Primer combination developed based on idesia polycarpa whole genome sequencing and application thereof

Abstract

The invention discloses a primer combination developed based on idesia polycarpa whole genome sequencing and application of the primer combination, and belongs to the technical field of molecular biology. The idesia polycarpa primer combination comprises 18 pairs of SSR primer pairs and / or 30 IRAP primers. Through multiple rounds of screening and verification, the idesia primer combination has the characteristics of codominance, high repeatability, high polymorphism, good stability and the like, can be applied to idesia genetic diversity analysis and genetic relationship analysis, and can also be used for idesia and related genus germplasm genetic identification, breeding and the like. The SSR primer pair and the IRAP primer in the primer combination can be used independently or combined for comprehensive analysis, a more reliable molecular marker is provided for idesia genetic diversity analysis and variety genetic relationship analysis, and the primer combination has important theoretical and practical values.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to a primer combination developed based on whole genome sequencing of Castanopsis japonici and an application thereof. Background Art

[0002] Idesia polycarpa Maxim. is a deciduous tree of the Salicaceae family. It is a high-quality and high-yield woody edible oil tree species and industrial oil raw material tree species native to my country. It has development and utilization value in gardening, bioenergy, medical care, beauty and skin care, etc. Idesia polycarpa oil contains a large amount of unsaturated fatty acids, and its linoleic acid content is as high as 52.5-81.4%. This is an unsaturated fatty acid that humans cannot synthesize on their own, but has the effect of lowering blood lipids, lowering blood pressure and preventing cardiovascular and cerebrovascular diseases in the human body. Therefore, the development of Idesia polycarpa is of great strategic significance for ensuring national energy and grain and oil security and protecting the ecological environment.

[0003] Due to the limited number of traditional plant morphological markers, selection through plant phenotypes is time-consuming and laborious, and is not conducive to genetic breeding and other applications because it is sensitive to factors such as the external environment, plant growth and development stage, etc. The development and application of molecular marker methods can not only produce a large number of markers, but also are not affected by the growth and development stage or the external environment, so they have high practical value in the field of genetic research.

[0004] At present, the research on the genetic diversity of Castanopsis chinensis mainly uses the tree height, leaves, flowers and fruits of Castanopsis chinensis to analyze the geographical distribution difference, provenance and family difference and germplasm resource identification. In terms of molecular, Wang Yanmei et al. (2014) used young leaves of Castanopsis chinensis from 12 different distribution areas as materials, used ISSR molecular marker technology to analyze the genetic differences of Castanopsis chinensis, and clustered the Castanopsis chinensis from 12 distribution areas; Dong Na et al. (2016) used the hairy-leaf Castanopsis chinensis as materials, and used ISSR molecular markers to screen out the primer UBC841 for identifying the sex of the hairy-leaf Castanopsis chinensis, making it possible to identify the sex of the hairy-leaf Castanopsis chinensis with molecular markers.

[0005] Although molecular-level research on the genetic diversity of Castanopsis chinensis has been carried out and some results have been achieved, the content and methods of research on the genetic diversity of Castanopsis chinensis are still relatively simple. Therefore, it is necessary to conduct more in-depth molecular-level research on Castanopsis chinensis in order to provide more reliable molecular markers for the analysis of genetic diversity and kinship of Castanopsis chinensis. Summary of the invention

[0006] The purpose of the present invention is to provide a primer combination developed based on the whole genome sequencing of Castanopsis schrenkiana and its application, so as to solve the problems existing in the above-mentioned prior art. The Castanopsis schrenkiana primer combination of the present invention has the characteristics of co-dominance, high repeatability, high polymorphism, good stability, etc. after multiple rounds of screening and verification, and can be applied to the genetic diversity analysis and kinship analysis of Castanopsis schrenkiana, and can also be used for the genetic identification and breeding of Castanopsis schrenkiana and its closely related genera.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a primer combination for Castanopsis japonici, comprising 18 pairs of SSR primer pairs and / or 30 IRAP primers;

