Chloroplast SNP molecular marker of Cunninghamia lanceolata and application thereof

Abstract

The application discloses a Chinese fir chloroplast SNP molecular marker and application thereof. The application develops a Chinese fir chloroplast SNP molecular marker combination for identifying Chinese fir individuals, and the combination comprises 60 Chinese fir chloroplast SNP sites. After primers capable of specifically amplifying the chloroplast SNP molecular marker combination are used to amplify a Chinese fir variety to be tested, sequencing is performed, and a SNP sequence characteristic of the Chinese fir variety to be tested is obtained. Therefore, by using the SNP molecular marker and the amplification primer thereof developed by the application, Chinese fir samples can be accurately and efficiently identified, and technical support is provided for high-quality Chinese fir individual resource protection and utilization.

Description

Technical Field

[0001] This invention relates to the field of plant molecular markers, and particularly to SNP molecular markers in cypress chloroplasts and their applications. Background Technology

[0002] Chinese fir (Cunninghamia lanceolata) is an evergreen coniferous tree species belonging to the genus Cunninghamia in the family Cupressaceae. It is one of the most important commercial timber and carbon sink species. For a long time, the allocation and mutual introduction of Chinese fir seeds have been characterized by individual naming practices and a lack of unified and standardized cataloging. This has resulted in unclear varietal origins and chaotic management of Chinese fir, which has greatly hindered Chinese fir breeding efforts. Therefore, it is urgent to establish an efficient and accurate individual identification technology system for Chinese fir within the same species.

[0003] DNA molecular markers, by their very nature, reflect individual differences and are a stable and reliable means of identifying individuals. Currently, commonly used molecular markers for identifying Chinese fir include simple repeat sequence markers, related sequence amplification polymorphism (RAPD) markers, random amplification polymorphism (RAPD) DNA markers, and simple repeat sequence interregion markers. However, these molecular markers have inherent drawbacks that limit their application in identifying Chinese fir germplasm resources; for example, SRAP, RAPD, and ISSR molecular markers exhibit poor reproducibility and stability. Compared to SRAP, RAPD, ISSR, and SSR, single nucleotide polymorphism (SNP), as a third-generation molecular marker, has advantages such as high density and strong stability, and has become an effective means of identifying individual plants.

[0004] However, there are few reports on the identification of Chinese fir germplasm resources based on chloroplast SNP marker typing technology. Utilizing chloroplast SNP loci for efficient identification of individual Chinese fir individuals is of great significance for Chinese fir breeding. Summary of the Invention

[0005] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a combination of SNP molecular markers for cypress chloroplasts.

[0006] The second objective of this invention is to provide primer combinations for amplifying the above-mentioned SNP molecular marker combinations in cypress chloroplasts;

[0007] The third objective of this invention is to provide the application of the above-mentioned combination of SNP molecular markers for chloroplasts of Cunninghamia lanceolata or the primer combination for amplifying the above-mentioned combination of SNP molecular markers for chloroplasts of Cunninghamia lanceolata in the identification of individual Cunninghamia lanceolata.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A molecular marker array of SNPs in Chinese fir chloroplasts for identifying individual Chinese fir trees, comprising 60 Chinese fir chloroplast SNP sites, is available in GenBank. ID:NC_021437.1 is the reference sequence for chloroplasts of *Cunninghamia lanceolata*. The 60 SNP sites are: 1341, 4865, 7726, 12335, 14207, 15429, 19699, 20673, 21821, 21962, 23183, 23533, 24087, 26116, 28929, 31064, 37419, 41947, 43696, 52890, 52891, 53049, 55698, 56636, 63572, 68775, 69253, 70883, 75... 549, 77989, 78958, 79416, 79493, 84262, 86464, 87557, 87900, 89547, 90014, 90759, 92672, 94179, 94448, 94614, 94625, 94708, 94823, 95022, 97731, 102900, 113243, 117311, 118850, 119384, 122865, 122937, 123103, 131298, 131852 and 134204 bases.

[0010] A primer set for amplifying the above-mentioned SNP molecular marker combination of cypress chloroplasts includes primers as shown in SEQ ID NO:1-57.

[0011] A kit for identifying individual Chinese fir trees, comprising the primer combination described above.

