A SNP molecular marker primer combination for identifying small-fruit camellia oleifera and common camellia oleifera and its application

Abstract

The present invention belongs to the field of molecular marker technology, and specifically relates to a SNP molecular marker primer combination for identifying small-fruit camellia oil-tea and common camellia oil-tea and its application. The primer combination provided by the present invention includes 14 pairs of primers, which can mark the molecular types of small-fruit camellia oil-tea and common camellia oil-tea, and realize interspecific identification in genetics. The primer development method is simple, fast and accurate, and its application is comprehensive, scientific and reasonable.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular markers, and in particular relates to a SNP molecular marker primer combination for identifying small-fruit camellia oleifera and common camellia oleifera and an application thereof. Background Art

[0002] Camellia oleifera is mainly produced and originated in China. It is a woody oil crop that China focuses on developing. It is one of the four major oil crops in China, along with rapeseed, peanuts, and soybeans. Camellia oleifera not only has aesthetic value for landscaping, but also has high economic value for cultivation. Camellia oleifera seed oil is rich in unsaturated fatty acids and has high nutritional value. It is a high-quality edible vegetable oil. Camellia oil and its by-products are also widely used in the chemical, pharmaceutical, and cosmetic industries. Camellia oleifera has a long history of cultivation and utilization in China, and the market demand for camellia oil is also growing. Therefore, tapping and utilizing high-quality genetic resources and cultivating new high-yield camellia oleifera varieties are the basis for the steady development of the camellia oleifera industry, and improved seedlings are the basic prerequisite for the sustained and healthy development of the camellia oleifera industry.

[0003] Camellia oleifera has abundant germplasm resources, among which common camellia oleifera has the longest cultivation history, the widest distribution area, and the highest total output of camellia oleifera. It is mainly distributed in Guangxi, Sichuan, Zhejiang, Anhui and other places; followed by small-fruit camellia oleifera, which ranks second in total output of camellia oleifera. It is mainly distributed in Fujian, Guangxi, Yichun City, Jiangxi and other places. At present, it is urgent to develop an accurate, fast and efficient molecular biology method to explore and utilize common camellia oleifera and small-fruit camellia oleifera resources, as well as to cultivate and promote improved varieties. SNP has great advantages over other molecular markers: it is suitable for faster and larger-scale screening and identification. Through the automation of analysis, there is no need to detect fragment length polymorphism, which can solve the problem of time-consuming and labor-intensive manual work. Therefore, it is necessary to use the polymorphism and versatility of SNP markers within and between species to develop a SNP marker that can realize interspecific identification of common camellia oleifera and small-fruit camellia oleifera. Summary of the invention

[0004] The present invention performs chloroplast whole genome sequencing and genome comparative analysis on small-fruit camellia and common camellia germplasm to obtain SNP polymorphic sites in small-fruit camellia and common camellia germplasm resources, and designs polymorphic primers based on the SNP polymorphic sites. The present invention provides the following technical solutions:

[0005] The first aspect of the present invention provides a SNP molecular marker primer combination for identifying small-fruit camellia oil-tea resources and common camellia oil-tea resources, which mainly consists of primer pair DJ01 to primer pair DJ14, and the primer nucleotide sequences are as follows:

[0006] DJ01 upstream primer: 5′-GCGAACGACGGGAATTGAAC-3′;

[0007] DJ01 downstream primer: 5′-TCAGGAAGGCGTTATTGTTCC-3′;

[0008] DJ02 upstream primer: 5′-TGCTTGCGGAGAAAAACGAA-3′;

[0009] DJ02 downstream primer: 5′-ATCTCAGCAACACGACTTCG-3′;

[0010] DJ03 upstream primer: 5′-TCGGACTTGATTAACCGCCC-3′;

[0011] DJ03 downstream primer: 5′-GGTTCGGGAAGGGATCATGG-3′;

[0012] DJ04 upstream primer: 5′-TAATGGGTGTCGCAATGGCT-3′;

[0013] DJ04 downstream primer: 5′-CGCGGTCGAACTACCAAAAC-3′;

[0014] DJ05 upstream primer: 5′-CGCCTACGGATTAGTCGACA-3′;

[0015] DJ05 downstream primer: 5′-ATGATTCAACCAGAGGGCGT-3′;