[0009] The 18 pairs of SSR primer pairs are composed of the nucleotide sequence of the forward primer as shown in SEQ ID NO.1, the nucleotide sequence of the reverse primer as shown in SEQ ID NO.2, IpSSR4, the nucleotide sequence of the forward primer as shown in SEQ ID NO.3, the nucleotide sequence of the reverse primer as shown in SEQ ID NO.4, IpSSR6, the nucleotide sequence of the forward primer as shown in SEQ ID NO.5, the nucleotide sequence of the reverse primer as shown in SEQ ID NO.6, IpSSR7, the nucleotide sequence of the forward primer as shown in SEQ ID NO.7, the nucleotide sequence of the reverse primer as shown in SEQ ID NO.8, IpSSR10, the nucleotide sequence of the forward primer as shown in SEQ ID NO.9, the nucleotide sequence of the reverse primer as shown in SEQ ID NO.10, IpSSR11, the nucleotide sequence of the forward primer as shown in SEQ ID NO.11, the nucleotide sequence of the reverse primer as shown in SEQ ID NO.12, IpSSR12, the nucleotide sequence of the forward primer as shown in SEQ ID NO.13, the nucleotide sequence of the reverse primer as shown in SEQ ID NO. The nucleotide sequence of IpSSR14 and the forward primer shown in SEQ ID NO.14 is shown in SEQ ID NO.15, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.16. The nucleotide sequence of IpSSR20 and the forward primer is shown in SEQ ID NO.17, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.18. The nucleotide sequence of IpSSR24 and the forward primer is shown in SEQ ID NO.19, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.20. The nucleotide sequence of IpSSR26 and the forward primer is shown in SEQ ID NO.21, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.22. The nucleotide sequence of IpSSR27 and the forward primer is shown in SEQ ID NO.23, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.24. The nucleotide sequence of IpSSR32 and the forward primer is shown in SEQ ID NO.25, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.26. The nucleotide sequence of IpSSR35 and the forward primer is shown in SEQ ID NO.27, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. The nucleotide sequence of IpSSR40 shown in SEQ ID NO.28 and the nucleotide sequence of the forward primer are shown in SEQ ID NO.29, and the nucleotide sequence of the reverse primer are shown in SEQ ID NO.30. The nucleotide sequence of IpSSR41 shown in SEQ ID NO.31 and the nucleotide sequence of the reverse primer are shown in SEQ ID NO.32. The nucleotide sequence of IpSSR44 shown in SEQ ID NO.32 and the nucleotide sequence of the forward primer are shown in SEQ ID NO.33, the nucleotide sequence of the reverse primer is IpSSR50 shown in SEQ ID NO.34 and the nucleotide sequence of the forward primer is IpSSR52 shown in SEQ ID NO.35, the nucleotide sequence of the reverse primer is IpSSR52 shown in SEQ ID NO.36;.

[0010] The 30 IRAP primers include IPRT4 as shown in SEQ ID NO.37, IPRT6 as shown in SEQ ID NO.38, IPRT7 as shown in SEQ ID NO.39, IPRT8 as shown in SEQ ID NO.40, IPRT9 as shown in SEQ ID NO.41, IPRT10 as shown in SEQ ID NO.42, IPRT11 as shown in SEQ ID NO.43, IPRT12 as shown in SEQ ID NO.44, IPRT15 as shown in SEQ ID NO.45, IPRT17 as shown in SEQ ID NO.46, IPRT18 as shown in SEQ ID NO.47, IPRT19 as shown in SEQ ID NO.48, IPRT20 as shown in SEQ ID NO.49, IPRT21 as shown in SEQ ID NO.50, IPRT23 as shown in SEQ ID NO.51, and IPRT24 as shown in SEQ ID NO.52. NO.52, IPRT24 as shown in SEQ ID NO.52, IPRT26 as shown in SEQ ID NO.53, IPRT27 as shown in SEQ ID NO.54, IPRT29 as shown in SEQ ID NO.55, IPRT30 as shown in SEQ ID NO.56, IPRT31 as shown in SEQ ID NO.57, IPRT33 as shown in SEQ ID NO.58, IPRT34 as shown in SEQ ID NO.59, IPRT37 as shown in SEQ ID NO.60, IPRT41 as shown in SEQ ID NO.61, IPRT47 as shown in SEQ ID NO.62, IPRT50 as shown in SEQ ID NO.63, IPRT51 as shown in SEQ ID NO.64, IPRT58 as shown in SEQ ID NO.65 and IPRT60 as shown in SEQ ID NO.66.

[0011] The present invention also provides a detection kit, comprising the above-mentioned Castanopsis castanopsis primer combination.

[0012] The present invention also provides the use of the above-mentioned Castanopsis japonici primer combination or the above-mentioned detection kit in the genetic diversity analysis and kinship analysis of Castanopsis japonici.

