Sambucus williamsii SSR molecular marker, primer group and application thereof in construction of Sambucus williamsii fingerprint spectrum

Abstract

The invention belongs to the technical field of plant germplasm resource identification, and particularly relates to an elderberry SSR molecular marker, a primer group and application of the elderberry SSR molecular marker in construction of an elderberry fingerprint spectrum. Specifically, the invention utilizes 16 groups of SSR molecular markers to carry out genetic variation and diversity analysis on collected 287 parts of elderberry germplasm resources (including 16 new varieties: 'Natural Red No.1', 'Natural Red No.2', 'Tianshan Maiyu', 'Qing ink', 'Yinhong', 'Shuidan', 'Liuyan Red', 'Zifeng', 'Jinjiang', 'Hongfeng', 'Hongdan', 'Hongdan', 'Baidan', 'Baidan', 'Baidan', 'Baidan', 'Baidan', 'Red agate' and 'Jinspike'). And fingerprint spectrum construction is carried out on 16 varieties, so that data reference is provided for management and utilization of elderberry germplasm resources and new variety breeding, and the method has good practical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of plant germplasm resource identification, and particularly relates to an elderberry SSR molecular marker, a primer set and an application thereof in constructing an elderberry fingerprint. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Elderberry (Sambucus williamsii Hance), also known as nine-section wind, bone grass, wood capsule, etc., is a deciduous shrub or small tree of the genus Sambucus in the Viburnum family. Elderberry has its unique medicinal value and ornamental characteristics, and its roots, stems, leaves, flowers and fruits all have certain medicinal value. It is sweet and bitter in nature, flat and non-toxic, contains a variety of effective ingredients such as anthocyanin glucoside, cyanol glycoside, iridoid ether terpenoid glycoside, and has multiple effects such as dispelling wind, promoting dampness, promoting blood circulation, and relieving pain. Its seeds have high oil content and are woody oil-bearing tree species; the fruit can be processed into jam, jelly and other foods with high nutritional value. The flowers and fruits of elderberry can also be used in spice production, and the fruit can also be used as a natural colorant for red food. At the same time, elderberry is also an excellent foliage, flower and fruit viewing tree species. Therefore, it is crucial to effectively and accurately identify the germplasm resources and varieties of elderberry for the protection and genetic management of elderberry germplasm resources.

[0004] Germplasm identification based on morphological characteristics is a traditional method, which has the disadvantages of being time-consuming, laborious, and susceptible to environmental influences. SSR (Simple Sequence Repeat) is a molecular marker technology commonly used in molecular biology, and has been widely used in germplasm identification, genetic diversity analysis, and kinship research. For the SSR research on elderberry germplasm resources, the genetic structure and kinship can be revealed by analyzing the SSR polymorphism of different varieties or populations, providing a scientific basis for the accurate identification of elderberry germplasm resources, the construction of core germplasm, and the creation of new varieties. However, the inventors found that there are still few reports on the analysis and research of elderberry germplasm resources based on SSR technology. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides an elderberry SSR molecular marker, a primer set and an application thereof in constructing an elderberry fingerprint map. Specifically, the present invention uses 16 groups of SSR molecular markers to analyze the genetic variation and diversity of 287 elderberry germplasm resources collected (including 16 new varieties: 'Natural Red No. 1', 'Natural Red No. 2', 'Tianshan Meiling', 'Qingmo', 'Wanhong', 'Shuidan', 'Liuyehong', 'Zifeng', 'Jinhuan', 'Hongfeng', 'Yandan', 'Quandan', 'Heimeiren', 'Heizhuzhu', 'Hongagate' and 'Jinsui'), and constructs a fingerprint map for the 16 varieties, thereby providing data reference for the management and utilization of elderberry germplasm resources and the selection and breeding of new varieties. Based on the above research results, the present invention is completed.

