A primer combination developed based on the whole-genome sequencing of Idesia polycarpa and its application

Abstract

The present invention discloses a primer combination developed based on the whole-genome sequencing of Idesia polycarpa and its application, belonging to the technical field of molecular biology. The Idesia polycarpa primer combination includes 18 pairs of SSR primer pairs and / or 30 IRAP primers. The Idesia polycarpa primer combination of the present invention has been screened and verified through multiple rounds, and has the characteristics of codominance, high repeatability, high polymorphism, good stability, etc. It can be applied to the analysis of genetic diversity and genetic relationship of Idesia polycarpa, and can also be used for genetic identification and breeding of Idesia polycarpa and its related germplasms. The SSR primer pairs and IRAP primers in the primer combination can be used alone or in combination for comprehensive analysis, providing more reliable molecular markers for the analysis of genetic diversity and varietal genetic relationship of Idesia polycarpa, and having important theoretical and practical values.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and particularly relates to a primer combination developed based on the whole-genome sequencing of Idesia polycarpa and its application. Background Art

[0002] Idesia polycarpa Maxim., a deciduous tree belonging to the genus Idesia in 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 China, and has development and utilization value in aspects such as landscape ornamentation, bioenergy, healthcare, and beauty skincare. Idesia polycarpa oil contains a large amount of unsaturated fatty acids, and the content of its linoleic acid is as high as 52.5 - 81.4%. This is an unsaturated fatty acid that humans cannot synthesize by themselves, but has the effects of reducing blood lipid, lowering blood pressure, and preventing the occurrence of cardiovascular and cerebrovascular diseases in the human body. Therefore, the development of Idesia polycarpa has important 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, the selection through plant phenotypes is both time-consuming and laborious, and because it is sensitive to factors such as the external environment, plant growth and development stages, etc., it is not conducive to applications such as genetic breeding. The development and application of molecular marker methods can not only generate a large number of markers, but also are not affected by the growth and development stages or external environment, so they have high practical value in the field of genetic research.

[0004] Currently, regarding the research on the genetic diversity of Idesia polycarpa, in terms of phenotypes, the tree height, leaves, flowers, fruits, etc. of Idesia polycarpa are mainly used for analyses such as geographical distribution differences, provenance and family differences, and germplasm resource identification. At the molecular level, Wang Yanmei et al. (2014) used the young leaves of Idesia polycarpa from 12 different distribution areas as materials, and used ISSR molecular marker technology to analyze the genetic differences of Idesia polycarpa and cluster the Idesia polycarpa from 12 distribution areas; Dong Na et al. (2016) used Idesia pubescens as materials and screened the primer UBC841 for identifying the gender of Idesia pubescens by using ISSR molecular markers, making it possible to identify the gender of Idesia pubescens by molecular markers.

[0005] Although research on the genetic diversity of Idesia polycarpa has been carried out at the molecular level and certain results have been obtained, the content and methods of current research on the genetic diversity of Idesia polycarpa are still relatively single. Therefore, it is necessary to carry out more in-depth research on the molecular level of Idesia polycarpa in order to provide more reliable molecular markers for the analysis of the genetic diversity of Idesia polycarpa and the analysis of the genetic relationship of varieties. Summary of the Invention

[0006] The object of the present invention is to provide a primer combination developed based on the whole-genome sequencing of Idesia polycarpa and its application to solve the problems existing in the above-mentioned prior art. The primer combination of Idesia polycarpa of the present invention has been screened and verified through multiple rounds, and has the characteristics of co-dominance, high repeatability, high polymorphism, good stability, etc., and can be applied to the analysis of genetic diversity and genetic relationship of Idesia polycarpa, and can also be used for genetic identification and breeding of Idesia polycarpa and its related germplasms.

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

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

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

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

[0011] The present invention also provides a detection kit, which includes the above-mentioned Idesia polycarpa primer combination.

[0012] The present invention also provides the application of the above-mentioned Idesia polycarpa primer combination or the above-mentioned detection kit in the analysis of genetic diversity and genetic relationship of Idesia polycarpa.

