CN115896329A - SSR molecular marker primer set and its application developed based on the whole genome of Osmanthus fragrans - Google Patents

Abstract

The invention discloses an SSR molecular marker primer set developed based on the whole genome of Osmanthus osmanthus and its application. The SSR molecular marker primer set of the invention has good versatility, is stable and efficient, and can quickly identify osmanthus germplasm resources from the molecular level , not affected by seasons, plant development periods and growth environment, utilizing the SSR molecular marker primer set of the present invention to carry out the variety identification technology of sweet-scented osmanthus has the characteristics of convenient material collection, high accuracy and stability of results, and is useful for the rapid identification of sweet-scented osmanthus germplasm resources, Research on new variety protection, genetic diversity evaluation and molecular marker-assisted breeding provides reference and basis.

Description

SSR molecular marker primer set and its application developed based on the whole genome of Osmanthus fragrans

technical field

The invention belongs to the technical field of molecular biology, and in particular relates to an SSR molecular marker primer set developed based on the whole genome of sweet-scented osmanthus fragrans and its application.

Background technique

Osmanthus fragrans, belonging to Oleaceae Oleaceae, is one of the top ten traditional famous flowers in my country. It is cultivated in the vast areas from the north of Nanling to the south of Huaihe River Basin in Qinling Mountains. It has a cultivation history of nearly 2,500 years. . In the process of perennial cultivation and domestication, osmanthus fragrans has formed a variety group with obvious differentiation in flower color and flowering period of Yingui, Jingui, Dangui and Sijigui. However, due to the small size of osmanthus fragrans, concentrated and short flowering period, it is difficult to quickly and accurately identify osmanthus fragrans varieties from the traditional morphological level. Seriously restricting the high-quality development of the sweet-scented osmanthus industry. Therefore, it is urgent to develop a more rapid and accurate detection method to provide a basis for the identification of sweet-scented osmanthus species and the development and utilization of germplasm resources.

Simple repeat sequence (SSR), also known as short tandem repeat (STR), microsatellite, is a short tandem repeat DNA sequence composed of 1-6 bases as repeating units. SSR markers are ideal co-dominant molecular markers that widely exist in the genome and have the advantages of high polymorphism, good repeatability, low cost, and easy operation. They are widely used in the variety identification and genetic diversity of horticultural plant germplasm resources. in the study of sex. Similarly, SSR markers are also an important tool for the identification of Osmanthus fragrans germplasm resources and the study of genetic diversity. However, the SSR primers for Osmanthus fragrans that have been developed so far are still relatively few, and the efficiency is low, which greatly limits the application of SSR markers in Osmanthus fragrans research, and cannot meet the needs of production and scientific research.

Contents of the invention

In order to solve the above-mentioned problems in the prior art, the present invention provides a universal and efficient SSR molecular marker primer set applicable to sweet-scented osmanthus and its application. The SSR molecular marker primer set of the present invention is obtained based on the screening of the whole genome of Osmanthus osmanthus, and at the same time, the corresponding SSR amplification technology system is established to be applied to the fields of germplasm resource identification, osmanthus osmanthus genetic diversity analysis, new variety protection and/or fingerprint construction, etc. .

The technical scheme adopted in the present invention is:

A SSR molecular marker primer set developed based on the whole genome of Osmanthus osmanthus, including the following 4 pairs of primer sequences:

P2-90:F:5'-GATCCCTGTCGCCTGAATGA-3',

R: 5'-CACCCCACGCCACATATACA-3';

P3-120:F:5'-CTCCTGACGCTTGGAGGAAA-3',

R: 5'-TGGTGATGGTCAATTCAGCCT-3';

P2-60:F:5'-TGGCTTCCATGAAAACTAGACGT-3',

R: 5'-TCACTTGACCCACATCGGTG-3'

P2-98:F:5'-CTGGAAACCACCGCGATAGT-3',

R: 5'-ACTACAGCACTGACGCTCAC-3'.

The application of the SSR molecular marker primer set developed based on the whole genome of Osmanthus osmanthus in identification of Osmanthus fragrans germplasm resources, analysis of genetic structure and genetic diversity, protection of new varieties and/or construction of fingerprints.

Described application comprises the following steps:

(1) extract the genomic DNA of sweet-scented osmanthus;

(2) taking the genomic DNA extracted in step (1) as a template, utilizing each primer pair in the SSR molecular marker primer set described in claim 1 to carry out PCR amplification respectively;

(3) typing the PCR product amplified in step (2);

(4) Perform data analysis on the typing results obtained in step (3) to realize identification of germplasm resources, analysis of genetic structure and genetic diversity, protection of new varieties and/or construction of fingerprints.

In step (2), the reaction system of the PCR amplification is: 30ng/μL template DNA 0.2μL, 10pm/μL forward primer 0.06μL, 10pm/μL reverse primer 0.3μL, 25pm/μL with FAM fluorescence Labeled M13 universal primer 0.12 μL, 2×TaqPCR MasterMix 7.5 μL, add double distilled water to 15 μL.