[0006] Specifically, the present invention relates to the following technical solutions:

[0007] In a first aspect of the present invention, there is provided an elderberry SSR molecular marker, wherein the SSR molecular marker comprises the following SSR loci: LOC003, LOC039, LOC043, LOC055, LOC067, LOC090, LOC092, LOC097, LOC114, LOC131, LOC141, LOC145, LOC163, LOC169, LOC177 and LOC186;

[0008] The above elderberry SSR molecular markers were obtained by amplifying the following primer pairs respectively: SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.9-10, SEQ ID NO.11-12, SEQ ID NO.13-14, SEQ ID NO.15-16, SEQ ID NO.17-18, SEQ ID NO.19-20, SEQ ID NO.21-22, SEQID NO.23-24, SEQ ID NO.25-26, SEQ ID NO.27-28, SEQ ID NO.29-30 and SEQ ID NO.31-32.

[0009] Furthermore, the elderberry SSR molecular marker is any one or more of LOC003, LOC039, LOC067, LOC090, LOC092, LOC114, LOC131, LOC145, LOC163, LOC169, LOC177 and LOC186. The elderberry SSR molecular marker has high quality, strong representativeness and high genetic variation, and is therefore very suitable for the construction of elderberry DNA fingerprint.

[0010] The second aspect of the present invention provides an elderberry SSR molecular marker primer set, which comprises any one or more of the following 16 pairs of specific primers, and the nucleotide sequences thereof are shown in SEQ ID NO.1-32 respectively.

[0011] Furthermore, the elderberry SSR molecular marker primer set comprises any one or more of the following 12 pairs of specific primer pairs, whose nucleotide sequences are SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ ID NO.9-10, SEQ ID NO.11-12, SEQ ID NO.13-14, SEQ ID NO.17-18, SEQ ID NO.19-20, SEQ ID NO.23-24, SEQID NO.25-26, SEQ ID NO.27-28, SEQ ID NO.29-30 and SEQ ID NO.31-32.

[0012] A third aspect of the present invention provides a product for detecting the elderberry SSR molecular marker, the product comprising at least the elderberry SSR molecular marker primer;

[0013] Furthermore, the product may be a detection kit, a gene chip, a detection device and equipment, etc., which are not specifically limited here.

[0014] The fourth aspect of the present invention provides the use of the elderberry SSR molecular marker, the elderberry SSR molecular marker primer, and the product for detecting the elderberry SSR molecular marker in any one or more of the following:

[0015] 1) Analysis of genetic diversity of elderberry;

[0016] 2) Construction of the elderberry genetic map;

[0017] 3) Identification of elderberry germplasm resources;

[0018] 4) Analysis of the relationship between elderberry;

[0019] 5) Mining of functional genes of elderberry;

[0020] 6) Molecular marker-assisted breeding of elderberry.

[0021] In another specific embodiment of the present invention, in said 2), constructing the elderberry genetic map is specifically constructing the elderberry SSR fingerprint map.

[0022] In another specific embodiment of the present invention, in said 3), the identification of elderberry germplasm at least includes the identification of elderberry varieties.

[0023] The fifth aspect of the present invention provides a method for constructing an elderberry SSR fingerprint, the steps comprising: extracting the genomic DNA of the elderberry to be tested, performing PCR amplification on the genomic DNA of the elderberry to be tested using the above-mentioned elderberry SSR molecular marker primers, performing capillary electrophoresis detection on the PCR products, and constructing the elderberry SSR fingerprint.

[0024] A sixth aspect of the present invention provides a method for identifying elderberry varieties, the method comprising: comparing the elderberry SSR fingerprint obtained above with the fingerprint of known elderberry varieties, and the elderberry varieties with the same fingerprints are the corresponding elderberry varieties.

[0025] The elderberry varieties include 'Natural Red No. 1', 'Natural Red No. 2', 'Tianshan Meiling', 'Qingmo', 'Wanhong', 'Shuidan', 'Liuyehong', 'Zifeng', 'Jinhuan', 'Hongfeng', 'Yandan', 'Quandan', 'Hei Meiren', 'Heizhuzhu', 'Hongmanyu' and 'Jinsui'. The present invention has found through research that the above-mentioned primers can be used to effectively distinguish the above-mentioned elderberry varieties.