[0016] DJ06 upstream primer: 5′-GATTCGTAGACCCGGTCGAC-3′;

[0017] DJ06 downstream primer: 5′-ATGGGTAGGGACACTGTTGC-3′;

[0018] DJ07 upstream primer: 5′-TGGTTGGTAAACAAGGATCGGT-3′;

[0019] DJ07 downstream primer: 5′-GATACACCAGATCCGGCTCG-3′;

[0020] DJ08 upstream primer: 5′-TTCGGGAGCAGTATGGGCTA-3′;

[0021] DJ08 downstream primer: 5′-AATTGTTCGTTGTACAATGCAAAA-3′;

[0022] DJ09 upstream primer: 5′-GCTACAGCGATTGCACCTAT-3′;

[0023] DJ09 downstream primer: 5′-CTGGTCAGGTCAACAAAGTCA-3′;

[0024] DJ10 upstream primer: 5′-ACCATGCATTGATGGGTGGT-3′;

[0025] DJ10 downstream primer: 5′-GGCAGTCGTTTCAGCATGTT-3′;

[0026] DJ11 upstream primer: 5′-TTGACCTTGTTCCCATTGCA-3′;

[0027] DJ11 downstream primer: 5′-ACAACACACATAAACATAAGGGCA-3′;

[0028] DJ12 upstream primer: 5′-CGGAATCCATTCTTCCTTGTTCTG-3′;

[0029] DJ12 downstream primer: 5′-TGGTATTAGTCTGGATACGGCA-3′;

[0030] DJ13 upstream primer: 5′-CCCTGACTTTGTCCGTGTCA-3′;

[0031] DJ13 downstream primer: 5′-TCTCCCTCCAAGCCGTACAT-3′;

[0032] DJ14 upstream primer: 5′-GATACACCAGATCCGGCTCG-3′;

[0033] DJ14 downstream primer: 5′-TGGTTGGTAAACAAGGATCGGT-3′.

[0034] The second aspect of the present invention proposes the use of the above SNP molecular marker primer combination in the identification of small-fruited camellia and common camellia species.

[0035] Among them, DJ01 upstream primer and DJ01 downstream primer identified site ZD01_SNP, and the site recognition sequence was TCCACTATTT[C / A]AATTATAAAA;

[0036] DJ02 upstream primer and DJ02 downstream primer identified locus ZD02_SNP, and the locus recognition sequence was CCCAAAAAT[G / A]GATTTTGGTT;

[0037] DJ03 upstream primer and DJ03 downstream primer identified site ZD03_SNP, and the site recognition sequence was TCAATGGCCC[C / T]CCTACGTAGT;

[0038] DJ04 upstream primer and DJ04 downstream primer identified site ZD04_SNP, and the site recognition sequence was ATTGAAAGCT[T / A]GGATTTCTAG;

[0039] DJ05 upstream primer and DJ05 downstream primer identified site ZD05_SNP, and the site recognition sequence was AATCCTTGTT[G / T]CGGAGTCGAT;

[0040] DJ06 upstream primer and DJ06 downstream primer identified site ZD06_SNP, and the site recognition sequence was ACCAAAAAAT[C / A]TTTTTTGCTT;

[0041] DJ07 upstream primer and DJ07 downstream primer identified locus ZD07_SNP, and the locus recognition sequence was TTCATCTATT[C / T]CATGACCGGA;

[0042] DJ08 upstream primer and DJ08 downstream primer identified site ZD08_SNP, and the site recognition sequence was ATAAAAAATT[T / A]CCCCCTGCAA;

[0043] DJ09 upstream primer and DJ09 downstream primer identified locus ZD09_SNP, and the locus recognition sequence was AAAATCATGT[G / A]TTGGTCCAGA;

[0044] DJ010 upstream primer and DJ010 downstream primer identified locus ZD010_SNP-1, and the locus recognition sequence was AGAATAATTT[G / T]AATCTTAAAA;

[0045] DJ010 upstream primer and DJ010 downstream primer identified locus ZD010_SNP-2, and the locus recognition sequence was AAAGAATAGT[C / A]AATTTTTGCA;

[0046] DJ011 upstream primer and DJ011 downstream primer identified the site ZD011_SNP, and the site recognition sequence was GTATAACCCC[T / C]TTTTGCTTTC;

[0047] DJ012 upstream primer and DJ012 downstream primer identified locus ZD012_SNP, and the locus recognition sequence was TAAGAATGGG[G / T]GACGGTATTC;

[0048] DJ013 upstream primer and DJ013 downstream primer identified locus ZD013_SNP, and the locus recognition sequence was GAATTCTGTG[A / G]AAAGCCGTAT;

[0049] DJ014 upstream primer and DJ014 downstream primer identified the site ZD014_SNP, and the site recognition sequence was TCCGGTCATG[C / T]AATAGATGAA.

