SSR primer for identifying bromegrass and application of SSR primer
By designing a combination of SSR molecular marker primers for Bromus spp., the problem of Bromus spp. invasion was solved, efficient detection of genetic diversity was achieved, and scientific prevention and control and the stability of agricultural production were supported.
Patent Information
- Application Number
- CN202511147288.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-15
- Publication Date
- 2025-10-17
AI Technical Summary
Existing technologies make it difficult to effectively control and prevent the invasion of Bromus arvense, especially in the Qinghai-Tibet Plateau where the effectiveness of chemical herbicides is limited and there is a lack of effective genetic diversity detection methods, which affects agricultural production and ecosystem balance.
An SSR molecular marker primer combination suitable for Bromus arvense was developed to amplify and analyze its genetic diversity. By designing multiple pairs of specific primers for PCR amplification and electrophoresis detection, a binary matrix was constructed to calculate the polymorphism index, and primer combinations with strong polymorphism were screened.
Efficient detection of the genetic diversity of Bromus arvense was achieved, providing a theoretical basis for scientific prevention and control strategies, improving the understanding of its spread and invasion mechanisms, and supporting the protection of agricultural production and ecosystems.
Smart Images
Figure BDA0005551353470000031 
Figure BDA0005551353470000042 
Figure BDA0005551353470000051
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of molecular biology, in particular to a SSR primer for identifying Bromus tectorum and application thereof. BACKGROUND
[0002] Bromus tectorum L. belongs to the annual herb of Bromus in Poaceae, also known as water oats, hairy brome, brome grass, etc., which has strong ecological adaptability and invasiveness. In the agricultural area of Qinghai-Tibet Plateau, Bromus tectorum has become one of the important weeds affecting crop yield and quality due to its rapid growth, short growth period, large seed setting amount and easy seed dissemination. In addition, the surface of the leaf and stem of Bromus tectorum has soft hair, which is not conducive to the absorption of herbicides, and the low temperature and dry environment of the plateau area further weaken the control effect of chemical herbicides, making the control of Bromus tectorum a big problem. Therefore, it is urgent to study the control of Bromus tectorum. SUMMARY
[0003] The present inventors believe that the transmission and adaptability of Bromus tectorum are closely related to its genetic diversity, and the in-depth study of the genetic diversity of Bromus tectorum not only helps to reveal its diffusion and invasion mechanism, but also provides a theoretical basis for formulating scientific control strategies. At the same time, by analyzing the genetic structure and diversity level of different populations, the population dynamics and environmental adaptability can be further understood, which has important significance for protecting the balance of plateau ecological system and ensuring agricultural production. Therefore, the present applicant developed a SSR molecular marker suitable for detecting the genetic diversity of Bromus tectorum.
[0004] In one aspect, the present application provides a primer composition for amplifying the SSR molecular marker of Bromus tectorum, which consists of the following primer pairs:
[0005] a primer pair of SEQ ID NO: 23 and 24; a primer pair of SEQ ID NO: 25 and 26; a primer pair of SEQ ID NO: 27 and 28; a primer pair of SEQ ID NO: 29 and 30; a primer pair of SEQ ID NO: 31 and 32; a primer pair of SEQ ID NO: 33 and 34; a primer pair of SEQ ID NO: 35 and 36; a primer pair of SEQ ID NO: 37 and 38; a primer pair of SEQ ID NO: 39 and 40; a primer pair of SEQ ID NO: 41 and 42; a primer pair of SEQ ID NO: 43 and 44; a primer pair of SEQ ID NO: 45 and 46; a primer pair of SEQ ID NO: 47 and 48; a primer pair of SEQ ID NO: 49 and 50; a primer pair of SEQ ID NO: 51 and 52; a primer pair of SEQ ID NO: 53 and 54; a primer pair of SEQ ID NO: 55 and 56; a primer pair of SEQ ID NO: 57 and 58; a primer pair of SEQ ID NO: 59 and 60; a primer pair of SEQ ID NO: 61 and 62; a primer pair of SEQ ID NO: 63 and 64; a primer pair of SEQ ID NO: 65 and 66.
[0006] The primer composition is used for amplifying the SSR marker.
[0007] The primer composition is used for amplifying the SSR marker of Bromus tectorum L.
[0008] In another aspect, the present application provides the use of any one of the following:
[0009] The primer composition is used for identifying the variety of Bromus tectorum L.
[0010] The primer composition is used for detecting and analyzing the genetic diversity of Bromus tectorum L.
[0011] The primer composition is used for screening the target gene in Bromus tectorum L.
[0012] The primer composition is used for breeding Bromus tectorum L.
[0013] In another aspect, the present application provides the SSR marker of Bromus tectorum L.
[0014] The SSR marker of Bromus tectorum L. consists of DNA fragments shown in SEQ ID NO: 1-22, and fragments corresponding to the DNA fragments in different Bromus tectorum L. individuals or different Bromus tectorum L. varieties.
[0015] Experiments prove that the primer combination of the SSR marker provided by the application has strong polymorphism detection capability, and the average polymorphism ratio (PIC) is 0.583. The application provides an advantageous tool for detecting the genetic diversity of Bromus tectorum L., and has important significance for solving Bromus tectorum L. prevention and control. DETAILED DESCRIPTION
[0016] The following are some specific embodiments of the application, wherein the technical means or reagents used are conventional methods and reagents known in the art unless otherwise specified.
[0017] EMBODIMENT
[0018] Bromus tectorum L. seeds were collected from 20 different regions in Qinghai Province. The seeds were planted in the greenhouse of the College of Agriculture and Forestry, Qinghai University, and after growing for a certain period of time, the leaves were taken for transcriptome sequencing. After sequencing, the original data was filtered using software, and the reads with joint contamination, low quality and unknown bases were deleted.