[0012] Application of the above-mentioned SNP molecular marker combination of chloroplasts in the identification of individual Chinese fir trees.

[0013] Furthermore, the application includes the following steps:

[0014] The bases of 60 SNP sites are identified, and the bases of each SNP site are compared to determine whether they belong to the same population. If the bases of the SNP sites are completely identical, they are considered to belong to the same population. If the bases of the SNP sites are different, they are considered not to belong to the same population.

[0015] The application of the primer combinations for amplifying the above-mentioned SNP molecular marker combinations of Cunninghamia lanceolata chloroplasts or the above-mentioned kits in the identification of individual Cunninghamia lanceolata.

[0016] Furthermore, the application includes the following steps:

[0017] The bases of 60 SNP sites were determined by amplification and sequencing using the above primer combinations. If the bases of the SNP sites are completely identical, they are considered to be from the same population. If the bases of the SNP sites are different, they are considered to be from different populations.

[0018] The present invention has the following advantages and effects compared with the prior art:

[0019] This invention utilizes chloroplast SNPs to accurately and efficiently identify Chinese fir samples through 60 SNP loci, providing technical support for the protection and utilization of high-quality Chinese fir individual resources. Attached Figure Description

[0020] Figure 1 It is a phylogenetic tree of SNP sequences from 87 samples. Detailed Implementation

[0021] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0022] Example 1

[0023] 1. DNA extraction and sample testing

[0024] Eighty-seven verified samples were collected (34 samples were used to confirm SNP loci, and the rest were used as verification samples, among which CX569 and CX837 were reported to be samples of the same parent, and CX574 and CX868 were also reported to be samples of the same parent). The samples came from 87 parental clones in the third round of genetic improvement breeding population of Cunninghamia lanceolata. The eighty-seven samples have been published in “Zheng, Huiquan, et al. Chinese fir breeding in the high-throughput sequencingera: Insights from SNPs. Forests 10.8(2019):681.” and “Huang Rong, Hu Dehuo, Deng Houyin, et al. Analysis of genetic diversity and structure of fast-growing high-quality Cunninghamia lanceolata population using SNP markers[J]. Molecular Plant Breeding, 2025(1)”. DNA extraction process: about 100 mg of fresh tissue was placed in a mortar and ground with liquid nitrogen. The ground tissue was then transferred to a 1.5 mL centrifuge tube for genomic DNA extraction. Genomic DNA was extracted using a universal column-based genomic DNA extraction kit (Universal Genomic DNA Kit). The kit manufacturer was CoWin Biosciences (China), and the kit model was CW2298. After extraction, the genomic DNA was stored at -20°C. Genomic DNA analysis included the following three methods: 1) 0.8% agarose gel electrophoresis to detect DNA degradation and impurities and estimate DNA concentration; 2) Nanodrop spectrophotometer (ThermoFisher Scientific, USA) to determine sample concentration and purity; 3) ... Sample concentration was determined using a 2.0 Flurometer (Life Technologies, USA). For sample concentrations ≥40 ng / μL, OD... 260 / OD 280 =1.8~2.0, OD 260 / OD 230 A DNA extraction sample with a value of 1.6 to 2.0 is considered acceptable.

[0025] 2. Library Construction

[0026] Samples that passed the initial testing were processed for genomic DNA fragmentation using NEBNext DNA double-strand fragmentation enzyme (NEBNext dsDNAFragmentase; NEB, #M0348), and libraries were constructed using the VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme, #ND607). Specific experimental procedures were performed according to the kit instructions. After library construction, quality control was performed using qPCR and an Agilent 2100 Bioanalyzer (Agilent Technologies, USA). Quality-controlled DNA libraries were then sequenced using an Illumina Novaseq 6000 (Illumina, USA) high-throughput sequencing platform with a PE150 (Pair-End 150) sequencing strategy.

[0027] 3. Sequencing data statistics and quality control

[0028] The original sequences were processed using Fastp v0.20.1 software for data quality control to obtain high-quality Clean Reads. Parameter settings: -l 150 -q 20, with the rest set to default. All subsequent analyses were performed based on the Clean Reads.

[0029] Bowtie2 v2.4.2 was used to align the quality-controlled sequencing reads with the reference sequence of Cunninghamia lanceolata chloroplasts (GenBank ID: NC_021437.1). Chloroplast genome sequencing sequences were isolated from the total DNA sequencing data. The captured chloroplast genome sequencing sequences were then statistically analyzed and quality-controlled. The results are shown in Table 1.