[0050] The third aspect of the present invention provides a method for applying a SNP molecular marker primer combination of Camellia oleifera and Camellia oleifera resources in interspecific identification, comprising the following steps:

[0051] (1) Extracting DNA from samples to be analyzed: Extracting total genomic DNA from the Camellia oleifera germplasm materials to be tested;

[0052] (2) PCR amplification reaction: using the above 14 pairs of SNP molecular marker primers to perform PCR amplification reaction on the genomic DNA extracted in step (1);

[0053] Among them, DJ01 upstream primer and DJ01 downstream primer identified site ZD01_SNP, and the site recognition sequence was TCCACTATTT[C / A]AATTATAAAA;

[0054] DJ02 upstream primer and DJ02 downstream primer identified locus ZD02_SNP, and the locus recognition sequence was CCCAAAAAT[G / A]GATTTTGGTT;

[0055] DJ03 upstream primer and DJ03 downstream primer identified site ZD03_SNP, and the site recognition sequence was TCAATGGCCC[C / T]CCTACGTAGT;

[0056] DJ04 upstream primer and DJ04 downstream primer identified site ZD04_SNP, and the site recognition sequence was ATTGAAAGCT[T / A]GGATTTCTAG;

[0057] DJ05 upstream primer and DJ05 downstream primer identified site ZD05_SNP, and the site recognition sequence was AATCCTTGTT[G / T]CGGAGTCGAT;

[0058] DJ06 upstream primer and DJ06 downstream primer identified site ZD06_SNP, and the site recognition sequence was ACCAAAAAAT[C / A]TTTTTTGCTT;

[0059] DJ07 upstream primer and DJ07 downstream primer identified locus ZD07_SNP, and the locus recognition sequence was TTCATCTATT[C / T]CATGACCGGA;

[0060] DJ08 upstream primer and DJ08 downstream primer identified site ZD08_SNP, and the site recognition sequence was ATAAAAAATT[T / A]CCCCCTGCAA;

[0061] DJ09 upstream primer and DJ09 downstream primer identified locus ZD09_SNP, and the locus recognition sequence was AAAATCATGT[G / A]TTGGTCCAGA;

[0062] DJ010 upstream primer and DJ010 downstream primer identified locus ZD010_SNP-1, and the locus recognition sequence was AGAATAATTT[G / T]AATCTTAAAA;

[0063] DJ010 upstream primer and DJ010 downstream primer identified locus ZD010_SNP-2, and the locus recognition sequence was AAAGAATAGT[C / A]AATTTTTGCA;

[0064] DJ011 upstream primer and DJ011 downstream primer identified the site ZD011_SNP, and the site recognition sequence was GTATAACCCC[T / C]TTTTGCTTTC;

[0065] DJ012 upstream primer and DJ012 downstream primer identified locus ZD012_SNP, and the locus recognition sequence was TAAGAATGGG[G / T]GACGGTATTC;

[0066] DJ013 upstream primer and DJ013 downstream primer identified locus ZD013_SNP, and the locus recognition sequence was GAATTCTGTG[A / G]AAAGCCGTAT;

[0067] DJ014 upstream primer and DJ014 downstream primer identified the site ZD014_SNP, and the site recognition sequence was TCCGGTCATG[C / T]AATAGATGAA;

[0068] (3) Detection of PCR amplification products: The PCR amplification products are subjected to genome sequencing.

[0069] Furthermore, the PCR amplification reaction procedure described in step (2) is: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 1 min, annealing at 58°C for 1 min, extension at 72°C for 2 min, for 35 cycles; extension at 72°C for 10 min.

