SNP (Single Nucleotide Polymorphism) marker combination for identifying strawberry germplasm resources and application of SNP marker combination

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) marker combination for identifying strawberry germplasm resources and application of the SNP marker combination. According to the present invention, strawberry core resource whole genome re-sequencing is performed, the SNP marker set is developed at the whole genome level, the KASP primer combination is designed and synthesized, and the DNA fingerprint database is established by using the SNP marker combination, such that the strawberry variety can be subjected to clustering analysis and variety identification so as to achieve the accurate and efficient detection result, and the method has characteristics of high specificity, high sensitivity and high sensitivity. Based on the primer combination marker, 162 germplasm can be effectively distinguished for the first time, which is the maximum population number capable of distinguishing and identifying strawberry germplasm resources in China at present.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to a group of SNP marker combinations for identifying strawberry germplasm resources and applications thereof. Background Art

[0002] Strawberry (Fragaria×ananassa Duchase) is a perennial herbaceous plant of the Rosaceae family. It has a delicious taste and high nutritional value and is an important horticultural economic crop. On the one hand, strawberry is mainly propagated by runners. After years of continuous vegetative propagation, there are common problems such as confusion of nursery resource types and inability to effectively distinguish them. In addition, since the growth of strawberries is easily affected by cultivation conditions and production environment, conventional plant morphological identification cannot effectively distinguish the complex and diverse strawberry planting resources.

[0003] The current conventional plant morphological identification method is limited to the macroscopic morphological judgment of the plant, and has the disadvantages of long identification time, large error in results, and difficulty in accurately judging subtle differences. Cultivated strawberry is an allo-octoploid plant with a highly heterozygous genome, and it is difficult to develop identification of natural populations at the genome level. So far, the development of markers reported for strawberry variety identification is limited to a small range of strawberry variety differentiation, and the development of identification markers for natural population germplasm resources of more than 100 strawberries has not been reported. At the same time, there are few reports on the development of strawberry variety identification markers, and the markers are only the screening and verification of existing markers, and no new polymorphic markers have been developed at the whole genome level. For example, Liu Lifeng et al. screened 9 polymorphic SSR markers to distinguish 60 strawberry varieties; Jia Yumeifeng et al. developed molecular marker primers for KASP to identify five-leaf strawberries. The development of strawberry markers retrieved so far is mainly focused on functional gene linkage and other aspects. The genome information of cultivated strawberries is complex. With the research on genome sequencing of cultivated strawberries, the genome information of strawberries has also been continuously decrypted. However, there is still a lack of systematic research on DNA fingerprinting and identification analysis of strawberry germplasm resources.

[0004] In summary, with the continuous development of the strawberry industry, more and more strawberries will apply for new variety rights, and more reliable marking methods are needed to identify strawberry varieties and protect variety rights. Summary of the invention

[0005] One of the purposes of the present invention is to provide a set of SNP marker combinations for identification of strawberry germplasm resources, wherein the SNP marker combination includes 26 SNP marker sites, the 26 SNP marker sites are numbered as Marker0-25, and the 26 SNP marker sites and the gene information thereof are as follows:

[0006] The SNP site Marker0The marker is the base T / C, located at position 12435672 on chromosome Fvb7-3 of the strawberry 'Camarosa' reference genome, on the annotated gene FxaC_26g24960;

[0007] The SNP site Marker1 The marker is the base C / T, located at position 890328 on chromosome Fvb5-3 of the strawberry 'Camarosa' reference genome, on the annotated gene FxaC_18g01421;

[0008] The SNP site Marker2 The marker is the base T / A, located at position 10652168 on chromosome Fvb6-4 of the strawberry 'Camarosa' reference genome, on the annotated gene FxaC_24g20050;

[0009] The SNP site Marker3 The marker is base C / A, located at position 8743239 on chromosome Fvb3-1 of the strawberry 'Camarosa' reference genome, on the annotated gene FxaC_12g13610;

[0010] The SNP site Marker4 The marker is the base T / A, located at position 20769821 on chromosome Fvb3-1 of the strawberry 'Camarosa' reference genome, on the annotated gene FxaC_12g30490;

[0011] The SNP site Marker5 The marker is the base T / G, located at position 5900876 on chromosome Fvb3-2 of the strawberry 'Camarosa' reference genome, on the annotated gene FxaC_12g30490;

[0012] The SNP site Marker6 The marker is the base C / T, located at position 8156949 on chromosome Fvb3-2 of the strawberry 'Camarosa' reference genome, on the annotated gene FxaC_10g16120;

