Combination of SNP molecular markers for identification of Chinese fir germplasm resources and its application

Abstract

The present invention discloses a SNP molecular marker combination for identifying Chinese fir germplasm resources and its application, including: SNP sites at the 501bp position of the sequences shown in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27. The present invention develops 9 SNP molecular markers for identifying Chinese fir germplasm resources, and after amplifying the Chinese fir varieties to be tested using primers that can specifically amplify the 9 SNP molecular markers, sequencing is performed to obtain a unique DNA fingerprint of the Chinese fir varieties to be tested. Therefore, using the SNP molecular markers and amplification primers developed by the present invention, Chinese fir samples can be quickly and efficiently and accurately identified, providing technical support for the protection and utilization of Chinese fir germplasm resources.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular marker-assisted crop genetic breeding, and more specifically, the invention relates to a SNP molecular marker combination and an amplification primer combination for identifying Chinese fir germplasm resources, and a method for identifying Chinese fir germplasm resources. Background Art

[0002] Cunninghamia lanceolata (Lamb.) Hook. is an evergreen coniferous tree species of the genus Cunninghamia in the Cupressaceae family. It is one of the most important native commercial timber and carbon sink tree species in my country. For a long time, the process of seed transfer and mutual introduction between provinces has resulted in different naming and lack of unified and standardized cataloging, which has caused unclear sources of Cunninghamia germplasm resources and chaotic management, bringing great obstacles to Cunninghamia breeding. Therefore, it is urgent to establish an efficient and accurate identification technology system for Cunninghamia germplasm resources.

[0003] DNA molecular markers essentially reflect individual differences and are a highly specific, stable, reliable, accurate and rapid means of identifying germplasm resources. Currently, the commonly used molecular markers for identifying Chinese fir germplasm resources include simple sequence repeats (SSR), sequence-related amplified polymorphism (SRAP), random amplified polymorphic DNA (RAPD), and inter-simple sequence repeat (ISSR). For example, researchers used SSR molecular markers to identify 52 Jiangxi Chinese fir core germplasms, used three pairs of SRAP primer combinations to identify six Chinese fir superior clones, used RAPD markers to effectively distinguish 28 different Chinese fir geographical provenances, and used ISSR molecular markers to successfully distinguish 24 Chinese fir provenances. However, some inherent shortcomings of these molecular markers make them have great limitations in the identification of Chinese fir germplasm resources, such as the poor reproducibility and stability of SRAP, RAPD and ISSR molecular markers. Compared with SRAP, RAPD, ISSR and SSR, single nucleotide polymorphism (SNP), as the third-generation molecular marker, has the advantages of high density and strong stability, and has become an effective means of identifying plant germplasm resources.

[0004] However, there are few reports on the identification of Chinese fir germplasm resources based on SNP marker typing technology. The efficient identification of Chinese fir germplasm resources using SNP loci is of great significance for Chinese fir breeding. Summary of the invention

[0005] Based on this, the purpose of the present invention is to provide a SNP molecular marker combination, amplification primer combination and a method for identifying Chinese fir germplasm resources. Using the 9 SNP molecular markers of the present invention, a fingerprint of Chinese fir germplasm resources can be constructed to identify Chinese fir germplasm resources.

[0006] The specific technical solutions for achieving the above-mentioned invention objectives include the following.

[0007] The first aspect of the present invention provides a SNP molecular marker combination for identifying Chinese fir germplasm resources, including: a SNP site at the 501bp position of the sequence shown in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27.