[0070] Furthermore, the PCR amplification reaction system described in step (2) is: 20 ng of Camellia oleifera or Camellia oleifera DNA, 0.30 mmol / L of dNTPs, 0.15 μmol / L of polymorphic primers, 2.00 U of Taq DNA polymerase, and ddH2O supplemented to a total volume of 30 μL.

[0071] Furthermore, the genome sequencing described in step (3) is conventional Sanger first generation sequencing.

[0072] Beneficial effects of the present invention:

[0073] By sequencing the chloroplast genomes of small-fruit camellia and common camellia oil trees and analyzing the polymorphic SNP site information of the nucleotide sequences, a SNP molecular marker primer for small-fruit camellia and common camellia oil tree resources was developed, providing an effective analytical technology tool for species identification of small-fruit camellia and common camellia oil trees. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 This is the result of polymorphic sequence alignment of primer DJ01 in two Camellia oleifera materials;

[0075] Figure 2 This is the result of polymorphic sequence alignment of primer DJ02 in two Camellia oleifera materials;

[0076] Figure 3 This is the result of polymorphic sequence alignment of primer DJ03 in two Camellia oleifera materials;

[0077] Figure 4 This is the result of polymorphic sequence alignment of primer DJ04 in two Camellia oleifera materials;

[0078] Figure 5 This is the result of polymorphic sequence alignment of primer DJ05 in two Camellia oleifera materials;

[0079] Figure 6 This is the result of polymorphic sequence alignment of primer DJ06 in two Camellia oleifera materials;

[0080] Figure 7 This is the result of polymorphic sequence alignment of primer DJ07 in two Camellia oleifera materials;

[0081] Figure 8 This is the result of polymorphic sequence alignment of primer DJ08 in two Camellia oleifera materials;

[0082] Fig. 9 This is the result of polymorphic sequence alignment of primer DJ09 in two Camellia oleifera materials;

[0083] Fig.10 This is the result of polymorphic sequence alignment of primer DJ10 in two Camellia oleifera materials;

[0084] Fig.11 This is the result of polymorphic sequence alignment of primer DJ11 in two Camellia oleifera materials;

[0085] Fig.12 This is the result of polymorphic sequence alignment of primer DJ12 in two Camellia oleifera materials;

[0086] Fig.13 This is the result of polymorphic sequence alignment of primer DJ13 in two Camellia oleifera materials;

[0087] Fig.14 The polymorphic sequence alignment results of primer DJ14 in two Camellia oleifera materials. DETAILED DESCRIPTION

[0088] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0089] Example 1: Development of SNP Molecular Marker Primers

[0090] (1) Extract genomic DNA from Camellia oleifera and Camellia oleifera germplasm, use Illumina second-generation sequencing technology to complete total DNA sequencing of the samples, establish sequencing libraries, and obtain SNP molecular markers of Camellia oleifera and Camellia oleifera resources based on the chloroplast genome sequences of Camellia oleifera and Camellia oleifera germplasm;

[0091] (2) Based on the SNP molecular markers of small-fruited camellia and common camellia resources obtained above, 14 pairs of primers were designed ( Figure 1 ), as shown in Table 1, the primer sequences are:

[0092] DJ01 upstream primer (SEQ ID NO .1): 5′-GCGAACGACGGGAATTGAAC-3′;

[0093] DJ01 downstream primer (SEQ ID NO .2): 5′-TCAGGAAGGCGTTATTGTTCC-3′;

[0094] DJ02 upstream primer (SEQ ID NO .3): ​​5′-TGCTTGCGGAGAAAAACGAA-3′;

[0095] DJ02 downstream primer (SEQ ID NO .4): 5′-ATCTCAGCAACACGACTTCG-3′;

[0096] DJ03 upstream primer (SEQ ID NO .5): 5′-TCGGACTTGATTAACCGCCC-3′;

[0097] DJ03 downstream primer (SEQ ID NO. 6): 5'-GGTTCGGGAAGGGATCATGG-3';

[0098] DJ04 upstream primer (SEQ ID NO .7): 5′-TAATGGGTGTCGCAATGGCT-3′;

[0099] DJ04 downstream primer (SEQ ID NO. 8): 5′-CGCGGTCGAACTACCAAAAC-3′;