[0026] The present invention has found through research that the elderberry SSR molecular marker primers screened by the present invention have high polymorphism, can effectively reveal the genetic diversity of elderberry, and thus are very beneficial to the identification of elderberry germplasm resources.

[0027] Beneficial technical effects of one or more of the above technical solutions:

[0028] The above technical scheme obtained 16 elderberry SSR molecular markers by screening, further evaluated their genetic diversity, and constructed fingerprint maps for 16 new elderberry varieties. The above technical scheme effectively improved the efficiency of elderberry variety identification, laid the foundation for research on elderberry kinship identification, systematic evolution, variety identification, genetic variation and genetic map construction, and therefore has good practical application value. DETAILED DESCRIPTION

[0029] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0030] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0031] The present invention is further described in conjunction with specific examples. The following examples are only for explaining the present invention and are not intended to limit the content thereof. If the specific experimental conditions are not specified in the examples, they are usually carried out under conventional conditions or under conditions recommended by the sales company; the materials and reagents used in the examples, unless otherwise specified, can be purchased through commercial channels.

[0032] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0033] Example 1

[0034] The applicant team designed and synthesized 200 pairs of SSR primers based on the elderberry transcriptome data and screened the identification effects of elderberry materials:

[0035] 1. First, 8 screening samples (TA10 (Tai'an No. 10), TA7 (Tai'an No. 7), TSML (Tianshan Meiling), Liuyehong, Zifeng, Jinhuan, Hongfeng) were used to amplify 200 pairs of primers, and the reaction was carried out on a Veriti384 PCR instrument. The PCR amplification program was set as follows: 95℃ pre-denaturation for 5min; 95℃ denaturation for 30s, 62-52℃ gradient annealing for 30s, 72℃ extension for 30s, run 10 cycles; 95℃ denaturation for 30s, 52℃ annealing for 30s, 72℃ extension for 30s, run 25 cycles; 72℃ extension for 20min, and finally stored at 4℃. After the PCR reaction, the amplified product was detected by fluorescent capillary electrophoresis. The results were analyzed using GeneMarker software, and 32 pairs of primers were preliminarily screened.

[0036] 2. The 32 pairs of primers screened in the previous step were screened again using 8 samples, and the reaction was performed on a Veriti384PCR instrument. The PCR amplification program was set as follows: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 62-52°C gradient annealing for 30 s, 72°C extension for 30 s, 10 cycles; 95°C denaturation for 30 s, 52°C annealing for 30 s, 72°C extension for 30 s, 25 cycles; 72°C extension for 20 min, and finally stored at 4°C. After the PCR reaction was completed, the amplified product was detected by fluorescent capillary electrophoresis. The results were analyzed using GeneMarker software, and 16 pairs of polymorphic primers were obtained as shown in Table 1.

[0037] 3. Twelve pairs of primers were selected from the alternative primers to detect 16 population samples, and the reaction was carried out on a Veriti 384 PCR instrument. The PCR amplification program was set as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, gradient annealing at 62 - 52°C for 30 s, extension at 72°C for 30 s, running for 10 cycles; denaturation at 95°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 30 s, running for 25 cycles; extension at 72°C for 20 min, and finally stored at 4°C. After the PCR reaction, the amplified products were detected by fluorescence capillary electrophoresis. The GeneMarker software was used to analyze the results to obtain the number of alleles, peak maps, and genotypes of each sample.

[0038] Table 1. Polymorphic primers

[0039]

[0040]

[0041] Among them, 0 < PIC < 0.25 indicates low polymorphism, 0.25 < PIC < 0.5 indicates moderate polymorphism, and PIC > 0.5 indicates high polymorphism. As can be seen from Table 1, the 12 pairs of SSR primers screened have relatively high polymorphism. Through repeated experiments, it was found that the identification effect of most SSR primers designed and synthesized based on genomic data is acceptable. Only using the 12 groups of primers recommended in the above table for the identification of the sites of Sambucus will have the best effect.