[0008] The second aspect of the present invention provides a primer combination for amplifying the SNP molecular marker combination for identifying Chinese fir germplasm resources, including: a primer pair with a sequence as shown in SEQ ID NO:1 and SEQ ID NO:2, a primer pair with a sequence as shown in SEQ ID NO:3 and SEQ ID NO:4, a primer pair with a sequence as shown in SEQ ID NO:5 and SEQ ID NO:6, a primer pair with a sequence as shown in SEQ ID NO:7 and SEQ ID NO:8, a primer pair with a sequence as shown in SEQ ID NO:9 and SEQ ID NO:10, a primer pair with a sequence as shown in SEQ ID NO:11 and SEQ ID NO:12, a primer pair with a sequence as shown in SEQ ID NO:13 and SEQ ID NO:14, a primer pair with a sequence as shown in SEQ ID NO:15 and SEQ ID NO:16, and a primer pair with a sequence as shown in SEQ ID NO:17 and SEQ ID NO:18.

[0009] The third aspect of the present invention provides the use of the above primer combination in identifying Chinese fir germplasm resources or constructing a Chinese fir fingerprint.

[0010] The fourth aspect of the present invention provides a method for identifying Chinese fir germplasm resources, comprising the following steps: using the genomic DNA of the Chinese fir sample to be tested as a template, and performing PCR amplification using the above primer combination.

[0011] The present invention has the following beneficial effects:

[0012] In the present invention, 78 core Chinese fir germplasm resources are used as materials to develop 9 SNP molecular markers for identification of Chinese fir germplasm resources. The Chinese fir varieties to be tested are amplified using primers that can specifically amplify the 9 SNP molecular markers, and then sequenced to obtain a unique DNA fingerprint of the Chinese fir varieties to be tested. Therefore, using the SNP molecular markers and amplification primers developed by the present invention, Chinese fir samples can be quickly and efficiently identified accurately, providing technical support for the protection and utilization of Chinese fir germplasm resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The electrophoresis diagram of DNA of 10 Chinese fir samples to be tested in Example 3 of the present invention is shown in FIG.

[0014] Figure 2 This is the electrophoresis diagram of the amplification of the tested Chinese fir sample cx552 using 9 pairs of primers (Pcl_1-Pcl_9) in Example 3 of the present invention, wherein M represents Mark, and lanes 1-9 represent the amplification results of primers Pcl_1-Pcl_9 respectively.

[0015] Figure 3 This is a peak graph of the results of amplifying 10 tested Chinese fir samples using primer Pcl_1 in Example 3 of the present invention. In the graph, the SNP is at the 235 bp position of the sequence.

[0016] Figure 4 This is a peak graph of the results of amplifying 10 tested Chinese fir samples using primer Pcl_2 in Example 3 of the present invention. In the figure, the SNP is at the 235 bp position of the sequence.

[0017] Figure 5 This is a peak graph of the results of amplifying 10 tested Chinese fir samples using primer Pcl_3 in Example 3 of the present invention. In the figure, the SNP is at the 496th bp of the sequence.

[0018] Figure 6 This is a peak graph of the results of amplifying 10 tested Chinese fir samples using primer Pcl_4 in Example 3 of the present invention. In the figure, the SNP is at the 173 bp position of the sequence.

[0019] Figure 7 This is a peak graph of the results of amplifying 10 tested Chinese fir samples using primer Pcl_5 in Example 3 of the present invention. In the figure, the SNP is at the 331 bp position of the sequence.

[0020] Figure 8 This is a peak graph of the results of amplifying 10 tested Chinese fir samples using primer Pcl_6 in Example 3 of the present invention. In the figure, the SNP is at the 318 bp position of the sequence.

[0021] Fig. 9This is a peak graph of the results of amplifying 10 tested Chinese fir samples using primer Pcl_7 in Example 3 of the present invention. In the figure, the SNP is at the 438 bp position of the sequence.

[0022] Fig.10 This is a peak graph of the results of amplifying 10 tested Chinese fir samples using primer Pcl_8 in Example 3 of the present invention. In the figure, the SNP is at the 365 bp position of the sequence.

[0023] Fig.11 This is a peak graph of the results of amplifying 10 tested Chinese fir samples using primer Pcl_9 in Example 3 of the present invention. In the figure, the SNP is at the 414 bp position of the sequence.