[0100] DJ05 upstream primer (SEQ ID NO .9): 5′-CGCCTACGGATTAGTCGACA-3′;

[0101] DJ05 downstream primer (SEQ ID NO .10): 5′-ATGATTCAACCAGAGGGCGT-3′;

[0102] DJ06 upstream primer (SEQ ID NO .11): 5′-GATTCGTAGACCCGGTCGAC-3′;

[0103] DJ06 downstream primer (SEQ ID NO .12): 5′-ATGGGTAGGGACACTGTTGC-3′;

[0104] DJ07 upstream primer (SEQ ID NO .13): 5′-TGGTTGGTAAACAAGGATCGGT-3′;

[0105] DJ07 downstream primer (SEQ ID NO .14): 5′-GATACACCAGATCCGGCTCG-3′;

[0106] DJ08 upstream primer (SEQ ID NO .15): 5′-TTCGGGAGCAGTATGGGCTA-3′;

[0107] DJ08 downstream primer (SEQ ID NO .16): 5′-AATTGTTCGTTGTACAATGCAAAA-3′;

[0108] DJ09 upstream primer (SEQ ID NO .17): 5′-GCTACAGCGATTGCACCTAT-3′;

[0109] DJ09 downstream primer (SEQ ID NO .18): 5′-CTGGTCAGGTCAACAAAGTCA-3′;

[0110] DJ10 upstream primer (SEQ ID NO .19): 5′-ACCATGCATTGATGGGTGGT-3′;

[0111] DJ10 downstream primer (SEQ ID NO .20): 5′-GGCAGTCGTTTCAGCATGTT-3′;

[0112] DJ11 upstream primer (SEQ ID NO .21): 5′-TTGACCTTGTTCCCATTGCA-3′;

[0113] DJ11 downstream primer (SEQ ID NO .22): 5′-ACAACACACATAAACATAAGGGCA-3′;

[0114] DJ12 upstream primer (SEQ ID NO .23): 5′-CGGAATCCATTCTTCCTTGTTCTG-3′;

[0115] DJ12 downstream primer (SEQ ID NO .24): 5′-TGGTATTAGTCTGGATACGGCA-3′;

[0116] DJ13 upstream primer (SEQ ID NO .25): 5′-CCCTGACTTTGTCCGTGTCA-3′;

[0117] DJ13 downstream primer (SEQ ID NO .26): 5′-TCTCCCTCCAAGCCGTACAT-3′;

[0118] DJ14 upstream primer (SEQ ID NO .27): 5′-GATACACCAGATCCGGCTCG-3′;

[0119] DJ14 downstream primer (SEQ ID NO. 28): 5'-TGGTTGGTAAACAAGGATCGGT-3'.

[0120] Table 1 Nucleotide sequences of 14 pairs of primers

[0121]

[0122] Example 2: Application of SNP Molecular Marker Primer Combinations in Camellia oleifera and Camellia oleifera Resources

[0123] Selection of experimental materials: Selected small-fruit camellia XGYC and common camellia NHWT as experimental materials;

[0124] (2) Extracting DNA from samples to be analyzed: Using the modified CTAB method, the total DNA of the above experimental samples was extracted respectively;

[0125] (3) PCR amplification reaction: The extracted DNA of Camellia oleifera XGYC and Camellia oleifera NHWT was used as amplification template and 14 pairs of SNP molecular marker primers were used as amplification primers (Table 1) for PCR amplification. The PCR reaction system was: Camellia oleifera or Camellia oleifera DNA 20 ng, dNTPs 0.30 mmol / L, polymorphic primers 0.15 μmol / L, Taq DNA polymerase 2.00 U, ddH 2 O to a total volume of 30 μL, PCR amplification program: 94 ° C pre-denaturation for 5 min; 94 ° C denaturation for 1 min, 60 ° C annealing for 1 min, 72 ° C extension for 2 min, 40 cycles; finally 72 ° C extension for 10 min, 4 ° C storage;

[0126] (4) Detection of PCR amplification products: PCR amplification products were subjected to conventional Sanger sequencing.