[0013] The SNP site Marker7 The marker is base A / G, located at position 4908958 on chromosome Fvb3-3 of the strawberry ‘Camarosa’ reference genome, on the annotated gene FxaC_11g10360;

[0014] The SNP site Marker8 The marker is the base A / T, located at position 31036635 on chromosome Fvb4-3 of the strawberry 'Camarosa' reference genome, on the annotated gene FxaC_13g54490;

[0015] The SNP site Marker9 The marker is the base T / C, located at position 20919719 on chromosome Fvb5-1 of the strawberry 'Camarosa' reference genome, on the annotated gene FxaC_17g40850;

[0016] The SNP site Marker10 The marker is the base G / C, located at position 9612529 on chromosome Fvb6-1 of the strawberry 'Camarosa' reference genome, on the annotated gene FxaC_21g20810;

[0017] The SNP site Marker11 The marker is the base G / A, located at position 34331392 on chromosome Fvb6-1 of the strawberry 'Camarosa' reference genome, on the annotated gene FxaC_21g67351;

[0018] The SNP site Marker12 The marker is the base G / T, located at position 11223473 on chromosome Fvb6-2 of the strawberry 'Camarosa' reference genome, on the annotated gene FxaC_23g16590;

[0019] The SNP site Marker13 The marker is base A / G, located at position 16863442 on chromosome Fvb6-4 of the strawberry ‘Camarosa’ reference genome, on the annotated gene FxaC_24g29440;

[0020] The SNP site Marker14 The marker is base A / G, located at position 24759158 on chromosome Fvb7-1 of the strawberry 'Camarosa' reference genome, on the annotated gene FxaC_27g37570;

[0021] The SNP site Marker15 The marker is base C / A, located at position 17209819 on chromosome Fvb1-2 of the strawberry ‘Camarosa’ reference genome, on the annotated gene FxaC_2g32680;

[0022] The SNP site Marker16 The marker is the base G / A, located at position 9937188 on chromosome Fvb2-2 of the strawberry 'Camarosa' reference genome, on the annotated gene FxaC_5g20800;

[0023] The SNP site Marker17The marker is the base G / A, located at position 30142967 on chromosome Fvb3-2 of the strawberry ‘Camarosa’ reference genome, on the annotated gene FxaC_10g49200;

[0024] The SNP site Marker18 The marker is the base C / T, located at position 16270624 on chromosome Fvb3-4 of the strawberry 'Camarosa' reference genome, on the annotated gene FxaC_9g29160;

[0025] The SNP site Marker19 The marker is the base G / C, located at position 16415540 on chromosome Fvb4-2 of the strawberry 'Camarosa' reference genome, on the annotated gene FxaC_15g29000;

[0026] The SNP site Marker20 The marker is the base T / C, located at position 1358988 on chromosome Fvb6-3 of the strawberry 'Camarosa' reference genome, on the annotated gene FxaC_22g02620;

[0027] The SNP site Marker21 The marker is the base G / A, located at position 4594448 on chromosome Fvb6-4 of the strawberry 'Camarosa' reference genome, on the annotated gene FxaC_24g09600;

[0028] The SNP site Marker22 The marker is base A / C, located at position 15710829 on chromosome Fvb2-3 of the strawberry ‘Camarosa’ reference genome, on the annotated gene FxaC8g28720 / FxaC8g28730;

[0029] The SNP site Marker23 The marker is the base T / C, located at position 7321585 on chromosome Fvb7-4 of the strawberry 'Camarosa' reference genome;

[0030] The SNP site Marker24 The marker is base A / G, located at position 31710521 on chromosome Fvb6-1 of the strawberry ‘Camarosa’ reference genome;

[0031] The SNP site Marker25 The marker is the base G / A, located at position 12136848 on chromosome Fvb1-1 of the strawberry 'Camarosa' reference genome, and on the annotated gene FxaC4g12680 / FxaC4g12700.

[0032] The second object of the present invention is to provide a set of primer sequence combinations for amplifying the above-mentioned SNP marker combination, including a forward primer and a reverse primer for amplifying the above-mentioned SNP site.

[0033] The third object of the present invention is to provide the use of the above-mentioned SNP marker combination or primer combination in any of the following aspects:

[0034] Genotyping of strawberry germplasm varieties;

[0035] Molecular identification of strawberry germplasm varieties;

[0036] Purity testing of strawberry germplasm resources or varieties;

[0037] Construction of SNP fingerprint library of strawberry germplasm resources;

[0038] Genetic evolution analysis of strawberry germplasm resources;

[0039] Molecular marker-assisted breeding of strawberry germplasm resources.