[0024] Figure 3 to Figure 11 In the middle, from left to right, from top to bottom, they represent the Chinese fir samples CX63, CX71, CX80, CX231, CX240, CX242, CX245, CX249, CX258, and CX552. DETAILED DESCRIPTION

[0025] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0026] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0027] Unless otherwise specified, the examples are based on conventional experimental conditions, such as Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001, or the conditions recommended by the manufacturer's instructions. The raw materials and reagents used in the present invention are all commercially available, and any biological germplasm material can be provided to the outside for scientific research.

[0028] In the present invention, 78 core Chinese fir germplasm resources are used as materials, and RNA-seq technology is used to develop a SNP molecular marker combination for identifying Chinese fir germplasm resources, which is composed of 9 SNP sites (SNP1 to SNP9) located in the gene coding region. The marker combination forms 78 different DNA fingerprints in the 78 core Chinese fir germplasm resources, that is, each resource has a unique DNA fingerprint composed of these 9 SNP sites. Therefore, the SNP molecular marker combination screened by the present invention can efficiently and accurately distinguish the 78 core Chinese fir germplasm resources. Furthermore, the SNP sites are aligned to the Chinese fir genome using a sequence alignment method to obtain the sequence information of the coding genes where the 9 SNP sites are located, and 9 pairs of efficient and highly specific PCR primers (Pcl_1 to Pcl_9) are designed. These 9 pairs of primers are used to amplify and sequence 10 samples to be tested, and the unique DNA fingerprints of the 10 Chinese fir samples to be tested are successfully obtained. Therefore, the 9 pairs of primers of the present invention can be used to quickly and accurately obtain the DNA fingerprint of the Chinese fir samples to be tested, thereby accurately and efficiently distinguishing the Chinese fir samples to be tested.

[0029] In some embodiments of the present invention, a SNP molecular marker combination for identifying Chinese fir germplasm resources is disclosed, including: the SNP site at the 501bp of the sequence shown in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27.

[0030] In other embodiments of the present invention, a primer combination for amplifying the above-mentioned SNP molecular marker combination for identifying Chinese fir germplasm resources is disclosed, including: a primer pair with sequences as shown in SEQ ID NO:1 and SEQ ID NO:2, a primer pair with sequences as shown in SEQ ID NO:3 and SEQ ID NO:4, a primer pair with sequences as shown in SEQ ID NO:5 and SEQ ID NO:6, a primer pair with sequences as shown in SEQ ID NO:7 and SEQ ID NO:8, a primer pair with sequences as shown in SEQ ID NO:9 and SEQ ID NO:10, a primer pair with sequences as shown in SEQ ID NO:11 and SEQ ID NO:12, a primer pair with sequences as shown in SEQ ID NO:13 and SEQ ID NO:14, a primer pair with sequences as shown in SEQ ID NO:15 and SEQ ID NO:16, and a primer pair with sequences as shown in SEQ ID NO:17 and SEQ ID NO:18.

[0031] In other embodiments of the present invention, the application of the above primer combination in identifying Chinese fir germplasm resources is disclosed.

[0032] In other embodiments of the present invention, a kit for identifying Chinese fir germplasm resources is disclosed, which comprises the above primer combination.

[0033] In other embodiments of the present invention, the application of the above primer combination in constructing a fingerprint map of Chinese fir is disclosed.

[0034] In other embodiments of the present invention, a kit for constructing a fingerprint of Chinese fir is disclosed, which comprises the above primer combination.

[0035] In other embodiments of the present invention, a method for identifying Chinese fir germplasm resources is disclosed, comprising the following steps: using the genomic DNA of the Chinese fir sample to be tested as a template, and performing PCR amplification using the above primer combination.

[0036] In some of the embodiments, the method further comprises sequencing the PCR amplification product to obtain a DNA fingerprint.

[0037] In some embodiments, the PCR amplification reaction system includes: 12.5±0.5 μL of PCR Mix, 1±0.1 μL of DNA template, 1±0.1 μL of upstream and downstream primers, and 25 μL of ddH2O.