[0013] The present invention also provides a method for analyzing the kinship of Castanopsis japonici, comprising extracting genomic DNA of the Castanopsis japonici to be tested, using the genomic DNA as a template, performing PCR amplification with each pair of SSR primers and / or each IRAP primer in the above-mentioned Castanopsis japonici primer combination, performing electrophoresis on the PCR products, and performing kinship analysis on the Castanopsis japonici according to the electrophoresis results.

[0014] Furthermore, when the SSR primer pair is used for PCR amplification, the reaction system of the PCR amplification is 3.0 μL ddH2O, 0.5 μL each of 10 μM forward and reverse primers, 1.0 μL of 20 ng / μL template DNA and 5.0 μL 2×TaqPCRMasterMixⅡ.

[0015] Furthermore, the reaction procedure of the PCR amplification is pre-denaturation at 94°C for 4 minutes; denaturation at 94°C for 30 seconds, annealing at 56.9-65°C for 30 seconds, extension at 72°C for 45 seconds, 35 cycles; and finally extension at 72°C for 5 minutes.

[0016] Furthermore, when PCR amplification was performed using IRAP primers, the reaction system for PCR amplification was 3.0 μL ddH2O, 0.5 μL of each 10 μM primer, 1.0 μL of 20 ng / μL template DNA, and 5.0 μL 2×TaqPCR Master MixⅡ.

[0017] Furthermore, the reaction procedure of the PCR amplification is pre-denaturation at 94°C for 4 minutes; denaturation at 94°C for 30 seconds, annealing at 40-46.2°C for 45 seconds, extension at 72°C for 1 minute, 35 cycles; and finally extension at 72°C for 7 minutes.

[0018] The present invention discloses the following technical effects:

[0019] The tung oil tree primer combination of the present invention has the characteristics of co-dominance, high repeatability, high polymorphism, good stability, etc. after multiple rounds of screening and verification, and can be applied to the genetic diversity analysis and kinship analysis of tung oil tree, and can also be used for the genetic identification and breeding of tung oil tree and its closely related genera. The SSR primer pair and IRAP primer in the primer combination can be used alone, or the two can be used in combination for comprehensive analysis, which provides more reliable molecular markers for the genetic diversity analysis and variety kinship analysis of tung oil tree, and has important theoretical and practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The electrophoresis pattern of PCR amplification at the optimal annealing temperature of some SSR primers in one sample of Castanopsis sylvestris; M is 2000bp loading marker; 1 is 65℃; 2 is 64.3℃; 3 is 63℃; 4 is 61.1℃; 5 is 58.8℃; 6 is 56.9℃; 7 is 55.7℃; 8 is 55℃;

[0021] Figure 2 The electrophoresis pattern of PCR amplification at the optimal annealing temperature of some IRAP primers in a sample of Castanopsis sylvestris; M is a 2000bp loading marker; 1 is 50℃; 2 is 49.2℃; 3 is 47.9℃; 4 is 46.2℃; 5 is 43.9℃; 6 is 41.9℃; 7 is 40.6℃; 8 is 40℃;

[0022] Figure 3 The genomic DNA gel electrophoresis patterns of 8 Castanopsis altissima resources are shown in Figure 1.

[0023] Figure 4 The PCR amplification electrophoresis patterns of some SSR primers in 8 samples of Castanopsis chinensis; M is a 500bp marker; 1 is Jiangkou No. 4 (JK4); 2 is Guiding No. 10 (GD10); 3 is Liupanshui No. 12 (LPS12); 4 is Yinjiang No. 5 (YJ5); 5 is Suiyang No. 6 (SY6); 6 is Jinping No. 1 (JP1); 7 is Xingyi No. 6 (XY6); 8 is Libo No. 1 (LB1);

[0024] Figure 5 The PCR amplification electrophoresis patterns of some SSR primers in 8 samples of Castanopsis chinensis were detected by Qsep 100 analyzer;

[0025] Figure 6 The PCR amplification electrophoresis patterns of some IRAP primers in 8 samples of Castanopsis chinensis; M is a 2000bp marker; 1 is Jiangkou No. 4 (JK4); 2 is Guiding No. 10 (GD10); 3 is Liupanshui No. 12 (LPS12); 4 is Yinjiang No. 5 (YJ5); 5 is Suiyang No. 6 (SY6); 6 is Jinping No. 1 (JP1); 7 is Xingyi No. 6 (XY6); 8 is Libo No. 1 (LB1);