[0040] A fourth object of the present invention is to provide a kit for identifying strawberry germplasm resources, comprising the above primer combination.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] The invention discloses a KASP primer combination for constructing a strawberry DNA fingerprint library. The primer combination is based on whole genome resequencing and combined with a bioinformatics method. Genotyping analysis is performed through a KASP PCR experiment, and 26 strawberry core SNP markers are selected for design. The DNA fingerprint library is established by using the SNP marker combination, so that cluster analysis and variety identification can be performed on strawberry varieties, so that the detection results are more accurate and efficient. Based on the primer combination marker, 162 germplasms are effectively distinguished for the first time, which is the largest number of groups for identification of strawberry germplasm resources in my country at present.

[0043] In addition, the KASP primer combination provided in the present invention has high accuracy and resolution for strawberry quality detection when the DNA quality meets the requirements of general KASP reaction (or conventional PCR reaction), the detection efficiency is 10-20 times that of SSR markers, the detection cost is comparable to that of SSR markers, and no toxic chemical reagents such as acrylamide are required during the detection process.

[0044] The present invention develops a core SNP molecular marker set of strawberry germplasm resources based on the KASP technology. The KASP genotyping technology is used to quickly and accurately identify and evaluate strawberry varieties or germplasm resources materials at the molecular level, which is an important basis for the current identification of strawberry germplasm resources.

[0045] The present invention has the following advantages:

[0046] 1. The present invention develops a SNP marker set at the whole genome level through whole genome resequencing of strawberry core resources. The markers are highly polymorphic, highly representative, and can effectively reflect the DNA polymorphism of different strawberry germplasm resources;

[0047] 2. The present invention provides a KASP marker primer combination that can be used to identify and evaluate 162 strawberry germplasm resources;

[0048] 3. The 171 strawberry germplasm resources identified in the present invention include diploid, octoploid and decaploid, the germplasm resources have diversified ploidy and the markers are more representative. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 It is the original SNP marker density map.

[0050] Figure 2 The results of some candidate SNP polymorphisms were verified in 5 resequencing core germplasm resources.

[0051] Figure 3 The present invention is a detection map of KASP genotyping technology for 171 strawberry varieties (or germplasm resources) using primers corresponding to the Mark8 site.

[0052] Figure 4 The KASP fingerprints of 171 strawberry varieties (or germplasm resources) are shown in Figure 2.

[0053] Figure 5 It is the marker identification efficiency of the KASP fingerprint of the present invention. DETAILED DESCRIPTION

[0054] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0055] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0056] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0057] Example 1

[0058] Resequencing of strawberry core germplasm resources and screening of polymorphic SNP markers

[0059] 1. Using the octoploid cultivated strawberry 'Camarosa' as the reference genome (https: / / www.rosaceae.org / Analysis / 9642085), whole genome resequencing (average coverage depth of 30×) was performed on five core strawberry germplasm resources (sample numbers: 1 Hongyan, 5 Christmas Red, 10 Zhangji, 14 Snow Rabbit, 32 Sweet Charlie) to analyze the SNP sites between the core strawberry germplasm resources (such as Figure 1 ), the markers at multiple positions in the alignment were removed, and 37738 SNP markers were obtained by filtering.

[0060] 2. Screening of polymorphic SNP markers in the genomes of five core strawberry germplasm resources

[0061] (1) Screen the conserved sequences greater than 50 bp before and after the DNA chain on the chromosome, and retain the following markers, average depth 5× and above, quality value greater than 30, minimum integrity greater than 0.9, and minimum allele frequency greater than 0.05 of the biallelic SNPs;

[0062] (2) 100 bp of sequence upstream and downstream of the SNP marker were intercepted, and then the sequence was aligned to the reference genome using blast software (version: 2.10.1+), markers at multiple positions in the alignment were removed, and specific and high-quality SNP sites were retained;

[0063] (3) Further retain markers with polymorphic information content (PIC) greater than 0.35. The PIC calculation formula is based on the references (Botstein D, et al. 1980 and Zhang, et al. 2020). Filter to obtain candidate SNP markers, and verify the PCR amplification polymorphism of the candidate SNP markers in the resequencing materials (such as Figure 2 shown).