[0030] Table 1. Statistical results of chloroplast genome data.

[0031]

[0032]

[0033]

[0034]

[0035]

[0036] Note:

[0037] Q20: The percentage of bases with a quality value greater than 20 (error rate less than 1%) out of the total number of bases;

[0038] Q30: The percentage of bases with a quality value greater than 30 (error rate less than 0.1%) out of the total number of bases;

[0039] GC%: The percentage of G and C bases in the total number of bases.

[0040] 4. Sequence alignment and SNP detection

[0041] Bowtie2 v2.4.2 alignment software was used to align 34 sequencing data points with the chloroplast reference genome (samples: CX67, CX70, CX71, CX73, CX82, CX190, CX200, CX520, CX522, CX523, CX525, CX537, CX541, CX545, CX549, CX561, CX567, CX569, CX571, CX574, CX619, CX652, CX708, CX735, CX770, CX805, CX822, CX830, CX841, CX844, CX845, CX846, CX848, CX861). SAMtools (1.14) software was used to sort the alignment results using the `sort` parameter to obtain the aligned BAM files. To improve alignment accuracy and reduce SNP detection errors caused by alignment mistakes, the SAMtools (1.14) view program with the -bF 12 parameter was used to screen paired-end aligned sequencing reads for subsequent analysis. Based on the alignment results, Deepvariant (version 1.3.0, https: / / github.com / google / deepvariant) software was used for SNP detection. SAMtools mpileup and Python programs were used to verify SNP genotypes, base sequencing quality, and read alignment quality, comprehensively determining SNP polymorphic sites. Based on the SNP location, annotation information from the corresponding position in the chloroplast reference genome was retrieved to obtain SNP annotation results. The SNP locations are as follows:

[0042] SNP1, located at base 1341 of the reference sequence of *Cunninghamia lanceolata* chloroplasts, is located in IGS; SNP2, located at base 4865 of the reference sequence of *Cunninghamia lanceolata* chloroplasts, is located in rps16; SNP3, located at base 7726 of the reference sequence of *Cunninghamia lanceolata* chloroplasts, is located in IGS; SNP4, located at base 12335 of the reference sequence of *Cunninghamia lanceolata* chloroplasts, is located in IGS; SNP5, located at base 14207 of the reference sequence of *Cunninghamia lanceolata* chloroplasts, is located in psaA; SNP6, located at base 15429 of the reference sequence of *Cunninghamia lanceolata* chloroplasts, is located in psaB; SNP7, located at base 19699 of the reference sequence of *Cunninghamia lanceolata* chloroplasts, is located in psbC; SNP8, located at base 20673 of the reference sequence of *Cunninghamia lanceolata* chloroplasts, is located in ps... bD; SNP9, located at base 21821 of the reference sequence of *Cunninghamia lanceolata* chloroplasts, is located in IGS; SNP10, located at base 21962 of the reference sequence of *Cunninghamia lanceolata* chloroplasts, is located in IGS; SNP11, located at base 23183 of the reference sequence of *Cunninghamia lanceolata* chloroplasts, is located in IGS; SNP12, located at base 23533 of the reference sequence of *Cunninghamia lanceolata* chloroplasts, is located in IGS; SNP13, located at base 24087 of the reference sequence of *Cunninghamia lanceolata* chloroplasts, is located in IGS; SNP14, located at base 26116 of the reference sequence of *Cunninghamia lanceolata* chloroplasts, is located in IGS; SNP15, located at base 28929 of the reference sequence of *Cunninghamia lanceolata* chloroplasts, is located in rpoB; SNP16, located at base 3106 of the reference sequence of *Cunninghamia lanceolata* chloroplasts. SNP17, located at base 37419 of the reference sequence for *Cunninghamia lanceolata* chloroplasts, is located at rps2; SNP18, located at base 41947 of the reference sequence for *Cunninghamia lanceolata* chloroplasts, is located at atpA; SNP19, located at base 43696 of the reference sequence for *Cunninghamia lanceolata* chloroplasts, is located at trnG; SNP20, located at base 52890 of the reference sequence for *Cunninghamia lanceolata* chloroplasts, is located at IGS; SNP21, located at base 52891 of the reference sequence for *Cunninghamia lanceolata* chloroplasts, is located at IGS; SNP22, located at base 53049 of the reference sequence for *Cunninghamia lanceolata* chloroplasts, is located at IGS; SNP23, located at base 55698 of the reference sequence for *Cunninghamia lanceolata* chloroplasts, is located at IGS; SNP24, located at base 55698 of the reference sequence for *Cunninghamia lanceolata* chloroplasts, is located at IGS; SNP24, located at base 55698 of the reference sequence for *Cunninghamia lanceolata* chloroplasts, is located at IGS; SNP25, located at ycf2, is the 56636th base of the chloroplast reference sequence; SNP26, located at 68775th base of the chloroplast reference sequence; SNP27, located at 69253rd base of the chloroplast reference sequence; SNP28, located at rpl2, is the 70883rd base of the chloroplast reference sequence; SNP29, located at 75549th base of the chloroplast reference sequence; SNP30, located at 77989th base of the chloroplast reference sequence; and SNP31, located at petD, is the 78958th base of the chloroplast reference sequence.SNP32, located at base 79416 of the reference sequence for chloroplasts of *Cunninghamia lanceolata*, is located in IGS; SNP33, located at base 79493 of the reference sequence for chloroplasts of *Cunninghamia lanceolata*, is located in IGS; SNP34, located at base 84262 of the reference sequence for chloroplasts of *Cunninghamia lanceolata*, is located in IGS; SNP35, located at base 86464 of the reference sequence for chloroplasts of *Cunninghamia lanceolata*, is located in IGS; SNP36, located at base 87557 of the reference sequence for chloroplasts of *Cunninghamia lanceolata*, is located in IGS; SNP37, located at base 87900 of the reference sequence for chloroplasts of *Cunninghamia lanceolata*, is located in IGS; SNP38, located at base 89547 of the reference sequence for chloroplasts of *Cunninghamia lanceolata*, is located in IGS; SNP39, located in ...0, located in the reference sequence for chloroplasts of *Cunninghamia lanceolata*, is located in IGS; SNP31, located in the reference sequence for chloroplasts of *Cunninghamia lanceolata*, is located in IGS; SNP32, located at base 79493 of the reference sequence for chloroplasts of *Cunninghamia lanceolata SNP40, located at base 90014 of the chloroplast reference sequence, is located in IGS; SNP40, located at base 90759 of the chloroplast reference sequence, is located in IGS; SNP41, located at base 92672 of the chloroplast reference sequence, is located in IGS; SNP42, located at base 94179 of the chloroplast reference sequence, is located in IGS; SNP43, located at base 94448 of the chloroplast reference sequence, is located in IGS; SNP44, located at base 94614 of the chloroplast reference sequence, is located in IGS; SNP45, located at base 94625 of the chloroplast reference sequence, is located in IGS; SNP46, located at base 94708 of the chloroplast reference sequence, is located in IGS. SNP47, located at base 94823 of the reference sequence for chloroplasts of *Cunninghamia lanceolata*, is located in IGS; SNP48, located at base 95022 of the reference sequence for chloroplasts of *Cunninghamia lanceolata*, is located in IGS; SNP49, located at base 97731 of the reference sequence for chloroplasts of *Cunninghamia lanceolata*, is located in ycf1; SNP50, located at base 102900 of the reference sequence for chloroplasts of *Cunninghamia lanceolata*, is located in IGS; SNP51, located at base 113243 of the reference sequence for chloroplasts of *Cunninghamia lanceolata*, is located in IGS; SNP52, located at base 117311 of the reference sequence for chloroplasts of *Cunninghamia lanceolata*, is located in trnL; SNP53, located at base 118850 of the reference sequence for chloroplasts of *Cunninghamia lanceolata*, is located in I... GS; SNP54, located at base 119384 of the reference sequence for chloroplasts of *Cunninghamia lanceolata*, is located in IGS; SNP55, located at base 122865 of the reference sequence for chloroplasts of *Cunninghamia lanceolata*, is located in IGS; SNP56, located at base 122937 of the reference sequence for chloroplasts of *Cunninghamia lanceolata*, is located in IGS; SNP57, located at base 123103 of the reference sequence for chloroplasts of *Cunninghamia lanceolata*, is located in rbcL; SNP58, located at base 131298 of the reference sequence for chloroplasts of *Cunninghamia lanceolata*, is located in IGS; SNP59, located at base 131852 of the reference sequence for chloroplasts of *Cunninghamia lanceolata*, is located in chlN; SNP60, located at base 134204 of the reference sequence for chloroplasts of *Cunninghamia lanceolata*, is located in IGS.

