Liriodendron was once widely distributed in the temperate regions of the northern hemisphere, and was affected by the climatic turmoil at the end of the Tertiary Period. It is currently only distributed in a small part of East Asia and North America, and has differentiated into Liriodendron and Liriodendron ( L. tulipifera). Lir...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/156
Inventor 杨爱红刘淑娟刘立盘钟永达刘腾云周华吴照祥李彦强余发新
Owner INST OF BIOLOGICAL RESOURCES JIANGXI ACAD OF SCI
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