[0064] Markers with polymorphic information content (PIC) greater than 0.35 were retained, and 1291 SNP markers were filtered. Primers were further designed for SNP markers and converted into KASP markers. 904 KASPs were successfully designed. Polymorphic markers were selected for first-generation sequencing. From the 137 markers that could be used in the first-generation sequencing results, the best 100 markers were selected for genotyping experiments, and 26 were successful.

[0065] 3. According to the screened high-quality SNP sites, 26 KASP primer combinations were successfully designed and synthesized. Each KASP primer combination consists of two forward primers F1 and F2 with different terminal bases and a reverse primer R. The 5'-ends of the forward primers F1 and F2 of the primer combination are connected to the FAM-tail: 5-GAAGGTGACCAAGTTCATGCT-3' universal fluorescent label sequence and the VIC-tail: 5'-GAAGGTCGGAGTCAACGGATT-3' universal fluorescent label sequence, respectively.

[0066] Specifically, the position information of 26 SNP sites 0 to 25 on the genome and the primer sequence information used to amplify the sites are shown in Table 1.

[0067] Table 1 The location information of Mark0-25 sites on the genome and their corresponding primer sequence information

[0068]

[0069]

[0070]

[0071]

[0072] Example 2

[0073] Construction of DNA fingerprint library of strawberry germplasm resources

[0074] The genomic DNA of 171 strawberry germplasm varieties was extracted and PCR amplification was performed in the strawberry germplasm resources using the designed KASP primer combination. The amplified products were subjected to agarose gel electrophoresis and first-generation sequencing for genotyping analysis.

[0075] The 171 strawberry germplasms of different ploidy were from China, Japan, South Korea, Europe, the United States, Canada, Australia and other places. The specific information is shown in Table 2.

[0076] Table 2 Information on 171 strawberry varieties (or germplasm resources)

[0077]

[0078]

[0079]

[0080] The above primers were diluted to 10 μM with TE (pH 8.0), and then mixed in a ratio of upstream typing primer 1 (F1): upstream typing primer 2 (F2): downstream universal primer (R) = 1:1:3 and loaded onto the machine. 1.25 μL of primer mixture was added to every 5 μL reaction system.

[0081] The DNA samples were diluted to a single-digit ratio according to the lowest concentration of the sample, and the entire batch of samples was diluted. Each 5 μL reaction system contained 1.25 μL of the diluted DNA sample.

[0082] When performing PCR amplification, the PCR reaction system used is shown in Table 3.

[0083] Table 3 PCR reaction system

[0084] Plate format 96 - well plate 2 * KASP master mix 2.5 μL primer mix 1.25 μL DNA samples 1.25 μL <![CDATA[H 2 The]]> 0 μL Total 5 μL

[0085] The 96-well PCR reaction plate was sealed, shaken, and centrifuged to ensure that the reaction system was evenly mixed.

[0086] After centrifugation, PCR reaction was performed. The reaction conditions are shown in Table 4.

[0087] Table 4 PCR reaction conditions

[0088] Step Description Tem Time No. of cycle 1 Activation 95℃ 10 min 1 2 Denaturation 95℃ 20 sec 10 Annealing / Elongation 61-55℃ 60 sec 3 Denaturation 95℃ 20 sec 27 Annealing / Elongation 55℃ 60 sec 4 Read 25℃ 30 sec 1

[0089] If the typing results are not ideal, you can optimize them by adding PCR reactions. The conditions are shown in Table 5.

[0090] Table 5 Optimized conditions for PCR reaction

[0091] Step Tem Time NO of cycle 1 95℃ 20 sec 3 55℃ 60 sec 55 °C 60 sec

[0092] The obtained 0 to 25 SNP markers were used to perform KASP typing detection on 171 strawberry varieties (or germplasm resources).

[0093] Determine the genotype of the gene in different strawberry germplasm resources: the horizontal axis FAM fluorescence accounts for a higher proportion, which is homozygous type 1 (yellow dots); the vertical axis VIC / HEX fluorescence accounts for a higher proportion, which is homozygous type 2 (blue squares); the two fluorescence proportions in the middle area are equal, which is heterozygous (green triangles); the black in the lower left corner is the negative control (NTC).

[0094] For example, Mark 8 (e.g. Figure 3 As shown in the figure, the FAM fluorescence on the horizontal axis accounts for a higher proportion, which is homozygous AA (yellow dots); the VIC / HEX fluorescence on the vertical axis accounts for a higher proportion, which is homozygous TT (blue squares); the two fluorescence proportions in the middle area are equal, which is heterozygous AT (green triangles); the black in the lower left corner is the negative control (NTC).