The reaction program of the PCR amplification is: pre-denaturation at 95°C for 5min; denaturation at 94°C for 30s, annealing for each primer pair at the annealing temperature for 45s, extension at 72°C for 45s, 30 cycles; denaturation at 94°C for 30s, annealing at 53°C for 45s, Extend at 72°C for 45s, 8 cycles, and terminate the reaction at 4°C after extending for 10min at 72°C.

In step (3), the amplified PCR product is typed by capillary electrophoresis.

In step (4), bioinformatics software is used to perform data analysis on the typing results obtained in step (3).

The beneficial effects of the present invention are:

The SSR molecular marker primer set developed based on the whole genome of osmanthus osmanthus has good versatility, is stable and efficient, and can quickly identify osmanthus germplasm resources from the molecular level without being affected by seasons, plant development periods and growth environments. The variety identification technology of Osmanthus osmanthus with molecular marker primer set has the characteristics of convenient material collection and highly accurate and stable results, which provides reference and basis for rapid identification of osmanthus germplasm resources, protection of new varieties, genetic diversity evaluation and molecular marker-assisted breeding. .

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only These are some embodiments of the present invention. Those skilled in the art can also obtain other drawings based on these drawings without creative work.

Fig. 1 is the cluster analysis result of different sweet-scented osmanthus varieties;

Fig. 2 is the capillary electrophoresis figure of the partial PCR amplification product of SSR primer P2-90;

Fig. 3 is capillary electrophoresis diagram of part of PCR amplification product of SSR primer P3-120;

Fig. 4 is the capillary electrophoresis figure of the partial PCR amplification product of SSR primer P2-60;

Fig. 5 is a capillary electrophoresis diagram of partial PCR amplification products of SSR primer P2-98.

Detailed ways

In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Apparently, the described embodiments are only some of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other implementations obtained by persons of ordinary skill in the art without making creative efforts fall within the protection scope of the present invention.

Embodiment 1: Osmanthus fragrans SSR marker and the acquisition of primers thereof

1. SSR site development

Using the existing whole genome data of Osmanthus fragrans, MISA software was used to detect simple repeat sequences in the whole genome sequence of Osmanthus fragrans, and the different types of SSRs and their distribution were counted. The parameters are set as follows: the minimum repetition times of single, di, tri, tetra, penta, and hexanucleotides are 16, 6, 5, 5, 5, and 5 times, respectively.

2. SSR primer design

Primer3 software was used to design batch primers for the screened SSR sites, and 131 primers were selected and synthesized by Shanghai Sangon Biotechnology Co., Ltd. Primer design follows the following core parameters:

(1) The length range of the primers is limited between 18-24bp. In order to detect the polymorphism of the SSR site by capillary electrophoresis in the next step, an M13 universal primer sequence (5') is added to the 5' end of the forward primer at all sites '-TGTAAAACGACGGCCAGT-3')

(2) In order to facilitate the establishment of a nested PCR amplification system and polymorphism detection in the next step, the PCR amplification length of each microsatellite site was set at 100-400bp in this experiment, and the internal structure of the primers, such as hairpin structures and Primer dimers, etc.

3. Extraction of osmanthus fragrans genomic DNA:

In this experiment, 32 osmanthus germplasm resources were used as test materials. The specific varieties are shown in Table 1. The young leaves of osmanthus plants were selected, and the plant genomic DNA extraction kit (DP305, TIANGEN, Beijing) was used to extract total For DNA, dilute the DNA of the test material to 30ng/μL with ddH ₂ O, and store it in a refrigerator at minus 20°C for subsequent primer screening and polymorphism detection.

Table 1-32 osmanthus germplasm resources

4. PCR amplification using SSR primers

Using the total DNA sample of sweet-scented osmanthus extracted in step 3 as a template, use the synthesized primers to carry out SSR-PCR amplification; it should be noted that the present invention uses M13 adapters for labeling, and can also use FAM, HEX, ROX or TAMRA four kinds of fluorescent Label the primers.

SSR-PCR reaction system 15μL: 0.2μL template DNA (30ng/μL), 0.06μL forward primer (10pm/μL), 0.3μL reverse primer (10pm/μL), 0.12μL M13 universal primer with FAM fluorescent label (25pm/μL), 7.5 μL 2×TaqPCR MasterMix (KT201, TIANGEN, Beijing), add ddH ₂ O to 15 μL.

SSR-PCR reaction program: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 30 s, annealing at 58°C for 45 s (the optimal annealing temperature depends on the annealing temperature of the primers), extension at 72°C for 45 s, 30 cycles; denaturation at 94°C for 30 s, 53 Refolding at ℃ for 45s, extension at 72℃ for 45s, 8 cycles, extension at 72℃ for 10min, and then terminate the reaction at 4℃.

5. Fluorescence detection of PCR products by capillary electrophoresis

The amplified product was sent to Shanghai Sangon Biological Co., Ltd. for fluorescence detection after it was sealed in shading. ABI3730XL sequencer was used for capillary electrophoresis. The molecular weight internal standard was GeneScan600. The data was automatically detected and stored, and GeneMapper 3.2 was used for data band analysis.