[0043] The SNP site characteristics of the 34 samples are as follows:

[0044] In sample CX67, SNP20 is T, SNP21 is C, and SNP36 is G; the bases at the remaining SNP sites are consistent with the reference sequence.

[0045] In sample CX70, SNP16 and SNP31 are T; the bases at the remaining SNP sites are consistent with the reference sequence.

[0046] In sample CX71, SNP20 is T, SNP21 is C, and SNP55 is A; the bases at the remaining SNP sites are consistent with the reference sequence.

[0047] In sample CX73, SNP6 is A, SNP12 is A, SNP24 is T, SNP37 is C, SNP38 is A, SNP40 is A, SNP49 is C, and SNP51 is T; the bases of the remaining SNP sites are consistent with the reference sequence.

[0048] In sample CX82, SNP44 is C; the bases at the remaining SNP sites are consistent with the reference sequence.

[0049] In sample CX190, SNP8 is T, SNP44 is C, and SNP46 is C; the bases at the remaining SNP sites are consistent with the reference sequence.

[0050] In sample CX200, SNP29 is T; the bases at the remaining SNP sites are consistent with the reference sequence.

[0051] In sample CX520, SNP20 is T, SNP21 is C, and SNP56 is T; the bases at the remaining SNP sites are consistent with the reference sequence.

[0052] In sample CX522, SNP34 is T and SNP44 is C; the bases at the remaining SNP sites are consistent with the reference sequence.

[0053] In sample CX523, SNP13 is T, SNP42 is G, and SNP60 is T; the bases at the remaining SNP sites are consistent with the reference sequence.

[0054] In sample CX525, SNP49 is C and SNP57 is T; the bases at the remaining SNP sites are consistent with the reference sequence.

[0055] In sample CX537, SNP25 is G; the bases at the remaining SNP sites are consistent with the reference sequence.

[0056] In sample CX541, SNP35 is A; the bases at the remaining SNP sites are consistent with the reference sequence.

[0057] Sample CX545 has SNP19 as C; the bases at the remaining SNP sites are consistent with the reference sequence.

[0058] In sample CX549, SNP23 is T, SNP27 is T, and SNP41 is A; the bases at the remaining SNP sites are consistent with the reference sequence.

[0059] In sample CX561, SNP21 is C; the bases at the remaining SNP sites are consistent with the reference sequence.

[0060] Sample CX567 has SNP1 as A; the bases at the remaining SNP sites are consistent with the reference sequence.

[0061] In sample CX569, the bases at the SNP sites are identical to those in the reference sequence.

[0062] In sample CX571, SNP26 is G, SNP44 is C, and SNP46 is C; the bases at the remaining SNP sites are consistent with the reference sequence.

[0063] In sample CX574, SNP20 is T and SNP21 is C; the bases at the remaining SNP sites are consistent with the reference sequence.

[0064] In sample CX619, SNP10 is C and SNP44 is C; the bases at the remaining SNP sites are consistent with the reference sequence.

[0065] In sample CX652, SNP7 is T, SNP44 is C, SNP45 is A, and SNP47 is A; the bases at the remaining SNP sites are consistent with the reference sequence.

[0066] In sample CX708, SNP2 is C and SNP31 is T; the bases at the remaining SNP sites are consistent with the reference sequence.

[0067] Sample CX735 has SNP3 as A; the bases at the remaining SNP sites are consistent with the reference sequence.

[0068] In sample CX770, SNP33 is T, SNP43 is T, SNP58 is T, and SNP59 is A; the bases of the remaining SNP sites are consistent with the reference sequence.

[0069] In sample CX805, SNP4 is A, SNP5 is A, SNP20 is T, SNP21 is C, SNP42 is G, and SNP60 is T; the bases of the remaining SNP sites are consistent with the reference sequence.

