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CN-119530433-B - Primer pair for identifying germplasm of ulmus pumila and ulmus parvifolia and application thereof

CN119530433BCN 119530433 BCN119530433 BCN 119530433BCN-119530433-B

Abstract

The invention discloses a primer pair for identifying the germplasm of Ulmus pumila and application thereof, wherein the primer pair is Ulmus_p104 or/and Ulmus_p40, fresh tissue samples of Ulmus pumila or Ulmus pumila to be detected are collected, DNA of the tissue samples is extracted, the DNA is used as a template, PCR amplification is carried out by using the primer pair, an amplification product is detected by electrophoresis, and the sample to be detected is judged to be the Ulmus pumila or the Ulmus pumila according to a detection result. The SSR molecular markers for transcriptome sequencing are used for identifying the germplasm resources of the ulmus pumila and the ulmus pumila, which is beneficial to promoting the fine variety breeding of the ulmus pumila and accelerating the utilization process of the fine variety. The specific primers of the invention amplify the ulmus pumila and ulmus pumila tissues respectively, and the difference of the product lengths of the two is obvious, which indicates that the specific primers can effectively identify the germplasm of the ulmus pumila and the ulmus pumila.

Inventors

  • DAI JIANFENG
  • Zheng Conghui
  • WANG YUZHONG
  • XU ZHENHUA
  • ZHANG MIAOMIAO
  • XU WENKAI
  • SUN RAN
  • WANG GUIXIN

Assignees

  • 河北省林业和草原科学研究院

Dates

Publication Date
20260505
Application Date
20241202
Priority Date
20240409

Claims (7)

  1. 1. The application of the primer group is characterized in that the primer group is used for identifying the ulmus pumila or ulmus pumila germplasm in the ulmus pumila and ulmus pumila, and consists of the following primer pairs: Ulmus_p104: An upstream primer 5'-CCCCATCTGGATTGGCAGTT-3'; a downstream primer 5'-CGCCCTTTGACGGTTGGATA-3'; Ulmus_p40: an upstream primer 5'-GCCAGTTGGGTGGATCAAGA-3'; and a downstream primer 5'-GTGGGGATGCGAGAGAACAA-3'.
  2. 2. A method for identifying the germplasm of ulmus pumila or ulmus parvifolia in ulmus pumila and ulmus parvifolia, characterized in that the identification steps are as follows: step S1, collecting fresh tissue samples of the ulmus pumila or ulmus pumila to be detected, and extracting DNA of the tissue samples; Step S2, using the DNA extracted in the step S1 as a template, and performing PCR amplification by using the primer set of claim 1; step S3, detecting an amplification product by electrophoresis; and S4, judging whether the sample to be detected is the elm or the elm parvifolia according to the detection result.
  3. 3. The method of claim 2, wherein the tissue sample is a young leaf.
  4. 4. The method of claim 2, wherein the electrophoresis is capillary electrophoresis or gel electrophoresis.
  5. 5. The method according to claim 4, wherein the capillary electrophoresis is carried out by taking 0.1 mu L of the PCR product obtained in the step S2, diluting to 1 mu L, mixing with 15 mu L of the mixture of formamide and the molecular weight internal standard 100:1, adding into a PCR plate, denaturing at 95 ℃ for 5min, cooling at 4 ℃, centrifuging, and detecting on a machine.
  6. 6. The method according to claim 2, wherein in step S4, the raw data obtained in step S3 is analyzed, and the positions of the internal molecular weight standards in each lane are compared with the positions of the peaks of each sample to obtain the fragment size.
  7. 7. The method according to claim 2, wherein the step S4 further comprises calculating a genetic distance matrix based on the original site data, and constructing a phylogenetic tree based on the GS value matrix by using a similarity coefficient method.