[0127] The test results are as follows: Table 2 is the analysis of the base results of polymorphic primer sequencing. The base results of 14 pairs of primers sequencing showed polymorphism, among which DJ01-SNP, DJ02-SNP, DJ03-SNP, DJ04-SNP, DJ05-SNP, DJ06-SNP, DJ07-SNP, DJ08-SNP, DJ09-SNP, DJ10-SNP-1, DJ10-SNP-2, DJ11-SNP, DJ12-SNP, DJ13-SNP, and DJ14-SNP were SNP polymorphic base sites.

[0128] Table 2 Analysis of base sequencing results of polymorphic primers

[0129]

[0130] like Figures 1 to 14 As shown:

[0131] When PCR amplification was performed with primers DJ01, base mutation occurred in XGYC of small-fruited tea oil tree to "A", and base mutation occurred in NHWT of common tea oil tree to "C"; when PCR amplification was performed with primers DJ02, base mutation occurred in XGYC of small-fruited tea oil tree to "A", and base mutation occurred in NHWT of common tea oil tree to "G"; when PCR amplification was performed with primers DJ03, base mutation occurred in XGYC of small-fruited tea oil tree to "T", and base mutation occurred in NHWT of common tea oil tree to "C"; when PCR amplification was performed with primers DJ04, base mutation occurred in XGYC of small-fruited tea oil tree to "A", and base mutation occurred in NHWT of common tea oil tree to "C" WT had a base mutation of "T"; PCR amplification with primers DJ05 resulted in a base mutation of "T" in Camellia oleifera XGYC and a base mutation of "G" in Camellia oleifera NHWT; PCR amplification with primers DJ06 resulted in a base mutation of "A" in Camellia oleifera XGYC and a base mutation of "C" in Camellia oleifera NHWT; PCR amplification with primers DJ07 resulted in a base mutation of "T" in Camellia oleifera XGYC and a base mutation of "C" in Camellia oleifera NHWT; PCR amplification with primers DJ08 resulted in a base mutation of "A" in Camellia oleifera XGYC and a base mutation of "C" in Camellia oleifera NHWT. The base mutation of the common tea oil NHWT was changed to "A"; the base mutation of the small fruit tea oil XGYC was changed to "A" and the base mutation of the common tea oil NHWT was changed to "G" by PCR amplification with primers DJ09; the base mutation of the small fruit tea oil XGYC was changed to "T" and the base mutation of the common tea oil NHWT was changed to "G" by PCR amplification with primers DJ10, the base mutation of the small fruit tea oil XGYC in ZD10_SNP-1 was changed to "T" and the base mutation of the common tea oil NHWT was changed to "G", or the base mutation of the small fruit tea oil XGYC in ZD10_SNP-2 was changed to "C" and the base mutation of the common tea oil NHWT was changed to "A" by PCR amplification with primers DJ11. When the primers DJ12 were used for PCR amplification, the base mutation of XGYC in small-fruited tea was "C", and the base mutation of NHWT in common tea was "G". When the primers DJ12 were used for PCR amplification, the base mutation of XGYC in small-fruited tea was "A", and the base mutation of NHWT in common tea was "C". When the primers DJ13 were used for PCR amplification, the base mutation of XGYC in small-fruited tea was "A", and the base mutation of NHWT in common tea was "T". When the primers DJ14 were used for PCR amplification, the base mutation of XGYC in small-fruited tea was "T", and the base mutation of NHWT in common tea was "C".

[0132] The present invention uses specific embodiments to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, for those skilled in the art, according to the idea of ​​the present invention, there will be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as a limitation on the present invention.