[0026] Figure 7 This is the phylogenetic relationship diagram of 8 Castanopsis sylvestris resources based on SSR markers;

[0027] Figure 8 This is the phylogenetic relationship diagram of 8 Castanopsis sylvestris resources based on IRAP markers. DETAILED DESCRIPTION

[0028] Example

[0029] 1. Identification of SSR primers and primer design and synthesis

[0030] The inventors previously used wild adult Castanopsis truncatula from Guiting County, Guizhou Province as samples to sequence the whole genome of Castanopsis truncatula. Based on the genome data, the krait software was used to identify and locate SSR sites. The search criteria were set as: mononucleotide repeats 10 or more times, dinucleotide repeats 7 or more times, tri-, tetra-, penta-, and hexa-nucleotide repeats 5 or more times. As a result, a total of 924,186 sites were found. 60 pairs of SSR primers were randomly designed using Primer5.0 software (see Table 1).

[0031] Table 1 60 pairs of randomly synthesized SSR primers

[0032]

[0033]

[0034]

[0035] 2. IRAP Primer Identification, Primer Design and Synthesis

[0036] Based on the genome data of Castanopsis sylvestris, the LTR retrotransposon sequences were obtained, and multiple sequence alignment was performed on these sequences. Combined with the known conserved regions of LTR retrotransposon RT sequences of other species, the conserved regions of Castanopsis sylvestris RT sequences were found, and 60 IRAP primers were randomly designed based on the conserved regions of RT sequences using Primer5.0 software (Table 2).

[0037] Table 2 60 randomly synthesized IRAP primers

[0038]

[0039]

[0040] 3. Screening of optimal primer annealing temperature

[0041] A random copy of the genome of Castanopsis schrenkiana was extracted, and the genome of Castanopsis schrenkiana was used as a template to perform PCR amplification temperature gradient screening using synthetic SSR primers and IRAP primers. The amplification products of SSR-PCR and IRAP-PCR were detected by 1.0% and 1.5% agarose gel electrophoresis, respectively. According to the band detection results, the amplification primers that can obtain clear and stable bands were selected for the next step of rescreening, and the optimal annealing temperature of the primers was determined.

[0042] SSR-PCR reaction procedure: pre-denaturation at 94°C for 4 min, denaturation at 94°C for 30 s, annealing at 55-65°C for 30 s, extension for 45 s, 35 cycles; and final extension at 72°C for 5 min.

[0043] SSR-PCR reaction system (10 μL): 3.0 μL ddH2O, 0.5 μL of upstream and downstream primers (10 μM), 1.0 μL template DNA (20 ng / μL) and 5.0 μL 2×Taq PCR Master MixⅡ (Tiangen Biochemical Technology Co., Ltd.). The PCR amplification electrophoresis results of some SSR primers in one sample of Castanopsis chinensis are shown in Figure 1 .

[0044] IRAP-PCR reaction program: pre-denaturation at 94°C for 4 min, denaturation at 94°C for 30 s, annealing at 40-50°C for 45 s, extension for 1 min, 35 cycles; and final extension at 72°C for 7 min.

[0045] IRAP-PCR reaction system (10 μL): 3.0 μL ddH2O, 1.0 μL primer (10 μM), 1.0 μL template DNA (20 ng / μL) and 5.0 μL 2×Taq PCR Master MixⅡ (Tiangen Biochemical Technology Co., Ltd.). The PCR amplification electrophoresis results of some IRAP primers in one sample of Castanopsis chinensis are shown in Figure 2 .

[0046] The SSR primer pairs and IRAP primers obtained in the initial screening that can amplify clear and stable target bands are shown in Table 3.

[0047] Table 3 Preliminary screening primer information

[0048]

[0049] 4. Primer rescreening

[0050] 4.1 Testing and sampling of Castanopsis chinensis

[0051] Eight samples of Achyranthes chinensis resources were collected in Tongren, Southeast Guizhou, and Liupanshui, Guizhou. Plants without pests and diseases were selected, and tender leaves of Achyranthes chinensis with good growth potential and no obvious pests and diseases were randomly collected and placed in 2-3 50mL sterilized centrifuge tubes and numbered. No. 1 was Jiangkou No. 4 (JK4); No. 2 was Guiding No. 10 (GD10); No. 3 was Liupanshui No. 12 (LPS12); No. 4 was Yinjiang No. 5 (YJ5); No. 5 was Suiyang No. 6 (SY6); No. 6 was Jinping No. 1 (JP1); No. 7 was Xingyi No. 6 (XY6); No. 8 was Libo No. 1 (LB1). Then they were placed in a liquid nitrogen tank, brought back to the laboratory, and stored at -80℃ for future use.