[0019] SSR site mining and primer design
[0020] The SSR sites from the transcriptome sequences were identified using the microsatellite (MISA) software. The configuration settings were as follows: the minimum number of single nucleotide repeats was set to 10 times, the minimum number of di-nucleotide repeats was set to 6 times, and the minimum number of other nucleotides was set to 5 times. The number, proportion and frequency of different repeat numbers of each repeat type in the Bromus tectorum L. transcriptome were counted. Primer 3.0 software was used to design primers for each single gene sequence containing SSR sites. The parameter settings were as follows: the primer length was about 20 bp, the GC content was 40% to 60%, the theoretical annealing temperature was 55.0℃ to 65.0℃, and the difference between the annealing temperatures of the forward and reverse primers was less than 2℃.
[0021] Polymorphic primer screening and verification
[0022] The 56 pairs of primers were selected and synthesized by Shanghai Sangon Biological Engineering Co. Ltd. The genomic DNA of 96 individuals of E. trachycaula was amplified by PCR. The PCR reaction system was 10 μL, including 5 μL of PCR Mix (green), 0.5 μL of forward and reverse primers, 1 μL of DNA (50 ng), and 3 μL of ddH2O. The PCR reaction procedure was as follows: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30 s, annealing for 45 s; extension at 72°C for 15 s, for 30 cycles; extension at 72°C for 10 min, and preservation at 4°C. The amplified products were detected by 8% polyacrylamide gel electrophoresis, using 1×TBE as the electrophoresis buffer, 2000 V voltage electrophoresis, and then silver staining for 30 min.
[0023] Data statistical method
[0024] The clear and consistent SSR amplification bands were analyzed, recorded as present (1) or absent (0), and a binary matrix was constructed. The polymorphism information index (PIC) of the selected SSR primers was calculated by the formula PIC = 1-∑(Pi 2 )2, wherein Pi is the frequency of the i th microsatellite allele.
[0025] It was found that 22 pairs of primers could solve the problem of polymorphism analysis, and the overall effect was good, the annealing temperature was uniform (the average Tm value was 54.2°C), the polymorphism detection ability was strong, and the average polymorphism ratio (PIC) was 0.583.
[0026] Table 1, Analysis results of 22 pairs of SSR primer amplification reactions
[0027]
[0028]
[0029] Table 2, Primer information table
[0030]
[0031]
[0032] Table 3, Sample distribution
[0033] latitude longitude single clone 37.13482 101.8029 5 37.0472 101.8165 5 36.9393 101.6936 5 37.09648 101.3669 5 36.8911 100.9944 4 36.93498 101.0205 5 37.08813 101.0937 5 36.5379 100.6552 5 36.8732 99.66697 4 37.03129 99.73395 5 36.59471 100.302 4 36.8044 102.0847 5 37.00216 102.1066 5 37.35397 101.6597 5 37.28244 101.8038 5 37.3368 101.9189 4 37.18274 102.1639 5 37.38797 101.8653 5 35.52065 102.2519 5 38.22014 100.0438 5
Claims
1. Primer composition, consisting of the following primer pairs: a primer pair set forth in SEQ ID NOs: 23 and 24; a primer pair set forth in SEQ ID NOs: 25 and 26; a primer pair set forth in SEQ ID NOs: 27 and 28; a primer pair set forth in SEQ ID NOs: 29 and 30; a primer pair set forth in SEQ ID NOs: 31 and 32; a primer pair set forth in SEQ ID NOs: 33 and 34; a primer pair set forth in SEQ ID NOs: 35 and 36; a primer pair set forth in SEQ ID NOs: 37 and 38; a primer pair set forth in SEQ ID NOs: 39 and 40; a primer pair set forth in SEQ ID NOs: 41 and 42; a primer pair set forth in SEQ ID NOs: 43 and 44; a primer pair set forth in SEQ ID NOs: 45 and 46; a primer pair set forth in SEQ ID NOs: 47 and 48; a primer pair set forth in SEQ ID NOs: 49 and 50; a primer pair set forth in SEQ ID NOs: 51 and 52; a primer pair set forth in SEQ ID NOs: 53 and 54; a primer pair set forth in SEQ ID NOs: 55 and 56; a primer pair set forth in SEQ ID NOs: 57 and 58; The primer pair shown in SEQ ID NOs: 57 and 58; the primer pair shown in SEQ ID NOs: 59 and 60; the primer pair shown in SEQ ID NOs: 61 and 62; the primer pair shown in SEQ ID NOs: 63 and 64; and the primer pair shown in SEQ ID NOs: 65 and 66.
2. The primer composition according to claim 1, wherein: The primer combination is used to amplify SSR markers.
3. The primer composition according to claim 1 or 2, characterized in that: The primer combination is used to amplify the SSR marker of Bromus tectorum L.
4. Use of the primer combination according to any one of claims 1 to 3 in identifying Bromus tectorum L. varieties.
5. Use of the primer combination according to any of claims 1 to 3 in detecting and analyzing the genetic diversity of Bromus tectorum L.
6. Use of the primer combination according to any one of claims 1 to 3 in screening target genes in Bromus tectorum L.
7. Use of the primer combination according to any one of claims 1 to 3 in the breeding of Bromus tectorum L.
8. The SSR markers of Bromus spp. are composed of the following markers: The DNA fragments shown in SEQ ID NOs: 1-22, respectively, and fragments corresponding to the DNA fragments in different Bromus tectorum L. individuals or different Bromus tectorum varieties.