[0013] The present invention also provides a method for analyzing the genetic relationship of Idesia polycarpa, which includes extracting genomic DNA of the Idesia polycarpa to be detected, using each pair of SSR primers and / or each IRAP primer in the above-mentioned Idesia polycarpa primer combination as templates to perform PCR amplification respectively, and then performing electrophoresis on the PCR products, and analyzing the genetic relationship of Idesia polycarpa according to the electrophoresis results.

[0014] Further, when performing PCR amplification using SSR primer pairs, the reaction system for the PCR amplification is 3.0 μL ddH 2 O, 0.5 μL of each of the forward and reverse primers at 10 μM, 1.0 μL of template DNA at 20 ng / μL, and 5.0 μL of 2× Taq PCR Master Mix Ⅱ.

[0015] Further, the reaction program for the PCR amplification is pre-denaturation at 94 °C for 4 min; denaturation at 94 °C for 30 s, annealing at 56.9 - 65 °C for 30 s, extension at 72 °C for 45 s, for 35 cycles; and finally extension at 72 °C for 5 min.

[0016] Further, when performing PCR amplification using IRAP primers, the reaction system for the PCR amplification is 3.0 μL ddH 2 O, 0.5 μL of each primer at 10 μM, 1.0 μL of template DNA at 20 ng / μL, and 5.0 μL of 2× Taq PCR Master Mix Ⅱ.

[0017] Further, the reaction program for the PCR amplification is pre-denaturation at 94 °C for 4 min; denaturation at 94 °C for 30 s, annealing at 40 - 46.2 °C for 45 s, extension at 72 °C for 1 min, for 35 cycles; and finally extension at 72 °C for 7 min.

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

[0019] The Idesia polycarpa primer combination of the present invention has been screened and verified through multiple rounds, and has characteristics such as co-dominance, high repeatability, high polymorphism, and good stability. It can be applied to the analysis of genetic diversity and genetic relationship of Idesia polycarpa, and can also be used for genetic identification and breeding of Idesia polycarpa and its related germplasms. The SSR primer pairs and IRAP primers in the primer combination can be used alone or in combination for comprehensive analysis, providing more reliable molecular markers for the analysis of genetic diversity and variety genetic relationship of Idesia polycarpa, and having important theoretical and practical values. Description of the Drawings

[0020] Figure 1 It is the PCR amplification electrophoresis pattern of the optimal annealing temperature of some SSR primers in one sample of Idesia polycarpa; among them, M is the 2000bp loading marker; 1 is 65°C; 2 is 64.3°C; 3 is 63°C; 4 is 61.1°C; 5 is 58.8°C; 6 is 56.9°C; 7 is 55.7°C; 8 is 55°C;

[0021] Figure 2 It is the PCR amplification electrophoresis pattern of the optimal annealing temperature of some IRAP primers in one sample of Idesia polycarpa; among them, M is the 2000bp loading marker; 1 is 50°C; 2 is 49.2°C; 3 is 47.9°C; 4 is 46.2°C; 5 is 43.9°C; 6 is 41.9°C; 7 is 40.6°C; 8 is 40°C;

[0022] Figure 3 It is the genomic DNA gel electrophoresis pattern of 8 Idesia polycarpa resources;

[0023] Figure 4 It is the PCR amplification electrophoresis pattern of some SSR primers in 8 samples of Idesia polycarpa; among them, M is the 500bp marker; 1 is Jiangkou No. 4 (JK4); No. 2 is Guiding No. 10 (GD10); No. 3 is Liupanshui No. 12 (LPS12); No. 4 is Yinjiang No. 5 (YJ5); No. 5 is Suiyang No. 6 (SY6); No. 6 is Jinping No. 1 (JP1); No. 7 is Xingyi No. 6 (XY6); No. 8 is Libo No. 1 (LB1);

[0024] Figure 5 It is the detection pattern of the PCR amplification electrophoresis pattern of some SSR primers in 8 samples of Idesia polycarpa on the Qsep 100 analyzer;

[0025] Figure 6 It is the PCR amplification electrophoresis pattern of some IRAP primers in 8 samples of Idesia polycarpa; among them, M is the 2000bp marker; 1 is Jiangkou No. 4 (JK4); No. 2 is Guiding No. 10 (GD10); No. 3 is Liupanshui No. 12 (LPS12); No. 4 is Yinjiang No. 5 (YJ5); No. 5 is Suiyang No. 6 (SY6); No. 6 is Jinping No. 1 (JP1); No. 7 is Xingyi No. 6 (XY6); No. 8 is Libo No. 1 (LB1);

