Method for identifying germplasm of pinus massoniana by utilizing SSR (simple sequence repeat) molecular marker based on transcriptome sequencing

A transcriptome sequencing and molecular marker technology, applied in the field of molecular biology, can solve the problems of low proportion of masson pine genome and complicated operation, and achieve the effects of good production process, good repeatability and short time consumption.

Inactive Publication Date: 2017-01-04
GUIZHOU UNIV
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Problems solved by technology

[0005] The technical problem to be solved by the present invention is: in order to overcome the shortcomings of existing molecular markers such as low coverage of Pine massoniana genome a

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  • Method for identifying germplasm of pinus massoniana by utilizing SSR (simple sequence repeat) molecular marker based on transcriptome sequencing
  • Method for identifying germplasm of pinus massoniana by utilizing SSR (simple sequence repeat) molecular marker based on transcriptome sequencing
  • Method for identifying germplasm of pinus massoniana by utilizing SSR (simple sequence repeat) molecular marker based on transcriptome sequencing

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Embodiment 1

[0026] Example 1: With the SSR primers of the present invention, a total of 72 Pinus masson germplasms in Guizhou, Fujian, and Guangxi were identified, and the primer pair PmS54 was used as an example to distinguish the tested materials.

[0027] 1. Design and synthesis of SSR primers

[0028] First, pre-process the sequence obtained by sequencing to obtain high-quality EST sequences without redundancy, and use MISA software to search for SSR sites in the transcription data. The search criteria are: single nucleotide, dinucleotide, trinucleotide , four nucleotides, five nucleotides and six nucleotides, the minimum number of repetitions were 10, 6, 5, 5, 5 and 5, and then use the Primer3.0 primer batch design program for the Unigene sequence containing the SSR site Primers were designed, and the length of the SSR site sequence was between 18 and 27 bp. Wherein, the main parameter of primer design is: annealing temperature (T m ) between 57-63°C, 60°C is the best; PCR product ...

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Abstract

The invention discloses a method for identifying germplasm of pinus massoniana by utilizing an SSR (simple sequence repeat) molecular marker based on transcriptome sequencing. The method is characterized by comprising steps as follows: (1) a Tiangen DNA secure Plant Kit optimization step is adopted for DNA extraction, and a pinus massoniana sample is amplified by the aid of a primer according to a PCR (polymerase chain reaction) system and a reaction procedure; (2) an SSR primer is designed on the basis of the sequence for transcriptome sequencing of pinus massoniana, and a PCR amplification system is optimized; (3) finally, the PCR primer is detected with a silver staining method through optimized 10% polyacrylamide denaturing gel electrophoresis, and a result is judged according to existence and the magnitude of a band.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to an SSR primer developed based on the sequencing sequence of the masson pine transcriptome and its application. Multiple pairs of SSR marker primers developed based on the transcriptome sequence are specially used for rapid identification and genetic relationship analysis of Pinus massoniana germplasm at the DNA level. Background technique [0002] Masson pine (Pinus massoniana) is one of the most important high-quality coniferous timber species in southern my country. It plays an important role in my country's forest resource development, forestry and paper integration, rosin forestry chemical industry and forest ecological service functions. Among them, masson pine is excellent. The discovery and protection of germplasm is crucial to the healthy development of the industry. Therefore, the accurate and efficient identification and genetic relationship analysis of Pinus massoniana g...

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6895C12Q1/686C12Q2600/156C12Q2565/125
Inventor 文晓鹏梅利那范付华崔博文
Owner GUIZHOU UNIV
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