[0070] In sample CX822, SNP48 is G; the bases at the remaining SNP sites are consistent with the reference sequence.

[0071] In sample CX830, SNP14 is T and SNP15 is A; the bases at the remaining SNP sites are consistent with the reference sequence.

[0072] In sample CX841, SNP28 is T, SNP44 is C, and SNP46 is C; the bases at the remaining SNP sites are consistent with the reference sequence.

[0073] In sample CX844, SNP17 is T, SNP39 is T, and SNP50 is A; the bases at the remaining SNP sites are consistent with the reference sequence.

[0074] In sample CX845, SNP22 is T, SNP32 is A, SNP44 is C, SNP52 is C, and SNP53 is A; the bases of the remaining SNP sites are consistent with the reference sequence.

[0075] In sample CX846, SNP10 is C, SNP18 is G, SNP44 is C, and SNP54 is A; the bases of the remaining SNP sites are consistent with the reference sequence.

[0076] In sample CX848, SNP20 is T, SNP21 is C, and SNP46 is C; the bases at the remaining SNP sites are consistent with the reference sequence.

[0077] In sample CX861, SNP9 is T, SNP11 is G, and SNP30 is T; the bases at the remaining SNP sites are consistent with the reference sequence.

[0078] 5. Genetic distance

[0079] The SNP information is converted into a format recognizable by PLINK software, using PLINK (v v1.90b4.3).

[0080] The genetic distances between 34 samples were calculated using the `--distance-matrix` parameter of the software (http: / / pngu.mgh.harvard.edu / purcell / plink / ). The results are shown in Table 2.

[0081]

[0082] Example 2

[0083] Probe design: Sequence capture probes were designed within a 150bp range upstream and downstream of 60 SNP sites. Probe design principles: 1. Probe sequences should be located within a 150bp range upstream or downstream of the SNP site; 2. Probe length should be controlled within the range of 40–50bp; 3. GC content of the probe sequence should be controlled within the range of 30–60%, with priority given to probes with GC content in the 40–50% range; 4. There should be no repetitive sequences or sequence overlap between probes.

[0084] Based on the above principles, a total of 57 probes covering 60 SNP sites were designed, as shown in Table 4. Among them, P94823F can cover SNPs 44 to 47.

[0085] Table 4

[0086]

[0087]

[0088] Example 3

[0089] 1. SNP sequence alignment

[0090] To verify the identification ability of the 60 SNP sites and the probes amplifying these sites, CX837, known to share the same parent as CX569, and CX868, known to share the same parent as CX574, were tested. The probes designed in Table 4 were used to capture the chloroplast genome target sequences of CX837 and CX868, and the bases of the 60 SNP sites were determined by high-throughput sequencing. The results are as follows:

[0091] In sample CX837, the bases at the SNP sites are identical to those in the reference sequence.

[0092] In sample CX868, SNP20 is T and SNP21 is C; the bases at the remaining SNP sites are consistent with the reference sequence.

[0093] The results for CX837 were consistent with those for CX569 in Example 1; the results for CX868 were consistent with those for CX574 in Example 1. It is evident that the 60 SNP loci and the probes amplifying these loci can be used to identify and confirm identical individuals (same parents) and to distinguish between different individuals.

[0094] 2. Genetic distance

[0095] The SNP information is converted into a format recognizable by PLINK software, using PLINK (v v1.90b4.3).

[0096] The genetic distance between CX837 and CX868 in the 34 samples screened for SNPs was calculated using the `--distance-matrix` parameter of the software (http: / / pngu.mgh.harvard.edu / purcell / plink / ). The results are shown in Table 3.

[0097] Example 4

[0098] 1. SNP sequence alignment

[0099] The probes designed in Table 4 were used to capture chloroplast genome target sequences from 87 samples, and the bases of 60 SNP sites were determined by high-throughput sequencing.

[0100] 2. Genetic distance

[0101] The SNP information is converted into a format recognizable by PLINK software, using PLINK (v v1.90b4.3).

[0102] The `--distance-matrix` parameter in the software (http: / / pngu.mgh.harvard.edu / purcell / plink / ) was used to calculate the genetic distance between the 87 samples that screened for SNPs.