[0052] 4.2 DNA extraction and quality control

[0053] The plant genomic DNA extraction kit (DP305-02) was used to extract the genomic DNA of the fruit of the mountain ash according to the instructions. After the genomic DNA was extracted, the quality of the DNA sample was detected by agarose gel electrophoresis. The DNA sample with a clear and single band was a qualified DNA sample. The DNA sample was placed on a UV spectrophotometer to detect its DNA concentration and OD 260 / OD 280 Value, OD 260 / OD 280 A ratio between 1.8 and 2.0 indicates no contamination; DNA samples that meet the test results are uniformly diluted to 20 ng / μL with Buffer TE and stored in a -20°C refrigerator for later use (such as Figure 3 ).

[0054] 4.3 PCR amplification of SSR and IRAP markers

[0055] Using the above 8 samples of Castanopsis castanopsis resource DNA as templates, PCR amplification was performed again using the SSR primers and IRAP primers obtained in the initial screening that could stably amplify clear target bands.

[0056] SSR-PCR amplification used a 10 μL reaction system, including 3.0 μL ddH2O, 0.5 μL (10 μM) of upstream and downstream primers, 1.0 μL template DNA (20 ng / μL) and 5.0 μL 2×Taq PCR Master MixⅡ (Tiangen Biochemical Technology Co., Ltd.). SSR-PCR program: 94℃ pre-denaturation for 4 min; 94℃ denaturation for 30 s, annealing at the optimal annealing temperature of each primer for 30 s, 72℃ extension for 45 s, 35 cycles; finally 72℃ extension for 5 min; PCR products were detected by 3.5% agarose gel electrophoresis, and the gel imaging system was used to take pictures after electrophoresis at 90 V for 50 min. DL500 was used as DNA marker, and the PCR products were detected by Qsep 100 analyzer. The PCR amplification electrophoresis patterns of some SSR primers in 8 samples of Castanopsis chinensis and the detection patterns of Qsep 100 analyzer are shown as follows: Figure 4 and Figure 5 shown.

[0057] IRAP-PCR amplification used a 10 μL reaction system, including 3.0 μL ddH2O, 1.0 μL of each primer (10 μM), 1.0 μL template DNA (20 ng / μL) and 5.0 μL 2×Taq PCR Master MixⅡ (Tiangen Biochemical Technology Co., Ltd.). IRAP-PCR program: 94℃ pre-denaturation for 4 min; 94℃ denaturation for 30 s, annealing at the optimal annealing temperature of each primer for 45 s, 72℃ extension for 1 min, 35 cycles; finally 72℃ extension for 7 min; PCR products were detected by 1.5% agarose gel electrophoresis, and the gel imaging system was used to take pictures after electrophoresis at 120V for 40 min. DL2000 was used as DNA marker. The PCR amplification electrophoresis patterns of some IRAP primers in 8 samples of Castanopsis sylvestris are shown in the figure. Figure 6 shown.

[0058] According to the results of PCR product gel electrophoresis and Qsep 100 analyzer detection, 18 pairs of SSR marker primers and 30 IRAP marker primers that can stably amplify clear target bands with high polymorphism and good repeatability were screened, as shown in Tables 4 and 5.

[0059] Table 4 18 pairs of SSR primers obtained by repeated screening

[0060]

[0061]

[0062] Table 5 30 IPAP primers obtained by rescreening

[0063]

[0064]

[0065] 5. Use multiple screening primers to analyze the relationship between varieties

[0066] According to the electrophoresis patterns of the amplified products of the eight A. truncatum samples amplified by the primers in Tables 4 and 5, the presence and absence of the bands were assigned values ​​of 1 and 0, respectively. The genetic distances among the eight samples were calculated using the SM similarity coefficient (Quanlitative date) in NTSYS-pc 2.10 software. The unweighted paired arithmetic mean (UPGMA) clustering method was used to construct a phylogenetic dendrogram. The phylogenetic relationships of the eight A. truncatum resources based on SSR markers and IRAP markers were shown in the following table. Figure 7 and Figure 8 shown.