[0042] Furthermore, for the genetic diversity analysis of the sites: parameters such as Na, Ne, I, Ho, He, F, and Fst were calculated using GenAIex software, and the PIC values of each site were calculated by Cervus software. The following are the specific analysis results and their explanations. The average number of alleles (Na), effective number of alleles (Ne), average observed heterozygosity (H0), average expected heterozygosity (He), etc. of each site are common indicators reflecting population genetic diversity. The fixation index F is used to measure the degree of deviation of the observed heterozygosity from the Hardy - Weinberg equilibrium.

[0043] The statistical results are shown in Table 2 below:

[0044] Table 2. Polymorphism of 12 pairs of SSR primers

[0045]

[0046] Note: Na: Observed allele; Ne: Effective allele; I: Shannon index; Ho: Observed heterozygosity; He: Expected heterozygosity; F: Fixation index, an index to evaluate the degree of deviation between the actual observed value and the theoretical value, PIC: Polymorphism information content

[0047] Table 3. Inbreeding coefficient and gene flow of 12 pairs of primers

[0048]

[0049] Note: Fis: inbreeding coefficient within a population, Fit: overall inbreeding coefficient, Fst: genetic differentiation coefficient, Nm: gene flow (Nm = 0.25 (1-Fst) / Fst).

[0050] As shown in Tables 2 and 3, 12 pairs of primers detected a total of 107 alleles (Na), of which the minimum number of alleles was 5, the maximum number of alleles was 16, and the average number of alleles per site was 8.917. The total number of effective alleles (Ne, the more evenly the alleles are distributed in the population, the closer Ne is to the number of alleles actually detected) was 59.847, with a range of values ​​of 2.876 (L0C163)-9.328 (LAC073), and the average number of effective alleles per site was 4.987. The range of Shannon index (I) values

[0051] 1.208 (L0C163)-2.496 (LOC067), with an average value of 1.766. The observed heterozygosity (Ho) ranged from

[0052] 0.500 (L0C169)-0.938 (L0C067), with an average value of 0.635. The expected heterozygosity (He) ranged from 0.652 (L0C163) to 0.893 (LOC067), with an average value of 0.769. The polymorphic information content (PIC) ranged from 0.635

[0053] (LOC177)-0.884(LOC067), with an average value of 0.738. The average value of the inbreeding coefficient was 0.055, with values ​​of -0.373(LOC163)-0.422(LOC114). This indicates that the 12 pairs of primers screened have high polymorphism and can effectively reveal the genetic diversity of elderberry.

[0054] Example 2 SSR fingerprint construction

[0055] 1.1 Experimental Instruments

[0056] Table 4. Experimental instruments

[0057]

[0058] 1.2 Experimental Materials and Reagents

[0059] Table 5. Experimental materials and reagents

[0060]

[0061] 1.3 Experimental Procedure

[0062] PCR amplification - PCR product capillary electrophoresis detection - data analysis and processing

[0063] 1.4 Experimental methods

[0064] 1.4.1 PCR amplification

[0065] The extracted elderberry total DNA was used as a template, and 12 pairs of primers provided by the present invention were used for SSR-PCR amplification, and the primers were labeled with FAM. The specific amplification system and amplification procedure are as follows.

[0066]

[0067]

[0068] 1.4.2 Agarose gel electrophoresis of PCR products

[0069] 1.4.3 Capillary electrophoresis on-machine testing

[0070] Take 0.3 μL of PCR product, 0.5 μL of molecular weight internal standard and 9.5 μL of deionized formamide, mix and add to the PCR plate, denature at 95℃ for 5 min, cool at 4℃ and centrifuge, and use 1× Buffer for detection. The specific parameters are shown in the table below:

[0071]

[0072] 1.4.4 Construction of SSR fingerprints and molecular identification: Genemarker 2.2.0 software was used for data collation. According to the peak value of each pair of SSR primers, the SSR fingerprints of the tested elderberry varieties were formed. According to the differences in the fingerprints, the elderberry varieties were distinguished and identified. The fingerprints constructed by the above 12 pairs of SSR primers are shown in Tables 6-8 below.