[0038] In some of the embodiments, the reaction procedure of the PCR amplification is: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s; annealing at 57°C for 30 s; extension at 72°C for 60 s-150 s; and extension at 72°C for 5 min.

[0039] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0040] Example 1 Obtaining SNP molecular markers for identifying Chinese fir germplasm resources

[0041] The following steps are involved:

[0042] 1. Extraction and detection of RNA from Chinese fir core germplasm

[0043] The core germplasm population of the third generation breeding population of Guangdong fir (78 samples) was used as the object, covering the main producing areas of fir including Guangdong, Fujian, Guangxi, Hunan, Jiangxi, Guizhou and other provinces (Table 1). The healthy branches of the current year were collected, and the RNA was extracted using an RNA extraction kit. Then, the RNA concentration and purity were preliminarily tested using Nanodro2000, and the RNA integrity and purity were tested and the concentration was quantified using Agilent4200Bioanalyzer. The concentration and purity of all extracted RNA samples met the library construction standards (Table 1), that is, the concentration ≥40ng / ul, the total amount ≥1ug, and the RIN ≥6.0.

[0044] Table 1 Information of Chinese fir samples and RNA concentration and integrity

[0045]

[0046]

[0047] Note: RIN stands for RNA Integrity Number (RIN) counted by the Agilent 4200 detector, which can accurately and intuitively evaluate the quality of RNA samples and screen out high-quality samples.

[0048] 2. RNA library construction and high-throughput sequencing

[0049] RNA with qualified quality enters the library construction process. Ultra TM RNALibrary Prep Kit for (NEB, USA) kit was used for construction. The process is as follows: eukaryotic mRNA is enriched using magnetic beads with Oligo (dT). Under the action of high temperature and metal ions, RNA is fragmented, and the first cDNA chain is synthesized using random hexamers, followed by the addition of enzymes, buffers, and dNTPs (dATP, dTTP, dGTP, dCTP) to synthesize the second cDNA chain. The synthesized double-stranded cDNA is purified by magnetic beads, then repaired and A-added, connected to sequencing adapters, and sorted by magnetic beads for fragment size sorting. The sorted fragments are enriched by PCR, and finally the PCR products are purified to obtain the final library.

[0050] Illumina novaseq sequencing platform was used for sequencing. The sequencing strategy was double-end 150bp sequencing. The sequencing volume was no less than 6Gb per sample. The sequencing quality requirements were Q20 no less than 90 and Q30 no less than 85. The original sequence was subjected to data quality control using Fastp (version 0.20.1) software to obtain high-quality Clean Reads. The parameter settings were: -l 150-q 20, and the rest were default parameters. The sample Clean Reads output and quality statistics are shown in Table 2.

[0051] Table 2 Statistics of sequencing data of Chinese fir samples

[0052]

[0053]

[0054]

[0055] Note: GC ratio (%): the ratio of G bases and C bases to the total number of bases; Q20: the ratio of bases with quality values ​​greater than 20 (error rate less than 1%) to the total number of bases; Q30: the ratio of bases with quality values ​​greater than 30 (error rate less than 0.1%) to the total number of bases.

[0056] 3. Development of SNP loci and screening of SNP molecular markers for identifying Chinese fir germplasm resources

[0057] The samtools (version 1.14) sort function was used to sort the sequences. The deepvariant (version 1.3.0, website: https: / / github.com / google / deepvariant) software was used for SNP detection, and the RNA model was used for detection. The sequencing data were aligned to the annotated Chinese fir genome (NCBI data ID: GCA_027924645.1, website: https: / / www.ncbi.nlm.nih.gov / data-hub / genome / GCA_027924645.1 / ) using STAR (version 2.7.10b) software. The SNP genotype, base sequencing quality, and read alignment quality were reviewed using samtools mpileup and python programs to comprehensively determine the SNP polymorphic sites. The following criteria were used for further SNP site filtering: non-biallelic sites were screened out; sites with a minimum allele frequency (MAF) < 0.05 were screened out; sites with a missing rate greater than 20% were screened out; a total of 207,859 SNP sites were obtained.