[0026] Figure 7 It is the genetic relationship diagram of 8 Idesia polycarpa resources based on SSR markers;

[0027] Figure 8 It is the genetic relationship diagram of 8 Idesia polycarpa resources based on IRAP markers. Detailed implementation mode

[0028] Example

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

[0030] Previously, the inventors used the wild adult Idesia polycarpa Maxim. in Guiding County, Guizhou Province as a sample for whole-genome sequencing of Idesia polycarpa Maxim. According to the genomic data, the krait software was used to identify and locate SSR loci, and the search criteria were set as follows: mononucleotide repeats 10 times or more, dinucleotide repeats 7 times or more, and trinucleotide, tetranucleotide, pentanucleotide, and hexanucleotide repeats 5 times or more. As a result, a total of 924,186 loci were found. Primer5.0 software was used to randomly design 60 pairs of SSR primers (see Table 1).

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

[0032]

[0033]

[0034]

[0035] 2. Identification of IRAP primers and primer design and synthesis

[0036] Based on the genomic data of Idesia polycarpa Maxim., LTR retrotransposon sequences were obtained, multiple sequence alignments were performed on these sequences, and combined with the conserved regions of LTR retrotransposon RT sequences of other known species, the conserved regions of RT sequences of Idesia polycarpa Maxim. were found, and Primer5.0 software was used to randomly design 60 IRAP primers according to the conserved regions of RT sequences (Table 2).

[0037] Table 2 60 randomly synthesized IRAP primers

[0038]

[0039]

[0040] 3. Screening of the optimal annealing temperature of primers

[0041] Randomly extract 1 copy of the genomic DNA of Idesia polycarpa Maxim., use the genomic DNA of Idesia polycarpa Maxim. as a template, and perform temperature gradient screening of PCR amplification using the synthesized SSR primers and IRAP primers respectively. The SSR-PCR and IRAP-PCR amplification products 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 target bands were screened for the next rescreening, and the optimal annealing temperature of the primers was determined at the same time.

[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 at 45 s, 35 cycles; finally, extension at 72°C for 5 min.

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

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

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

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

[0047] Table 3 Information table of primers for primary screening

[0048]

[0049] 4. Rescreening of primers

[0050] 4.1 Idesia polycarpa test materials and sampling

[0051] Collect 8 samples of Idesia polycarpa resources from Tongren, Southeast Guizhou, Liupanshui and other places in Guizhou. Select pest- and disease-free plants, and randomly collect 2 - 3 tubes of young leaves of Idesia polycarpa with good growth potential and no obvious pests and diseases and number them. No. 1 is Jiangkou No. 4 (JK4); No. 2 is Guiding No. 10 (GD10); No. 3 is Liupanshui No. 12 (LPS12); No. 4 is Yinjiang No. 5 (YJ5); No. 5 is Suiyang No. 6 (SY6); No. 6 is Jinping No. 1 (JP1); No. 7 is Xingyi No. 6 (XY6); No. 8 is Libo No. 1 (LB1). Then put them into a liquid nitrogen tank, take them back to the laboratory, and store them at -80°C for later use.

[0052] 4.2 DNA Extraction and Quality Inspection

[0053] Use a plant genomic DNA extraction kit (DP305-02) to extract the genomic DNA of Idesia polycarpa Maxim. according to the instructions. After the genomic DNA is extracted, use agarose gel electrophoresis to detect the quality of the DNA sample. Among them, the DNA sample with clear and single bands is a qualified DNA sample. Take the DNA sample and detect its DNA concentration and OD 260 / OD 280 value on a UV spectrophotometer. The OD 260 / OD 280 ratio between 1.8 and 2.0 indicates no pollution; Dilute the DNA samples with qualified test results to 20 ng / μL uniformly with BufferTE and store them at -20 °C in the refrigerator for standby (such as Figure 3 ).

[0054] 4.3 SSR and IRAP Marker PCR Amplification

[0055] Using the DNA of the above 8 Idesia polycarpa Maxim. resources as templates, perform PCR amplification again with the SSR primers and IRAP primers that can stably amplify clear target bands obtained by preliminary screening.