[0103] 3. Phylogenetic Tree

[0104] SNP bases obtained from 87 samples were tandemly linked into SNP sequences. IQ-TREE v2.2.0-beta (http: / / www.iqtree.org / ) software was used for model selection and construction of maximum likelihood (ML) phylogenetic trees. The optimal model selection was based on the Bayesian information criterion scores (BIC) rule. The tree construction parameters were set to: -m MFP-B 1000 -alrt 1000. The results are as follows... Figure 1 As shown.

[0105] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A primer set for amplifying SNP molecular marker combinations in Cunninghamia lanceolata chloroplasts, characterized in that, The aforementioned assemblage of SNP molecular markers for *Cunninghamia lanceolata* chloroplasts includes 60 SNP sites, using GenBank ID:NC_021437.1 as the reference sequence for *Cunninghamia lanceolata* chloroplasts. The 60 SNP sites are as follows: SNP1 is located at base 1341 of the reference sequence of cypress chloroplasts, and is A relative to the reference sequence SNP1. SNP2 is located at base 4865 of the reference sequence of cypress chloroplasts, and is C relative to the reference sequence SNP2. SNP3 is located at base 7726 of the reference sequence of cypress chloroplasts, and is A relative to the reference sequence SNP3. SNP4 is located at base 12335 of the reference sequence of cypress chloroplasts, and is A relative to the reference sequence SNP4. SNP5 is located at base 14207 of the reference sequence of cypress chloroplasts, and is A relative to the reference sequence SNP5. SNP6 is located at base 15429 of the reference sequence of cypress chloroplasts, and is A relative to the reference sequence SNP6. SNP7 is located at base 19699 of the reference sequence of cypress chloroplasts, and is T relative to the reference sequence SNP7. SNP8 is located at base 20673 of the reference sequence of cypress chloroplasts, and is T relative to the reference sequence SNP8. SNP9 is located at base 21821 of the reference sequence of cypress chloroplasts, and is T relative to the reference sequence SNP9; SNP10 is located at 21962 bases of the reference sequence of cypress chloroplasts, and is C relative to the reference sequence SNP10; SNP11 is located at base 23183 of the reference sequence of cypress chloroplasts, and is G relative to the reference sequence SNP11; SNP12 is located at base 23533 of the reference sequence of cypress chloroplasts, and is A relative to the reference sequence SNP12. SNP13 is located at 24087 bases of the reference sequence of cypress chloroplasts, and is T relative to the reference sequence SNP13; SNP14 is located at base 26116 of the reference sequence of cypress chloroplasts, and is T relative to the reference sequence SNP14; SNP15 is located at base 28929 of the reference sequence of cypress chloroplasts, and is A relative to SNP5 in the reference sequence; SNP16 is located at base 31064 of the reference sequence of cypress chloroplasts, and is T relative to the reference sequence SNP16. SNP17 is located at base 37419 of the reference sequence of cypress chloroplasts, and is T relative to the reference sequence SNP17. SNP18 is located at base 41947 of the reference sequence of cypress chloroplasts, and is G relative to the reference sequence SNP18; SNP19 is located at base 43696 of the reference sequence of cypress chloroplasts, and is C relative to the reference sequence SNP19. SNP20 is located at base 52890 of the reference sequence of cypress chloroplasts, and is T relative to the reference sequence SNP20. SNP21 is located at base 52891 of the reference sequence of cypress chloroplasts, and is C relative to the reference sequence SNP21. SNP22 is located at base 53049 of the reference sequence of cypress chloroplasts, and is T relative to the reference sequence SNP22; SNP23 is located at base 55698 of the reference sequence of cypress chloroplasts, and is T relative to the reference sequence SNP23; SNP24 is located at base 56636 of the reference sequence of cypress chloroplasts, and is T relative to the reference sequence SNP24. SNP25 is located at base 63572 of the reference sequence of cypress chloroplasts, and is G relative to the reference sequence SNP25; SNP26 is located at base 68775 of the reference sequence of cypress chloroplasts, and is G relative to the reference sequence SNP26. SNP27 is located at base 69253 of the reference sequence of cypress chloroplasts, and is T relative to the reference sequence SNP27. SNP28 is located at base 70883 of the reference sequence of cypress chloroplasts, and is T relative to the reference sequence SNP28. SNP29 is located at base 75549 of the reference sequence of cypress chloroplasts, and is T relative to SNP29 in