[0058] Principles for screening SNP molecular markers: (1) Select SNP sites in the functional region of the gene; (2) Select SNP sites with only two alleles; (3) The alleles of the SNP site include three genotypes: AA, Aa, and aa; (4) The minimum allele frequency (MAF) of the SNP site is greater than or equal to 0.05; (5) Ensure that the SNP fingerprint of each sample is unique; (6) There are no other SNP sites within 300bp upstream and downstream of the SNP site to avoid interference with primer specificity; (7) There are flanking sequences within 300bp upstream and downstream of the SNP site that can be used to design specific primers, and the amplification band is specific; (8) The GC ratio of the sequence segment within 300bp upstream and downstream of the SNP site is between 30% and 50%, ensuring that the sequence where the SNP site is located is easy to amplify; (9) The peak diagram of conventional sequencing is clear and easy to judge at the location of the SNP site. Since each SNP site may have three genotypes: AA, Aa, and aa, the discrimination of a single SNP site is 3. The relationship between the number of SNP sites n and the maximum number of samples that can be distinguished by molecular map design is: the number of distinguishable samples = 3 n (According to the above formula, the SNP marker combination of the present invention is expected to distinguish up to 19683 different samples.) Finally, 9 SNP sites (SNP1 to SNP9) located in the gene coding region were screened out from 207,859 SNP sites, and 78 core Chinese fir germplasm resources formed a unique DNA fingerprint composed of these 9 sites (Table 3). It can be seen that these 9 SNP sites can be used as efficient and accurate methods to distinguish 78 core Chinese fir germplasm resources.

[0059] SNP molecular markers of Chinese fir core germplasm resources.

[0060] Table 3 DNA fingerprints of 78 Chinese fir samples based on 9 SNP loci

[0061]

[0062]

[0063] Note: R, Y, M, S, and W in the table are degenerate bases, and their corresponding bases are A / G, C / T, A / C, G / C, and A / T respectively.

[0064] 4. Align these 9 SNP sites to the Cunninghamia lanceolata genome to obtain the corresponding gene reference sequences (Table 4) for subsequent amplification primer design.

[0065] Table 4 Gene sequence information of SNP sites

[0066]

[0067]

[0068]

[0069]

[0070] Note: The bases in bold capital letters in the sequence are the target SNPs.

[0071] Example 2 Amplification primers for identifying SNP molecular markers of Chinese fir germplasm resources

[0072] The following steps are involved:

[0073] For the 9 selected SNP sites, the gene sequence where the SNP is located was used as a template (sequence information is shown in Table 4), and Primer3 (https: / / primer3.org / ) was used to design SNP primers. The primer design principles include: (1) primer length 20-24 bp; (2) primer amplification length 600-800 bp; (3) the primer amplification band is required to be single and clear, without non-specific amplification. Finally, 9 pairs of optimal primers (primer 1 to primer 9) were obtained. The primer information is shown in Table 5, and the amplified target sequence information is shown in Table 6.

[0074] Table 5 PCR primer information for amplifying 9 SNP sites

[0075]

[0076] Table 6 Information of sequences amplified by 9 pairs of primers

[0077]

[0078]

[0079]

[0080]

[0081] Note: The bases in bold capital letters in the sequence are the target SNPs.