[0095] The fluorescence signal analysis results of KASP genotyping are shown in Table 6, which are the KASP typing results of 171 strawberry varieties (or germplasm resources) at 26 SNP sites. It can be seen that the 26 sets of SNP primers of the present invention can be applied to the genotyping identification of strawberry germplasm resources and the genotyping identification of germplasm resources.

[0096] Table 6 KASP genotyping results of 171 strawberry varieties (or germplasm resources) at 26 SNP loci

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105] KASP fingerprint Figure 4 As shown, one row is one marker, one column is one sample, and different genotypes are represented by different colors: C / C-yellow, A / A-green, T / T-blue, G / G-purple, A / T-orange, A / G-pink, A / C-dark pink, T / G-brown, T / C-dark green, C / G-light blue, C / T-gray, G / T-dark red, N / N-white (genotype missing).

[0106] The method for identifying strawberry varieties based on the polymorphic SNP sites and strawberry fingerprint spectrum comprises the following steps:

[0107] F1. Extract DNA of the strawberry variety (germplasm resources) to be tested;

[0108] F2. The genes of 26 SNP sites in the DNA of the strawberry variety (germplasm resources) to be tested are determined by PCR amplification, and the amplified products are compared with the genes of all strawberry varieties in the above fingerprint library and KASP genotyping results;

[0109] F3. If the comparison result of the fingerprint of the strawberry to be tested is inconsistent with that of any strawberry in the strawberry fingerprint, the strawberry to be tested is a new variety, and the new variety is added to the fingerprint library; if the comparison result of the fingerprint of the strawberry to be tested is completely consistent with that of a certain strawberry in the strawberry fingerprint, further comparison is required to determine whether the strawberry to be tested is that variety of strawberry.

[0110] The further comparison process includes comparing the origin of the strawberry varieties, the appearance and taste of the fruit, the growth characteristics of the plant, etc.

[0111] In order to demonstrate the identification capability of the strawberry fingerprint library developed based on KASP markers in the present invention, the genotype data of 171 strawberry germplasms detected by 26 KASP markers were analyzed for identification efficiency in this example.

[0112] By comparing the genotype data, it was found that the genotypes of 9 of the 171 germplasms were consistent and could not be completely distinguished, namely 157 Kinugan, 146 Fuyuan, 54 Shengxue, 95 Zhengyuan No. 2, 136 Zhangji (B1), 143 Banana, 130 Hongyan (A1), 149 Hongyan and 158 Jingyao. After removing the strawberry germplasms with consistent genotypes, using the Perl language program, we finally calculated a set of 26 markers that can completely distinguish 162 strawberry germplasms, and the identification efficiency reached 98.8%. Figure 5 shown.

[0113] Example 3

[0114] This embodiment provides a method for clustering analysis of strawberry varieties

[0115] G1 extracted genomic DNA based on 26 SNP loci of all strawberry varieties to be grouped;

[0116] G2 used PowerMarker's neighbor-joining algorithm to calculate the genetic distance of strawberry germplasm, construct a cluster diagram, and perform cluster analysis on strawberries.