[0056] The SSR-PCR amplification uses a 10 μL reaction system, which includes 3.0 μL ddH 2 O, 0.5 μL of each upstream and downstream primer (10 μM), 1.0 μL of template DNA (20 ng / μL), and 5.0 μL of 2×Taq PCR Master MixⅡ (Tiangen Biochemical Technology Co., Ltd.). SSR-PCR program: Pre-denature at 94 °C for 4 min; Denature at 94 °C for 30 s, anneal at the optimal annealing temperature corresponding to each primer for 30 s, extend at 72 °C for 45 s, for 35 cycles; Finally, extend at 72 °C for 5 min; The PCR products are detected by 3.5% agarose gel electrophoresis. After electrophoresis at 90 v for 50 min, take pictures with a gel imaging system. Use DL500 as the DNA marker, and then detect the PCR products with a Qsep 100 analyzer. The PCR amplification electrophoresis maps of some SSR primers in 8 Idesia polycarpa Maxim. samples and the detection maps of the Qsep 100 analyzer are respectively as Figure 4 and Figure 5 shown.

[0057] The IRAP-PCR amplification uses a 10 μL reaction system, which includes 3.0 μL ddH 21.0 μL of each primer (10 μM), 1.0 μL of template DNA (20 ng / μL), and 5.0 μL of 2× Taq PCR Master Mix Ⅱ (TIANGEN BIOTECH (BEIJING) CO., LTD.). IRAP-PCR program: pre-denaturation at 94 °C for 4 min; denaturation at 94 °C for 30 s, annealing at the optimal annealing temperature corresponding to each primer for 45 s, extension at 72 °C for 1 min, 35 cycles; finally, extension at 72 °C for 7 min; the PCR products were detected by 1.5% agarose gel electrophoresis, and after electrophoresis at 120 v for 40 min, the gel imaging system was used to take pictures. Using DL2000 as the DNA marker, the PCR amplification electrophoresis patterns of some IRAP primers in 8 samples of Idesia polycarpa are as 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, have high polymorphism and good repeatability were re-screened, as shown in Table 4 and Table 5.

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

[0060]

[0061]

[0062] Table 5 30 IPAP primers obtained by re-screening

[0063]

[0064]

[0065] 5. Analysis of varietal genetic relationships using re-screened primers

[0066] According to the electrophoresis patterns of the amplification products of the above 8 Idesia polycarpa test materials with the primers in Table 4 and Table 5, 1 and 0 were respectively assigned according to the presence and absence of the bands. The genetic distances between 8 samples were calculated using the SM similarity coefficient (Quanlitative date) in NTSYS-pc 2.10 software, and the unweighted pair-group method with arithmetic means (UPGMA) was used for clustering to construct a phylogenetic dendrogram. The phylogenetic relationship diagrams of 8 Idesia polycarpa resources based on SSR markers and IRAP markers are respectively as Figure 7 and Figure 8 shown.

[0067] The results of SSR molecular markers showed that the genetic diversity coefficients of the 8 varieties were between 0.18 and 0.77. Taking 0.62 as the threshold, the 8 resources could be further divided into 3 groups. Among them, Group I contained 5 resources, namely JK4, YJ5, JP1, SY6, and XY6. Except for XY6, according to geographical analysis, the other 4 trees were geographically contiguous and were all in the northeast of Guizhou; Group II contained 2 resources, both collected from Qiannan Prefecture, Guizhou Province, namely GD10 and LB1, located in the southeast of Guizhou; Group III (GLPS12) had a significantly distant genetic relationship with the first two major groups. This sample was collected in Liupanshui, where there were rich wild resources.

[0068] The results of IRAP molecular markers showed that the genetic diversity coefficients of the 8 varieties were between 0.48 and 0.72. Taking 0.66 as the threshold, the 8 resources could be further divided into 3 groups. Among them, Group I contained 5 resources, mainly JK4, GD10, JP1, YJ5, and SY6. Similarly, these 5 trees were geographically contiguous; Group II contained 2 resources, collected from Xingyi City, Qianxinan Prefecture (XY6) and Libo County, Qiannan Prefecture (LB1), both in the south of Guizhou; Group III was GLPS12.