Description

Primer pair for identifying germplasm of ulmus pumila and ulmus parvifolia and application thereof Technical Field The invention belongs to the technical field of genetic engineering, and relates to a primer pair, in particular to a primer pair for identifying the germplasm of ulmus pumila and ulmus parvifolia. Background Ulmus pumila L. and Ulmus pumila Ulmus parvifolia are Ulmus (Ulmus L.) trees of Ulmus genus (Ulmaceae) of Ulmaceae family, which are broad-leaved tree species in rural area with relatively wide China distribution. The elm has more than 30 species and generates northern hemisphere. There are 25 varieties of 6 in our country, which are distributed throughout the country, more in the Yangtze river basin and more in the north. The white elm and the hammer elm are all light tree species, have developed root systems and strong resistance, are excellent tree species for protecting soil and fixing sand, which are important in severe ecological environment areas, are edible (oil content is 20% -40%), are excellent in material quality, are important raw material tree species for medicine and light industry, are resistant to pruning of branches and leaves, have high ornamental value, and are widely used for modeling of garden plants. Therefore, the ulmus pumila and the ulmus pumila are multifunctional tree species which integrate ecological, economic and ornamental values. In the process of implementing the present invention, the inventor finds that at least one of the following technical problems exists in the prior art: the white elm and the hammer elm in China have abundant germplasm resources, have similar phenotype characters except obvious difference of flowering time (flowering in spring and autumn), are difficult to distinguish, and are difficult to identify in early germplasm. Disclosure of Invention In view of the above, the present invention aims to provide an SSR molecular marker using transcriptome sequencing to rapidly identify the germplasm of ulmus pumila and ulmus pumila. The inventor continuously reforms and innovates through long-term exploration and trial and repeated experiments and efforts, and in order to solve the technical problems, the invention provides a primer pair for identifying the germplasm of ulmus pumila or ulmus parvifolia, wherein the primer pair is one or more of the following primer pairs: Ulmus_p104: an upstream primer 5'-CCCCATCTGGATTGGCAGTT-3'; A downstream primer 5'-CGCCCTTTGACGGTTGGATA-3'; Ulmus_p40: An upstream primer 5'-GCCAGTTGGGTGGATCAAGA-3'; And a downstream primer 5'-GTGGGGATGCGAGAGAACAA-3'. The invention also provides application of the primer pair, which is used for identifying the germplasm of ulmus pumila or ulmus parvifolia; The primer pair is Ulmus_p104: the sample with the amplified product fragment size of 212bp is elm parvifolia; The amplified product contains a sample with a fragment size of 209bp, which is elm; the primer pair is Ulmus_p40: The sample with 253bp of amplified product fragment size is Ulmus parvifolia; the sample with the amplified product fragment size of 241-251bp is elm. The invention also discloses a method for identifying the germplasm of ulmus pumila and ulmus parvifolia by using the primer pair, which comprises the following steps: step S1, collecting fresh tissue samples of the ulmus pumila or ulmus pumila to be detected, and extracting DNA of the tissue samples; S2, performing PCR amplification by using the DNA extracted in the step S1 as a template and using the primer pair; step S3, detecting an amplification product by electrophoresis; step S4, judging whether the sample to be detected is the elm or the elm parvifolia according to the detection result; The primer pair is Ulmus_p104: the sample with the amplified product fragment size of 212bp is elm parvifolia; The amplified product contains a sample with a fragment size of 209bp, which is elm; the primer pair is Ulmus_p40: The sample with 253bp of amplified product fragment size is Ulmus parvifolia; the sample with the amplified product fragment size of 241-251bp is elm. According to one embodiment of the method of the invention, the tissue sample is a young leaf. According to one embodiment of the method of the invention, the electrophoresis is capillary electrophoresis or gel electrophoresis. According to one embodiment of the method, the capillary electrophoresis is carried out by taking 0.1 mu L of the PCR product in the step S2, diluting to 1 mu L, uniformly mixing with 15 mu L of the mixture of formamide and the molecular weight internal standard 100:1, adding into a PCR plate, denaturing at 95 ℃ for 5min, cooling at 4 ℃, centrifuging, and loading into a machine for detection. According to one embodiment of the method of the present invention, in the step S4, the raw data obtained in the step S3 is analyzed, and the positions of the internal molecular weight standards in each lane are compared with the positions of the peak values of each sample, so as to o