[0067] The results of SSR molecular markers showed that the genetic diversity coefficients of the eight varieties were between 0.18 and 0.77. With a threshold of 0.62, the eight resources can be divided into three categories. The first group contains five resources, namely JK4, YJ5, JP1, SY6, and XY6. Except for XY6, according to regional analysis, the other four trees are adjacent to each other and are located in the northeast of Guizhou. The second group contains two resources, both collected from Qiannan Prefecture, Guizhou Province, namely GD10 and LB1, located in the southeast of Guizhou. The third group (GLPS12) is obviously more distantly related to the first two categories. The sample was collected in Liupanshui, where local wild resources are abundant.

[0068] The results of IRAP molecular markers showed that the genetic diversity coefficients of the eight varieties ranged from 0.48 to 0.72. With a threshold of 0.66, the eight resources can be further divided into three categories, of which the first group contains five resources, mainly JK4, GD10, JP1, YJ5, and SY6. Similarly, these five trees are geographically adjacent; the second group contains two resources, collected from Xingyi City, Qianxinan Prefecture (XY6) and Libo County, Qiannan Prefecture (LB1), both in the south of Guizhou; the third group is GLPS12.

[0069] In summary, the clustering results of the two molecular markers show that the relationship of the affine tree is consistent with the geographical distribution of resources, variety sources and other characteristics. In addition, it was found that the affine tree in Liupanshui is different from that in other places in Guizhou. The back of the leaves of the affine tree in various places in Guizhou is smooth, while the back of the leaves of the affine tree in Liupanshui has villi, so the relationship is obviously farther than the other two categories. This shows that the SSR and IRAP primers screened by the present invention have good polymorphism and can be used for genetic diversity analysis and affine relationship analysis of affine tree.

[0070] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A primer combination for Castanopsis chinensis, characterized in that: The invention comprises 18 pairs of SSR primer pairs and / or 30 IRAP primers; the 18 pairs of SSR primer pairs are composed of a forward primer having a nucleotide sequence as shown in SEQ ID NO.1, a reverse primer having a nucleotide sequence as shown in SEQ ID NO.2, IpSSR4, a forward primer having a nucleotide sequence as shown in SEQ ID NO.3, a reverse primer having a nucleotide sequence as shown in SEQ ID NO.4, IpSSR6, a forward primer having a nucleotide sequence as shown in SEQ ID NO.5, a reverse primer having a nucleotide sequence as shown in SEQ ID NO.6, IpSSR7, a forward primer having a nucleotide sequence as shown in SEQ ID NO.7, a reverse primer having a nucleotide sequence as shown in SEQ ID NO.8, IpSSR10, a forward primer having a nucleotide sequence as shown in SEQ ID NO.9, a reverse primer having a nucleotide sequence as shown in SEQ ID NO.10, IpSSR11, a forward primer having a nucleotide sequence as shown in SEQ ID NO.11, a reverse primer having a nucleotide sequence as shown in SEQ ID NO.12, IpSSR12, a forward primer having a nucleotide sequence as shown in SEQ ID NO. The nucleotide sequence of the reverse primer is shown in SEQ ID NO.13, the nucleotide sequence of the reverse primer is IpSSR14 shown in SEQ ID NO.14, and the nucleotide sequence of the forward primer is shown in SEQ ID NO.15, the nucleotide sequence of the reverse primer is IpSSR20 shown in SEQ ID NO.16, and the nucleotide sequence of the forward primer is shown in SEQ ID NO.17, the nucleotide sequence of the reverse primer is IpSSR24 shown in SEQ ID NO.18, and the nucleotide sequence of the forward primer is shown in SEQ ID NO.19, the nucleotide sequence of the reverse primer is IpSSR26 shown in SEQ ID NO.20, and the nucleotide sequence of the forward primer is shown in SEQ ID NO.21, the nucleotide sequence of the reverse primer is IpSSR27 shown in SEQ ID NO.22, and the nucleotide sequence of the forward primer is shown in SEQ ID NO.23, the nucleotide sequence of the reverse primer is IpSSR32 shown in SEQ ID NO.24, and the nucleotide sequence of the forward primer is shown in SEQ ID NO.25, the nucleotide sequence of the reverse primer is IpSSR35 shown in SEQ ID NO.26, and the nucleotide sequence of the forward primer is shown in SEQ ID NO. The nucleotide sequence of the reverse primer is shown in SEQ ID NO.27, the nucleotide sequence of the forward primer is shown in SEQ ID NO.28, the nucleotide sequence of the reverse primer is shown in SEQ ID NO.29, the nucleotide sequence of the reverse primer is shown in SEQ ID NO.30, the nucleotide sequence of the forward primer is shown in SEQ ID NO.31, the nucleotide sequence of the reverse primer is shown in SEQ ID NO.32, the nucleotide sequence of the forward primer is shown in SEQ ID NO.33, the nucleotide sequence of the reverse primer is shown in SEQ ID NO.34, IpSSR50, and the nucleotide sequence of the forward primer is shown in SEQ ID NO.35, the nucleotide sequence of the reverse primer is shown in SEQ ID NO.36, IpSSR52;. The 30 IRAP primers include IPRT4 as shown in SEQ ID NO.37, IPRT6 as shown in SEQ ID NO.38, IPRT7 as shown in SEQ ID NO.39, IPRT8 as shown in SEQ ID NO.40, IPRT9 as shown in SEQ ID NO.41, IPRT10 as shown in SEQ ID NO.42, IPRT11 as shown in SEQ ID NO.43, IPRT12 as shown in SEQ ID NO.44, IPRT15 as shown in SEQ ID NO.45, IPRT17 as shown in SEQ ID NO.46, IPRT18 as shown in SEQ ID NO.47, IPRT19 as shown in SEQ ID NO.48, IPRT20 as shown in SEQ ID NO.49, IPRT21 as shown in SEQ ID NO.50, IPRT23 as shown in SEQ ID NO.51, and IPRT24 as shown in SEQ ID NO.