[0073] Table 6. Fingerprints constructed by primer pairs LOC003, LOC039, LOC067 and LOC090

[0074]

[0075]

[0076] Table 7 Fingerprints constructed by primer pairs LOC092, LOC131, LOC169 and LOC186

[0077]

[0078] Table 8 Fingerprints constructed by primer pairs LOC145, LOC114, LOC163 and LOC177

[0079]

[0080]

[0081] The results show that the above 16 elderberry materials can be completely distinguished by using the two pairs of SSR primer combinations provided by the present invention (primer pair LOC090 and the remaining 11 pairs of primers (excluding LOC090); primer pair LOC177 and the remaining 11 pairs of primers (excluding LOC003 and LOC163), etc.). The test results of these primer combinations have good repeatability and high polymorphism, and can quickly, accurately and efficiently complete the identification of elderberry varieties.

[0082] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An elderberry SSR molecular marker, characterized in that: An elderberry SSR molecular marker, the SSR molecular marker comprising the following SSR loci: LOC003, LOC039, LOC043, LOC055, LOC067, LOC090, LOC092, LOC097, LOC114, LOC131, LOC141, LOC145, LOC163, LOC169, LOC177 and LOC186; The above elderberry SSR molecular markers were obtained by amplifying the following primer pairs respectively: SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.9-10, SEQ ID NO.11-12, SEQ ID NO.13-14, SEQ ID NO.15-16, SEQ ID NO.17-18, SEQ ID NO.19-20, SEQ ID NO.21-22, SEQID NO.23-24, SEQ ID NO.25-26, SEQ ID NO.27-28, SEQ ID NO.29-30 and SEQ ID NO.31-32.

2. The elderberry SSR molecular marker according to claim 1, characterized in that Any one or more of LOC003, LOC039, LOC067, LOC090, LOC092, LOC114, LOC131, LOC145, LOC163, LOC169, LOC177 and LOC186.

3. An elderberry SSR molecular marker primer, characterized in that: The elderberry SSR molecular marker primers include any one or more of the following 16 pairs of specific primer pairs, and their nucleotide sequences are shown in SEQ ID NO.1-32 respectively.

4. The elderberry SSR molecular marker primer according to claim 3, characterized in that: The elderberry SSR molecular marker primers include any one or more of the following 12 pairs of specific primer pairs, whose nucleotide sequences are SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ ID NO.9-10, SEQ ID NO.11-12, SEQ ID NO.13-14, SEQ ID NO.17-18, SEQ ID NO.19-20, SEQ ID NO.23-24, SEQ ID NO.25-26, SEQ ID NO.27-28, SEQ ID NO.29-30 and SEQ ID NO.31-32.

5. A product for detecting the elderberry SSR molecular marker according to claim 1 or 2, characterized in that: The product at least comprises the elderberry SSR molecular marker primer according to claim 3 or 4; Furthermore, the product is a detection kit, a gene chip, a detection device or equipment.

6. Use of the elderberry SSR molecular marker according to any one of claims 1 to 2, the elderberry SSR molecular marker primer according to any one of claims 3 to 4, or the product according to claim 5 in any one or more of the following: 1) Analysis of genetic diversity of elderberry; 2) Construction of the elderberry genetic map; 3) Identification of elderberry germplasm resources; 4) Analysis of the relationship between elderberry; 5) Mining of functional genes of elderberry; 6) Molecular marker-assisted breeding of elderberry.

7. The use according to claim 6, characterized in that In the above 2), constructing the elderberry genetic map specifically involves constructing the elderberry SSR fingerprint map.

8. The use according to claim 6, characterized in that In the above 3), the identification of elderberry germplasm at least includes the identification of elderberry varieties.

9. A method for constructing an elderberry SSR fingerprint, characterized in that: The steps include: Extract the genomic DNA of the elderberry to be tested, perform PCR amplification on the genomic DNA of the elderberry to be tested using the elderberry SSR molecular marker primers described in claim 3 or 4, perform capillary electrophoresis detection on the PCR products, and construct an elderberry SSR fingerprint map.

10. A method for identifying elderberry varieties, characterized in that: The method comprises: comparing the elderberry SSR fingerprint obtained by the construction method of claim 9 with the fingerprint of a known elderberry variety, and the elderberry variety with the same fingerprint is the corresponding elderberry variety.

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