[0082] Example 3 Identification of different germplasm resources of Chinese fir using the SNP molecular markers of the present invention

[0083] In this example, 10 Chinese fir provenances (cx63, cx71, cx80, cx231, cx240, cx242, cx245, cx249, cx258, and cx552) from Guangdong, Guizhou, and other provinces were used as test materials, and the SNP molecular markers of Example 1 were used to identify Chinese fir germplasm resources, including the following steps:

[0084] 1. DNA extraction and PCR amplification of test samples

[0085] Refer to the DNAsecure Plant Kit (TIANGEN) instructions for extracting DNA from the sample to be tested (DNA extraction results are shown in Figure 1 The concentration (ng / μL) and purity (OD 260 / OD 280 ), and run electrophoresis to check the quality before use. The specific steps are as follows: (1) Take 100 mg of fir needles or 20 mg of dry weight tissue, add liquid nitrogen and grind thoroughly. Add 400 μL buffer LP1 and 6 μL RNase A (10 mg / ml), vortex for 1 minute, and leave at room temperature for 10 minutes; (2) Add 130 μL buffer LP2, mix thoroughly, and vortex for 1 minute; (3) Centrifuge at 12,000rpm (~13,400xg) for 5 minutes, and transfer the supernatant to a new centrifuge tube; (4) Add 1.5 times the volume of buffer LP3 (for example, 500 μL supernatant plus 750 μL buffer LP3) (please check whether anhydrous ethanol has been added before use), immediately vortex and mix thoroughly for 15 seconds, then you can A flocculent precipitate may appear; (5) Add the solution and flocculent precipitate obtained in the previous step to an adsorption column CB3 (the adsorption column is placed in a collection tube), centrifuge at 12,000 rpm (~13,400 x g) for 30 seconds, discard the waste liquid, and place the adsorption column CB3 in a collection tube; (6) Add 600 μL of rinse solution PW to the adsorption column CB3 (please check whether anhydrous ethanol has been added before use), centrifuge at 12,000 rpm (~13,400 x g) for 30 seconds, discard the waste liquid, and place the adsorption column CB3 in a collection tube. Note: If the adsorption column membrane appears green, add 500 μL of anhydrous ethanol to the adsorption column CB3, centrifuge at 12,000 rpm (~13,400 x g) for 30 seconds, discard the waste liquid, and place the adsorption column CB3 in the collection tube; (7) Repeat step 6; (8) Place the adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm (~13,400 x g) for 2 minutes, and discard the waste liquid. Leave the adsorption column CB3 at room temperature for several minutes to completely dry the adsorption material. (9) Transfer the adsorption column CB3 to a clean centrifuge tube, add 50-200 μL of elution buffer TE to the middle part of the adsorption membrane, place at room temperature for 2-5 minutes, centrifuge at 12,000 rpm (~13,400 x g) for 2 minutes, and collect the solution into a centrifuge tube; (10) DNA concentration and purity detection: The recovered DNA fragments can be tested for concentration and purity by agarose gel electrophoresis and UV spectrophotometer. DNA should be at OD 260 There is a significant absorption peak at OD 260 A value of 1 is equivalent to approximately 50 μg / ml double-stranded DNA and 40 μg / ml single-stranded DNA. 260 / OD 280The ratio should be 1.7-1.9. If elution buffer is not used but ddH2O is used for elution, the ratio will be lower because the pH value and the presence of ions will affect the light absorption value, but it does not mean that the purity is low.

[0086] The DNA of the sample to be tested was used as a template and amplified using the 9 pairs of primers (Pcl_1-Pcl_9) in Table 5. A 25 μL reaction system was used for amplification, including 12.5 μL of PCRMix (containing dye), 1 μL of the sample DNA template to be tested, 1 μL of each of the upstream and downstream primers of SNP, and 9.5 μL of ddH2O. The PCR amplification procedure was as follows: (1) pre-denaturation at 94°C for 3 min; (2) denaturation at 94°C for 30 s; (3) annealing at 57°C for 30 s; (4) extension at 72°C for 60 s-150 s; (5) extension at 72°C for 5 min. Steps (2), (3), and (4) were repeated for 30 cycles and stored at 4°C. The SNP-PCR reaction product was electrophoresed using 1% agarose gel with a buffer of 1×TAE. Figure 2 The electrophoresis diagram of the Chinese fir sample cx552 to be tested was shown using 9 pairs of primers to amplify a clear specific band, and the size of the amplified band was consistent with the designed fragment size.