the reference sequence; SNP30 is located at base 77989 of the reference sequence of cypress chloroplasts, and is T relative to the reference sequence SNP30. SNP31 is located at base 78958 of the reference sequence of cypress chloroplasts, and is T relative to the reference sequence SNP31. SNP32 is located at base 79416 of the reference sequence of cypress chloroplasts, and is A relative to the reference sequence SNP32. SNP33 is located at base 79493 of the reference sequence of cypress chloroplasts, and is T relative to the reference sequence SNP33. SNP34 is located at base 84262 of the reference sequence of cypress chloroplasts, and is T relative to the reference sequence SNP34. SNP35 is located at base 86464 of the reference sequence of cypress chloroplasts, and is A relative to the reference sequence SNP35. SNP36 is located at base 87557 of the reference sequence of cypress chloroplasts, and is G relative to the reference sequence SNP36. SNP37 is located at base 87900 of the reference sequence of cypress chloroplasts, and is C relative to the reference sequence SNP37. SNP38 is located at base 89547 of the reference sequence of cypress chloroplasts, and is A relative to SNP38 in the reference sequence. SNP39 is located at base 90014 of the reference sequence of cypress chloroplasts, and is T relative to the reference sequence SNP39. SNP40 is located at base 90759 of the reference sequence of cypress chloroplasts, and is A relative to the reference sequence SNP40. SNP41 is located at base 92672 of the reference sequence of cypress chloroplasts, and is A relative to the reference sequence SNP41. SNP42 is located at base 94179 of the reference sequence of cypress chloroplasts, and is G relative to the reference sequence SNP42. SNP43 is located at base 94448 of the reference sequence of cypress chloroplasts, and is T relative to the reference sequence SNP43. SNP44 is located at base 94614 of the reference sequence of cypress chloroplasts, and is C relative to the reference sequence SNP44. SNP45 is located at base 94625 of the reference sequence of cypress chloroplasts, and is A relative to the reference sequence SNP45. SNP46 is located at base 94708 of the reference sequence of cypress chloroplasts, and is C relative to the reference sequence SNP46. SNP47 is located at base 94823 of the reference sequence of cypress chloroplasts, and is A relative to the reference sequence SNP47. SNP48 is located at base 95022 of the reference sequence of cypress chloroplasts, and is G relative to the reference sequence SNP48. SNP49 is located at base 97731 of the reference sequence of cypress chloroplasts, and is C relative to the reference sequence SNP49. SNP50 is located at 102,900 bases in the reference sequence of cypress chloroplasts, and is A relative to the reference sequence SNP50. SNP51 is located at base 113243 of the reference sequence of cypress chloroplasts, and is T relative to the reference sequence SNP51. SNP52 is located at base 117311 of the reference sequence of cypress chloroplasts, and is C relative to the reference sequence SNP52. SNP53 is located at base 118850 of the reference sequence of cypress chloroplasts, and is A relative to the reference sequence SNP53. SNP54 is located at base 119384 of the reference sequence of cypress chloroplasts, and is A relative to the reference sequence SNP54. SNP55 is located at base 122865 of the reference sequence of cypress chloroplasts, and is A relative to the reference sequence SNP55. SNP56 is located at base 122937 of the reference sequence of cypress chloroplasts, and is T relative to the reference sequence SNP56. SNP57 is located at base 123103 of the reference sequence of cypress chloroplasts, and is T relative to the reference sequence SNP57. SNP58 is located at base 131298 of the reference sequence of cypress chloroplasts, and is T relative to SNP58 in the reference sequence. SNP59 is located at base 131852 of the reference sequence of cypress chloroplasts, and is A relative to the reference sequence SNP59. SNP60 is located at base 134204 of the reference sequence of cypress chloroplasts, and is T relative to the reference sequence SNP60.

2. The primer combination according to claim 1, characterized in that, Includes primers as shown in SEQ ID NO: 1-57.

3. A reagent kit for identifying individual Chinese fir trees, characterized in that, Includes the primer combination as described in any one of claims 1 or 2.

4. The primer combination for amplifying SNP molecular marker combinations of Cunninghamia lanceolata chloroplasts as described in claim 1 or 2, or the kit described in claim 3, for the identification of individual Cunninghamia lanceolata.

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