52. NO.52, IPRT24 as shown in SEQ ID NO.52, IPRT26 as shown in SEQ ID NO.53, IPRT27 as shown in SEQ ID NO.54, IPRT29 as shown in SEQ ID NO.55, IPRT30 as shown in SEQ ID NO.56, IPRT31 as shown in SEQ ID NO.57, IPRT33 as shown in SEQ ID NO.58, IPRT34 as shown in SEQ ID NO.59, IPRT37 as shown in SEQ ID NO.60, IPRT41 as shown in SEQ ID NO.61, IPRT47 as shown in SEQ ID NO.62, IPRT50 as shown in SEQ ID NO.63, IPRT51 as shown in SEQ ID NO.64, IPRT58 as shown in SEQ ID NO.65 and IPRT60 as shown in SEQ ID NO.

66.

2. A detection kit, characterized in that: It includes the Castanopsis japonici primer combination described in claim 1.

3. Use of the Castanopsis japonici primer combination of claim 1 or the detection kit of claim 2 in genetic diversity analysis and kinship analysis of Castanopsis japonici.

4. A method for analyzing the kinship of Castanopsis sylvestris, characterized in that: The method comprises extracting genomic DNA of the Castanopsis affine to be tested, using the genomic DNA as a template, performing PCR amplification respectively with each pair of SSR primers and / or each IRAP primer in the Castanopsis affine primer combination of claim 1, then performing electrophoresis on the PCR products, and performing kinship analysis on the Castanopsis affine according to the electrophoresis results.

5. The method for kinship analysis according to claim 4, characterized in that: When the SSR primer pair is used for PCR amplification, the reaction system of the PCR amplification is 3.0 μL ddH2O, 0.5 μL each of 10 μM forward and reverse primers, 1.0 μL of 20 ng / μL template DNA and 5.0 μL 2×TaqPCR Master MixⅡ.

6. The method for kinship analysis according to claim 5, characterized in that: The reaction procedure of the PCR amplification is pre-denaturation at 94°C for 4 minutes; denaturation at 94°C for 30 seconds, annealing at 56.9-65°C for 30 seconds, extension at 72°C for 45 seconds, 35 cycles; and finally extension at 72°C for 5 minutes.

7. The method for kinship analysis according to claim 4, characterized in that: When PCR amplification is performed using IRAP primers, the reaction system for PCR amplification is 3.0 μL ddH2O, 0.5 μL of each 10 μM primer, 1.0 μL of 20 ng / μL template DNA and 5.0 μL 2×Taq PCR Master MixⅡ.

8. The method for kinship analysis according to claim 7, characterized in that: The reaction procedure of the PCR amplification is pre-denaturation at 94°C for 4 minutes; denaturation at 94°C for 30 seconds, annealing at 40-46.2°C for 45 seconds, extension at 72°C for 1 minute, 35 cycles; and finally extension at 72°C for 7 minutes.

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