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Pinus massoniana cpSSR polymorphism primer and identification method of pine sibling species of primer

A technology of polymorphic primers and masson pine, which is applied in the field of tree molecular biology, can solve problems such as incompleteness, complicated experimental procedures, and safety hazards for experimenters, and achieve the effect of simple structure, multi-copy molecular weight, and difficult recombination

Active Publication Date: 2018-01-09
NANJING FORESTRY UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In the process of realizing the present invention, the inventors have found that the prior art has at least the following problems: the prior art method for distinguishing pine germplasm resources and identifying kinship is only aimed at the nuclear DNA of pine, because in plant cells, except for nuclear inheritance, , also has cytoplasmic inheritance, but there are few records in the prior art that are used for the germplasm resource differentiation and kinship identification of pine trees other than the nuclear DNA of pine trees, so only using the nuclear DNA identification method is not comprehensive
This method has to go through steps such as electrophoresis, fixation, silver staining, color development, and washing. harm
Although this method can theoretically achieve a separation rate of 5 bp, it is difficult to achieve in practice, and the size of the fragment cannot be accurately read out. It can only be roughly estimated artificially, and the amplified fragment with a length of less than 50 bp cannot be analyzed.

Method used

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  • Pinus massoniana cpSSR polymorphism primer and identification method of pine sibling species of primer
  • Pinus massoniana cpSSR polymorphism primer and identification method of pine sibling species of primer
  • Pinus massoniana cpSSR polymorphism primer and identification method of pine sibling species of primer

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Experimental program
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Embodiment 1

[0027] Acquisition of chloroplast microsatellite molecular marker polymorphism primer (cpSSR) of Pinus massoniana: the process is as follows:

[0028] 1) Polymorphic primer screening of chloroplast microsatellite molecular markers

[0029] Download the masson pine (P.massioniana) chloroplast genome sequence (MF564195) from the GenBank public database (http: / / www.ncbi.nlm.nih.gov / ), and use MicroSAtellite (http: / / pgrc.ipk-gatersleben.de / misa / mi sa.hrml)(Thiel T,Michalek W,Varshney R K,etal.Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley(Hordeumvulg are L.).[J].Theoretical&AppliedGenetics, 2003,106(3):411-422.) Analyze the chloroplast genome sequence of Pinus massoniana, and screen the chloroplast microsatellite molecular sequence.

[0030] 2) Design of primers for chloroplast microsatellite molecular markers

[0031] Based on the chloroplast microsatellite molecular sequences obtained above, the primer design software P...

Embodiment 3

[0048] The method that 7 pairs of pine cpSSR polymorphism primers identify 8 kinds of pine closely related species in application embodiment 1 comprises the following steps:

[0049] 1) Use the plant genome DNA extraction kit of Beijing Zhuangmeng International Biogene Technology Co., Ltd. to extract the total DNA.

[0050] 2) Carry out PCR amplification sequentially according to the primer sequence in Table 1 according to the following PCR reaction system and reaction program until the identification is completed.

[0051] The PCR amplification system includes: 10×PCR Buffer (10mM Tris-HCl); 0.25mM dNTP; 2.5mMMgCl2; 0.3mM described Pinus massoniana cpSSR primer; 0.5U Taq DNA polymerase and 50ng DNA template, the total amount of the PCR amplification system The volume is 10 μL;

[0052] The PCR amplification program was: pre-denaturation at 94°C for 5min; 25 cycles of 94°C for 30s, annealing for 30s, and 72°C for 40s; extension at 72°C for 5min.

[0053] 3) Perform capillary...

Embodiment 4

[0056] The present invention provides a kind of method that utilizes the polymorphic primer of the chloroplast microsatellite molecular marker of Pine massoniana provided in Example 1 to identify 8 kinds of pine close relatives, specifically as follows:

[0057] First, sample genomic DNA is extracted. The samples were the 8 pine relative species provided in Example 2 of the present invention, wherein 15 individuals were sampled for each tree species, and the DNA of the samples was extracted. Then, according to the mixture of 7 pairs of masson pine cpSSR polymorphic primers provided in Example 1 and the genomic DNA of the sample, the amplified product was obtained, and the amplified product was subjected to capillary electrophoresis. The processing method of capillary electrophoresis data is as follows: figure 2 As shown, wherein code is the name of the tree species, 15 seedlings are sampled for each tree species, and marker represents the primers. Since the present invention...

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Abstract

The invention discloses a pinus massoniana cpSSR polymorphism primer and an identification method of pine sibling species of the primer. According to design of a chloroplast genome of pinus massoniana, 16 pairs of cpSSR primers are obtained, further, 7 pairs of primers which are stable in difference and good in polymorphism are screened from the 16 pairs of cpSSR primers, and through utilization of the 7 pairs of primers, a finger print and phylogenetic tree of 8 kinds of pine sibling species are successfully established. Based on the 7 pairs of cpSSR primers, a PCR amplification reaction is carried out. Capillary electrophoresis technology is used to analyze a PCR product, 8 kinds of pine sibling species trees can be accurately identified. The primer and method are efficient, accurate, and convenient and are not prone to be affected by the environment. The polymorphism primer and an identification method thereof can reflect the genetic information of the 8 kinds of pine plasmons, andcan be used for research of identification of 8 kinds of pine sibling species and genetic relationship.

Description

technical field [0001] The invention belongs to the technical field of tree molecular biology, in particular to polymorphic primers of chloroplast microsatellite molecular markers (cpS SR) of pine pine and its application in identification of eight pine relative species. Background technique [0002] Pinus (Pinaceae) is an important group of gymnosperms, and it is one of the most widely distributed and economically valuable tree species in the world. In China, there are about 22 species of Pinus species, which are widely distributed and play an extremely important role in China's forestry production and ecological greening. In addition, China has introduced some important pine species from abroad, including loblolly pine (P.taeda), slash pine (P.elliottii), Caribbean pine (P.caribaea) and so on. These rich resources provide an important material basis for the development of research on pine genetics and breeding in my country. [0003] Accurate differentiation and identifi...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 徐立安荣浩倪州献胥猛韩炘祁铭
Owner NANJING FORESTRY UNIV
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