The 131 pairs of primers synthesized above were used for capillary electrophoresis fluorescence detection of 32 osmanthus varieties, and finally 4 pairs of primers with high polymorphism and stable amplification were screened out for subsequent identification of osmanthus germplasm resources and genetic diversity research. The results are shown in Figure 2-Figure 5. The abscissa is the size of the amplified fragment, and the ordinate is the fluorescence intensity of the amplified product. It can be seen from the figure that the overall signal is clear, and the electrophoresis peak types of DNA samples are different, showing better polymorphism.

Example 2: Polymorphism Analysis of Microsatellite Primers

Use PowerMarker v3.25 software to calculate primer polymorphism information content (PIC), use POPGENEv1.32 software to calculate the number of observed alleles (Na), effective number of alleles (Ne), and expected heterogeneity of each Osmanthus osmanthus SSR locus. Heterozygosity (He), observed heterozygosity (Ho), and genetic diversity information of microsatellite loci are shown in Table 2.

Table 2 - Genetic diversity information of microsatellite loci

4 pairs of SSR primers were used to amplify 32 varieties of Osmanthus fragrans, and a total of 56 alleles were obtained. Using Popgene v1.32 software to analyze the polymorphism of the screened SSR, the number of detected alleles (Na): 12-15, with an average of 14; the effective number of alleles (Ne): 6.8267-8.192, with an average of 7. 3989. The average observed heterozygosity (Ho) was 0.6563, and the average expected heterozygosity (He) was 0.878.

Using PowerMarkerv3.25 software to calculate the polymorphism information content PIC of primers: 0.837-0.8665, with an average of 0.851. Taking PIC≥0.5 as highly polymorphic primers, 0.25<PIC<0.5 as moderately polymorphic primers, and PIC<0.25 as lowly polymorphic primers as the standard, the primers announced by the present invention are highly polymorphic primers. Morphological primers.

Embodiment 3: the identification of Osmanthus fragrans germplasm resources

According to the band data amplified by the four SSR markers, the genetic similarity coefficients among the varieties were calculated, and clustered according to the UPGMA method (Fig. 1). The genetic similarity coefficients among the 32 varieties were 0.714-1.00, with an average value of 0.807. The above Osmanthus osmanthus SSR core primer combination can completely distinguish 30 other osmanthus varieties except SJG01 and SJG04, and the identification efficiency reaches 93.75%.

The present invention is not limited to the above-mentioned best implementation mode, anyone can draw other various forms of products under the inspiration of the present invention, but no matter make any changes in its shape or structure, all those with the same or similar features as the present application Approximate technical solutions all fall within the protection scope of the present invention.

Claims (7)

1. a kind of SSR molecular marker primer set based on the whole genome development of Osmanthus osmanthus, is characterized in that, comprises following 4 pairs of primer sequences: P2-90:F:5'-GATCCCTGTCGCCTGAATGA-3', R: 5'-CACCCCACGCCACATATACA-3'; P3-120:F:5'-CTCCTGACGCTTGGAGGAAA-3', R: 5'-TGGTGATGGTCAATTCAGCCT-3'; P2-60:F:5'-TGGCTTCCATGAAAACTAGACGT-3', R: 5'-TCACTTGACCCACATCGGTG-3' P2-98:F:5'-CTGGAAACCACCGCGATAGT-3', R: 5'-ACTACAGCACTGACGCTCAC-3'. 2. the application of the SSR molecular marker primer set based on the whole genome of osmanthus osmanthus according to claim 1 in the identification of osmanthus germplasm resources, genetic structure and genetic diversity analysis, new variety protection and/or fingerprint map construction. 3. The application according to claim 2, characterized in that, comprising the steps of: (1) extract the genomic DNA of sweet-scented osmanthus; (2) taking the genomic DNA extracted in step (1) as a template, utilizing each primer pair in the SSR molecular marker primer set described in claim 1 to carry out PCR amplification respectively; (3) typing the PCR product amplified in step (2); (4) Perform data analysis on the typing results obtained in step (3) to realize identification of germplasm resources, analysis of genetic structure and genetic diversity, protection of new varieties and/or construction of fingerprints. 4. application according to claim 3, is characterized in that, in step (2), the reaction system of described PCR amplification is: template DNA 0.2 μ L of 30ng/μL, 10pm/μL forward primer 0.06 μ L, 10pm /μL reverse primer 0.3μL, 25pm/μL M13 universal primer with FAM fluorescent label 0.12μL, 2×Taq PCR MasterMix 7.5μL, add double distilled water to 15μL. 5. The application according to claim 3, wherein the reaction procedure of the PCR amplification is: pre-denaturation at 95°C for 5 minutes; denaturation at 94°C for 30s; 30 cycles; denaturation at 94°C for 30s, renaturation at 53°C for 45s, extension at 72°C for 45s, 8 cycles, extension at 72°C for 10min and termination at 4°C. 6. The application according to claim 3, characterized in that in step (3), the amplified PCR product is typed by capillary electrophoresis. 7. The application according to claim 3, characterized in that in step (4), data analysis is performed on the typing results obtained in step (3) using bioinformatics software.

Images