Claims

1. A SNP molecular marker primer combination for identifying small-fruit camellia and common camellia resources, characterized in that: It is mainly composed of primer pair DJ01 to primer pair DJ14, and the primer nucleotide sequences are as follows: DJ01 upstream primer: 5′-GCGAACGACGGGAATTGAAC-3′; DJ01 downstream primer: 5′-TCAGGAAGGCGTTATTGTTCC-3′; DJ02 upstream primer: 5′-TGCTTGCGGAGAAAAACGAA-3′; DJ02 downstream primer: 5′-ATCTCAGCAACACGACTTCG-3′; DJ03 upstream primer: 5′-TCGGACTTGATTAACCGCCC-3′; DJ03 downstream primer: 5′-GGTTCGGGAAGGGATCATGG-3′; DJ04 upstream primer: 5′-TAATGGGTGTCGCAATGGCT-3′; DJ04 downstream primer: 5′-CGCGGTCGAACTACCAAAAC-3′; DJ05 upstream primer: 5′-CGCCTACGGATTAGTCGACA-3′; DJ05 downstream primer: 5′-ATGATTCAACCAGAGGGCGT-3′; DJ06 upstream primer: 5′-GATTCGTAGACCCGGTCGAC-3′; DJ06 downstream primer: 5′-ATGGGTAGGGACACTGTTGC-3′; DJ07 upstream primer: 5′-TGGTTGGTAAACAAGGATCGGT-3′; DJ07 downstream primer: 5′-GATACACCAGATCCGGCTCG-3′; DJ08 upstream primer: 5′-TTCGGGAGCAGTATGGGCTA-3′; DJ08 downstream primer: 5′-AATTGTTCGTTGTACAATGCAAAA-3′; DJ09 upstream primer: 5′-GCTACAGCGATTGCACCTAT-3′; DJ09 downstream primer: 5′-CTGGTCAGGTCAACAAAGTCA-3′; DJ10 upstream primer: 5′-ACCATGCATTGATGGGTGGT-3′; DJ10 downstream primer: 5′-GGCAGTCGTTTCAGCATGTT-3′; DJ11 upstream primer: 5′-TTGACCTTGTTCCCATTGCA-3′; DJ11 downstream primer: 5′-ACAACACACATAAACATAAGGGCA-3′; DJ12 upstream primer: 5′-CGGAATCCATTCTTCCTTGTTCTG-3′; DJ12 downstream primer: 5′-TGGTATTAGTCTGGATACGGCA-3′; DJ13 upstream primer: 5′-CCCTGACTTTGTCCGTGTCA-3′; DJ13 downstream primer: 5′-TCTCCCTCCAAGCCGTACAT-3′; DJ14 upstream primer: 5′-GATACACCAGATCCGGCTCG-3′; DJ14 downstream primer: 5′-TGGTTGGTAAACAAGGATCGGT-3′.

2. Use of the SNP molecular marker primer combination according to claim 1 in the identification of small-fruit camellia and common camellia species, in, DJ01 upstream primer and DJ01 downstream primer identified site ZD01_SNP, and the site recognition sequence was TCCACTATTT[C / A]AATTATAAAA; DJ02 upstream primer and DJ02 downstream primer identified locus ZD02_SNP, and the locus recognition sequence was CCCAAAAAT[G / A]GATTTTGGTT; DJ03 upstream primer and DJ03 downstream primer identified site ZD03_SNP, and the site recognition sequence was TCAATGGCCC[C / T]CCTACGTAGT; DJ04 upstream primer and DJ04 downstream primer identified site ZD04_SNP, and the site recognition sequence was ATTGAAAGCT[T / A]GGATTTCTAG; DJ05 upstream primer and DJ05 downstream primer identified site ZD05_SNP, and the site recognition sequence was AATCCTTGTT[G / T]CGGAGTCGAT; DJ06 upstream primer and DJ06 downstream primer identified site ZD06_SNP, and the site recognition sequence was ACCAAAAAAT[C / A]TTTTTTGCTT; DJ07 upstream primer and DJ07 downstream primer identified locus ZD07_SNP, and the locus recognition sequence was TTCATCTATT[C / T]CATGACCGGA; DJ08 upstream primer and DJ08 downstream primer identified site ZD08_SNP, and the site recognition sequence was ATAAAAAATT[T / A]CCCCCTGCAA; DJ09 upstream primer and DJ09 downstream primer identified locus ZD09_SNP, and the locus recognition sequence was AAAATCATGT[G / A]TTGGTCCAGA; DJ010 upstream primer and DJ010 downstream primer identified locus ZD010_SNP-1, and the locus recognition sequence was AGAATAATTT[G / T]AATCTTAAAA; DJ010 upstream primer and DJ010 downstream primer identified locus ZD010_SNP-2, and the locus recognition sequence was AAAGAATAGT[C / A]AATTTTTGCA; DJ011 upstream primer and DJ011 downstream primer identified the site ZD011_SNP, and the site recognition sequence was GTATAACCCC[T / C]TTTTGCTTTC; DJ012 upstream primer and DJ012 downstream primer identified locus ZD012_SNP, and the locus recognition sequence was TAAGAATGGG[G / T]GACGGTATTC; DJ013 upstream primer and DJ013 downstream primer identified locus ZD013_SNP, and the locus recognition sequence was GAATTCTGTG[A / G]AAAGCCGTAT; DJ014 upstream primer and DJ014 downstream primer identified the site ZD014_SNP, and the site recognition sequence was TCCGGTCATG[C / T]AATAGATGAA.