[0087] The PCR products were sent for sequencing, and the sequencing results were compared with the reference sequences (SEQ ID NO: 28 to SEQ ID NO: 36) to determine the location of the SNP site. Subsequently, the peak map software CodonCode Aligner was used to view the peak map results of the SNP location, and finally the SNP base information of the 10 tested Chinese fir samples was obtained to form its DNA fingerprint map.

[0088] The results are shown in Table 7 and Figure 3 to Figure 11 shown.

[0089] Table 7 DNA fingerprints of the 10 Chinese fir samples to be tested

[0090] sample SNP1 SNP2 SNP3 SNP4 SNP5 SNP6 SNP7 SNP8 SNP9 cx63 G A T C A T C C Y cx71 R A T G R W C Y Y cx80 R G T C A A M C Y cx231 G R T G A T M C T cx240 A R Y G G W M C T cx242 R A Y C R W A C T cx245 A G T S A T C Y T cx249 A R T C A T M C T cx258 R A Y G A A M C T cx552 G R T G A A A Y Y

[0091] Note: A, T, C and G indicate a single peak during sequencing, R, Y, M, S, W indicate a superimposed double peak during sequencing, where the bases corresponding to R, Y, M, S, W are A / G, C / T, A / C, G / C, and A / T, respectively.

[0092] The results showed that all 10 samples had unique fingerprints, indicating that the SNP molecular markers of the present invention can effectively distinguish the 10 samples to be tested.

[0093] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0094] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A SNP molecular marker combination for identifying Chinese fir germplasm resources, characterized in that: include: Sequences as shown in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27, wherein the 501 bp position of the sequence is a SNP site.

2. A primer combination for amplifying the SNP molecular marker combination for identifying Chinese fir germplasm resources according to claim 1, characterized in that: include: The primer pair whose sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2, The primer pair whose sequences are shown in SEQ ID NO:3 and SEQ ID NO:4, The primer pair whose sequences are shown in SEQ ID NO:5 and SEQ ID NO:6, The primer pair whose sequences are shown in SEQ ID NO:7 and SEQ ID NO:8, The primer pair whose sequences are shown in SEQ ID NO:9 and SEQ ID NO:10, The primer pair whose sequences are shown in SEQ ID NO: 11 and SEQ ID NO: 12, The primer pair whose sequences are shown in SEQ ID NO: 13 and SEQ ID NO: 14, The primer pair whose sequences are shown in SEQ ID NO: 15 and SEQ ID NO: 16, and The primer pair has sequences shown as SEQ ID NO:17 and SEQ ID NO:

18.

3. Use of the primer combination according to claim 2 in identifying Chinese fir germplasm resources.

4. A kit for identifying Chinese fir germplasm resources, characterized in that: It comprises the primer combination described in claim 2.

5. Use of the primer combination described in claim 2 in constructing a fingerprint map of Chinese fir.

6. A kit for constructing a fingerprint of Chinese fir, characterized in that: It comprises the primer combination described in claim 2.

7. A method for identifying Chinese fir germplasm resources, characterized in that: The method comprises the following steps: using the genome DNA of the Chinese fir sample to be tested as a template and using the primer combination of claim 2 to perform PCR amplification.

8. The method for identifying Chinese fir germplasm resources according to claim 7, characterized in that: The method also includes the step of sequencing the PCR amplification product to obtain a DNA fingerprint map.

9. The method for identifying Chinese fir germplasm resources according to claim 7 or 8, characterized in that: The reaction system of the PCR amplification includes: 12.5±0.5 μL of PCR Mix, 1±0.1 μL of DNA template, 1±0.1 μL of upstream and downstream primers, and 25 μL of ddH2O.

10. The method for identifying Chinese fir germplasm resources according to claim 7 or 8, characterized in that: The reaction procedure of the PCR amplification is: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s; annealing at 57°C for 30 s; extension at 72°C for 60 s-150 s; and extension at 72°C for 5 min.

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