[0069] Based on the clustering results of the above two molecular markers, the genetic relationship of Idesia polycarpa Maxim. was consistent with characteristics such as the geographical distribution of resources and the variety origin. Moreover, it was found that the Idesia polycarpa Maxim. in Liupanshui was different from that in other places in Guizhou. The back of the leaves of Idesia polycarpa Maxim. in other places in Guizhou was smooth, while the back of the leaves of Idesia polycarpa Maxim. in Liupanshui had villi, so its genetic relationship was significantly more distant from the other two major groups. This indicated that the SSR and IRAP primers screened in the present invention had good polymorphism and could be used for the genetic diversity analysis and genetic relationship analysis of Idesia polycarpa Maxim.

[0070] The embodiments described above are only for describing the preferred mode of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solution of the present invention shall 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 is composed of 18 pairs of SSR primer pairs and 30 IRAP primers; the 18 pairs of SSR primer pairs are composed of IpSSR4 whose nucleotide sequence of the forward primer is shown in SEQ ID NO.1 and whose nucleotide sequence of the reverse primer is shown in SEQ ID NO.2, IpSSR6 whose nucleotide sequence of the forward primer is shown in SEQ ID NO.3 and whose nucleotide sequence of the reverse primer is shown in SEQ ID NO.4, IpSSR7 whose nucleotide sequence of the forward primer is shown in SEQ ID NO.5 and whose nucleotide sequence of the reverse primer is shown in SEQ ID NO.6, IpSSR10 whose nucleotide sequence of the forward primer is shown in SEQ ID NO.7 and whose nucleotide sequence of the reverse primer is shown in SEQ ID NO.8, IpSSR11 whose nucleotide sequence of the forward primer is shown in SEQ ID NO.9 and whose nucleotide sequence of the reverse primer is shown in SEQ ID NO.10, IpSSR12 whose nucleotide sequence of the forward primer is shown in SEQ ID NO.11 and whose nucleotide sequence of the reverse primer is shown in SEQ ID NO.12, and IpSSR13 whose nucleotide sequence of the forward primer is shown in SEQ ID NO.

14. NO.13, and the nucleotide sequence of the reverse primer is IpSSR14 shown in SEQ ID NO.14, the nucleotide sequence of the forward primer is IpSSR20 shown in SEQ ID NO.15, and the nucleotide sequence of the reverse primer is IpSSR24 shown in SEQ ID NO.17, and the nucleotide sequence of the reverse primer is IpSSR26 shown in SEQ ID NO.20, the nucleotide sequence of the forward primer is IpSSR27 shown in SEQ ID NO.21, and the nucleotide sequence of the reverse primer is IpSSR28 shown in SEQ ID NO.29, the nucleotide sequence of the forward primer is IpSSR30 shown in SEQ ID NO.31, and the nucleotide sequence of the reverse primer is IpSSR31 shown in SEQ ID NO.32, the nucleotide sequence of the forward primer is IpSSR32 shown in SEQ ID NO.33, and the nucleotide sequence of the reverse primer is IpSSR33 shown in SEQ ID NO.34, the nucleotide sequence of the forward primer is IpSSR35 shown in SEQ ID NO.36, and the nucleotide sequence of the reverse primer is IpSSR36 shown in SEQ ID NO.

37. The nucleotide sequence of the forward primer is shown in SEQ ID NO.29, and 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, and 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.33.33, the nucleotide sequence of the reverse primer is IpSSR50 as shown in SEQ ID NO.34, and the nucleotide sequence of the forward primer is IpSSR52 as shown in SEQ ID NO.35, the nucleotide sequence of the reverse primer is IpSSR52 as shown in SEQ ID NO.36;. 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 schrenkiana to be tested, using the genomic DNA as a template, performing PCR amplification respectively with each pair of SSR primers and each IRAP primer in the Castanopsis schrenkiana primer combination described in claim 1, then performing electrophoresis on the PCR products, and performing kinship analysis on the Castanopsis schrenkiana 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×Taq PCR 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 min; denaturation at 94°C for 30 s, annealing at 56.9-65°C for 30 s, extension at 72°C for 45 s, 35 cycles; and finally extension at 72°C for 5 min.

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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