3. The method for using the primer combination of SNP molecular markers as claimed in claim 1 for interspecific identification of Camellia oleifera and Camellia oleifera resources, characterized in that: The steps include: (1) Extracting DNA from samples to be analyzed: Extracting total genomic DNA from the Camellia oleifera germplasm materials to be tested; (2) PCR amplification reaction: using the 14 pairs of SNP molecular marker primers in claim 1 to perform PCR amplification reaction on the genomic DNA extracted in step (1); Among them, DJ01 upstream primer and DJ01 downstream primer identified site ZD01_SNP, and the site recognition sequence was TCCACTATTT[C / A]AATTATAAAA; DJ02 upstream primer and DJ02 downstream primer identified locus ZD02_SNP, and the locus recognition sequence was CCCAAAAAT[G / A]GATTTTGGTT; DJ03 upstream primer and DJ03 downstream primer identified site ZD03_SNP, and the site recognition sequence was TCAATGGCCC[C / T]CCTACGTAGT; DJ04 upstream primer and DJ04 downstream primer identified site ZD04_SNP, and the site recognition sequence was ATTGAAAGCT[T / A]GGATTTCTAG; DJ05 upstream primer and DJ05 downstream primer identified site ZD05_SNP, and the site recognition sequence was AATCCTTGTT[G / T]CGGAGTCGAT; DJ06 upstream primer and DJ06 downstream primer identified site ZD06_SNP, and the site recognition sequence was ACCAAAAAAT[C / A]TTTTTTGCTT; DJ07 upstream primer and DJ07 downstream primer identified locus ZD07_SNP, and the locus recognition sequence was TTCATCTATT[C / T]CATGACCGGA; DJ08 upstream primer and DJ08 downstream primer identified site ZD08_SNP, and the site recognition sequence was ATAAAAAATT[T / A]CCCCCTGCAA; DJ09 upstream primer and DJ09 downstream primer identified locus ZD09_SNP, and the locus recognition sequence was AAAATCATGT[G / A]TTGGTCCAGA; DJ010 upstream primer and DJ010 downstream primer identified locus ZD010_SNP-1, and the locus recognition sequence was AGAATAATTT[G / T]AATCTTAAAA; DJ010 upstream primer and DJ010 downstream primer identified locus ZD010_SNP-2, and the locus recognition sequence was AAAGAATAGT[C / A]AATTTTTGCA; DJ011 upstream primer and DJ011 downstream primer identified the site ZD011_SNP, and the site recognition sequence was GTATAACCCC[T / C]TTTTGCTTTC; DJ012 upstream primer and DJ012 downstream primer identified locus ZD012_SNP, and the locus recognition sequence was TAAGAATGGG[G / T]GACGGTATTC; DJ013 upstream primer and DJ013 downstream primer identified locus ZD013_SNP, and the locus recognition sequence was GAATTCTGTG[A / G]AAAGCCGTAT; DJ014 upstream primer and DJ014 downstream primer identified the site ZD014_SNP, and the site recognition sequence was TCCGGTCATG[C / T]AATAGATGAA; (3) Detection of PCR amplification products: The PCR amplification products are subjected to genome sequencing.

4. The method according to claim 3, characterized in that The procedure of the PCR amplification reaction described in step (2) is: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 1 min, annealing at 58°C for 1 min, extension at 72°C for 2 min, for 35 cycles; extension at 72°C for 10 min.

5. The method according to claim 3, characterized in that: The system of the PCR amplification reaction described in step (2) is: 20 ng of Camellia oleifera or Camellia oleifera DNA, 0.30 mmol / L of dNTPs, 0.15 μmol / L of polymorphic primers, 2.00 U of Taq DNA polymerase, and ddH2O to make up the total volume to 30 μL.

6. The method according to claim 3, characterized in that The genome sequencing described in step (3) is conventional Sanger first generation sequencing.

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