Claims

1. A set of SNP marker combinations for identification of strawberry germplasm resources, characterized in that: It includes 26 SNP marker sites, which are numbered as Marker0 to Marker25. The 26 SNP marker sites and the gene information are as follows: The SNP site Marker0 The marker is the base T / C, located at position 12435672 on chromosome Fvb7-3 of the strawberry 'Camarosa' reference genome, on the annotated gene FxaC_26g24960; The SNP site Marker1 The marker is the base C / T, located at position 890328 on chromosome Fvb5-3 of the strawberry 'Camarosa' reference genome, on the annotated gene FxaC_18g01421; The SNP site Marker2 The marker is the base T / A, located at position 10652168 on chromosome Fvb6-4 of the strawberry 'Camarosa' reference genome, on the annotated gene FxaC_24g20050; The SNP site Marker3 The marker is base C / A, located at position 8743239 on chromosome Fvb3-1 of the strawberry 'Camarosa' reference genome, on the annotated gene FxaC_12g13610; The SNP site Marker4 The marker is the base T / A, located at position 20769821 on chromosome Fvb3-1 of the strawberry 'Camarosa' reference genome, on the annotated gene FxaC_12g30490; The SNP site Marker5 The marker is the base T / G, located at position 5900876 on chromosome Fvb3-2 of the strawberry 'Camarosa' reference genome, on the annotated gene FxaC_12g30490; The SNP site Marker6 The marker is the base C / T, located at position 8156949 on chromosome Fvb3-2 of the strawberry 'Camarosa' reference genome, on the annotated gene FxaC_10g16120; The SNP site Marker7 The marker is base A / G, located at position 4908958 on chromosome Fvb3-3 of the strawberry ‘Camarosa’ reference genome, on the annotated gene FxaC_11g10360; The SNP site Marker8 The marker is the base A / T, located at position 31036635 on chromosome Fvb4-3 of the strawberry 'Camarosa' reference genome, on the annotated gene FxaC_13g54490; The SNP site Marker9 The marker is the base T / C, located at position 20919719 on chromosome Fvb5-1 of the strawberry 'Camarosa' reference genome, on the annotated gene FxaC_17g40850; The SNP site Marker10 The marker is the base G / C, located at position 9612529 on chromosome Fvb6-1 of the strawberry 'Camarosa' reference genome, on the annotated gene FxaC_21g20810; The SNP site Marker11 The marker is the base G / A, located at position 34331392 on chromosome Fvb6-1 of the strawberry 'Camarosa' reference genome, on the annotated gene FxaC_21g67351; The SNP site Marker12 The marker is the base G / T, located at position 11223473 on chromosome Fvb6-2 of the strawberry 'Camarosa' reference genome, on the annotated gene FxaC_23g16590; The SNP site Marker13 The marker is base A / G, located at position 16863442 on chromosome Fvb6-4 of the strawberry ‘Camarosa’ reference genome, on the annotated gene FxaC_24g29440; The SNP site Marker14 The marker is base A / G, located at position 24759158 on chromosome Fvb7-1 of the strawberry 'Camarosa' reference genome, on the annotated gene FxaC_27g37570; The SNP site Marker15 The marker is base C / A, located at position 17209819 on chromosome Fvb1-2 of the strawberry ‘Camarosa’ reference genome, on the annotated gene FxaC_2g32680; The SNP site Marker16 The marker is the base G / A, located at position 9937188 on chromosome Fvb2-2 of the strawberry 'Camarosa' reference genome, on the annotated gene FxaC_5g20800; The SNP site Marker17 The marker is the base G / A, located at position 30142967 on chromosome Fvb3-2 of the strawberry ‘Camarosa’ reference genome, on the annotated gene FxaC_10g49200; The SNP site Marker18 The marker is the base C / T, located at position 16270624 on chromosome Fvb3-4 of the strawberry 'Camarosa' reference genome, on the annotated gene FxaC_9g29160; The SNP site Marker19 The marker is the base G / C, located at position 16415540 on chromosome Fvb4-2 of the strawberry 'Camarosa' reference genome, on the annotated gene FxaC_15g29000; The SNP site Marker20 The marker is the base T / C, located at position 1358988 on chromosome Fvb6-3 of the strawberry 'Camarosa' reference genome, on the annotated gene FxaC_22g02620; The SNP site Marker21 The marker is the base G / A, located at position 4594448 on chromosome Fvb6-4 of the strawberry 'Camarosa' reference genome, on the annotated gene FxaC_24g09600; The SNP site Marker22 The marker is base A / C, located at position 15710829 on chromosome Fvb2-3 of the strawberry ‘Camarosa’ reference genome, on the annotated gene FxaC8g28720 / FxaC8g28730; The SNP site Marker23 The marker is the base T / C, located at position 7321585 on chromosome Fvb7-4 of the strawberry 'Camarosa' reference genome; The SNP site Marker24 The marker is base A / G, located at position 31710521 on chromosome Fvb6-1 of the strawberry ‘Camarosa’ reference genome; The SNP site Marker25 The marker is the base G / A, located at position 12136848 on chromosome Fvb1-1 of the strawberry 'Camarosa' reference genome, and on the annotated gene FxaC4g12680 / FxaC4g12700.

2. A set of primer sequence combinations for amplifying the SNP marker combination according to claim 1, characterized in that: The forward primer and reverse primer for amplifying the 26 SNP sites are as follows:

3. Use of the SNP marker combination according to claim 1 or the primer combination according to claim 2 in any of the following aspects: Genotyping of strawberry germplasm varieties; Molecular identification of strawberry germplasm varieties; Purity testing of strawberry germplasm resources or varieties; Construction of SNP fingerprint library of strawberry germplasm resources; Genetic evolution analysis of strawberry germplasm resources; Molecular marker-assisted breeding of strawberry germplasm resources.

4. A kit for identifying strawberry germplasm resources, characterized in that: Comprising the primer combination described in claim 2.

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