SNP (Single Nucleotide Polymorphism) site combination related to growth and material properties of Chinese fir, liquid phase chip and application of SNP site combination

Abstract

The invention relates to a Chinese fir growth and material character related SNP site combination. The SNP site combination comprises a combination of SNP sites shown in at least one table from a table 4 to a table 8 and a combination of SNP sites shown in a table 9. According to the invention, an SNP site combination related to growth traits (tree height, diameter at breast height and volume of single plant) and material traits (basic density of wood and heartwood ratio) of Chinese fir is obtained, and 13 and 530 SNP sites are included in total; according to the SNP loci, the SNP locus combined probe related to the growth material character of the cedarwood is obtained, and a liquid phase chip of the probe is loaded. The SNP site combination and the liquid chip related to the growth and material properties of the Chinese fir can be well used for early selection, genetic relationship analysis and genetic structure analysis of Chinese fir parents with excellent growth and material properties, the information can be used for breeding of the Chinese fir, and breeding efficiency is improved.

Description

Technical Field

[0001] The present invention belongs to biotechnology and relates to plant molecular breeding technology, in particular to a combination of SNP sites related to fir growth and material traits, a liquid phase chip and applications thereof. Background Art

[0002] As a traditionally dominant commercial forest species in my country, Chinese fir (Cunninghamia lanceolata (Lamb.) Hook.) has long provided a vital source of raw material for the country's timber supply, holding an undisputedly important position in my country's timber industry and strategic reserves. In recent years, domestic and international market demand for timber has shifted from a purely quantitative approach to a multi-purpose approach that prioritizes both quantity and quality. Consequently, simultaneously improving growth and wood properties has become a key challenge in Chinese fir breeding.

[0003] Traditional forest tree breeding techniques suffer from drawbacks such as long breeding cycles, high economic costs, and poor stability, significantly impacting their efficiency and economic benefits. With the rapid development of sequencing technology, molecular breeding techniques offer a highly promising and efficient means for early tree selection and accelerated breeding processes. Currently, traditional breeding methods are still the primary method for Chinese fir breeding, and research on molecular marker-assisted selection (MAS) is still in its infancy. Currently, the main markers used in Chinese fir MAS breeding include Random Amplified Polymorphic DNA (RAPD), Sequence-Related Amplified Polymorphism (SRAP), Simple Sequence Repeats (SSR), and Inter-Simple Sequence Repeat (ISSR). However, these markers are limited in number, have low automation levels, are cumbersome to operate, and some are dominant markers with unknown target sequences. These drawbacks significantly limit their application in Chinese fir breeding. With the rise of next-generation sequencing technology and microarray chips, a number of high-throughput marker detection technologies have emerged, such as chips containing thousands, tens of thousands, or even hundreds of thousands of single nucleotide polymorphism (SNP) sites. These chips offer the advantages of large-scale site coverage and automated analysis, making them suitable for genetic diversity analysis and molecular breeding.

[0004] Currently, there are no reports on gene chips for Chinese fir. Therefore, it is essential to develop SNP loci combinations related to Chinese fir growth and wood properties, as well as liquid-phase microarrays, for use in Chinese fir genetic improvement. This could significantly shorten the time required to improve growth and wood properties and select fast-growing, high-quality varieties. Summary of the Invention

[0005] Based on this, the purpose of the present invention is to provide SNP sites or their combinations related to Chinese fir growth and material traits, liquid phase chips and their applications.

[0006] The first aspect of the present invention is to provide a SNP site combination related to the growth and material traits of Chinese fir, which is selected from the SNP site combination shown in at least one of Tables 4 to 8, and the SNP site combination shown in Table 9.

[0007] In some embodiments, the SNP site combination includes the combination of SNP sites shown in Table 5 and the combination of SNP sites shown in Table 9.

[0008] In some embodiments, the SNP site combination includes the combination of SNP sites shown in Table 7 and the combination of SNP sites shown in Table 9.

[0009] In some embodiments, the SNP site combination includes the combination of SNP sites shown in Table 8 and the combination of SNP sites shown in Table 9.

[0010] In some embodiments, any of the above combinations further includes a combination of SNP sites shown in at least one group in Table 4 or Table 6.

[0011] In some embodiments, the SNP site combination includes all the SNP site combinations shown in Tables 4 to 9.

[0012] The second aspect of the present invention is to provide the application of any of the above-mentioned SNP site combinations in detecting the growth and material traits of Chinese fir, or to provide the application of any of the above-mentioned SNP site combinations in Chinese fir kinship analysis and / or population genetic structure analysis.

[0013] The third aspect of the present invention is to provide the use of any of the above-mentioned SNP site combinations in Chinese fir breeding.

[0014] A fourth aspect of the present invention is to provide a reagent for detecting any of the above-mentioned SNP site combinations for use in detecting growth and material properties of Chinese fir.

[0015] Application of a reagent for detecting any of the above SNP site combinations in Chinese fir kinship analysis and / or population genetic structure analysis.

[0016] Application of a reagent for detecting any of the above SNP site combinations in Chinese fir breeding.

[0017] A fifth aspect of the present invention is to provide a kit for detecting growth and material properties of Chinese fir, comprising reagents for detecting any combination of the above-mentioned SNP sites.

[0018] In some embodiments, the kit is a liquid phase chip loaded with detection probes for detecting any of the above-mentioned SNP sites.

[0019] In some embodiments, the detection probe of the SNP site has the following characteristics:

[0020] (1) The probe is located within 150 bp upstream and downstream of the target SNP site;

[0021] (2) Probe length range is 80–120 bp;

[0022] (3) The GC ratio of the probe is 40%-60%;

[0023] (4) Avoid designing probes on repetitive structures;

[0024] (5) Avoid the formation of secondary structures within the probe;

[0025] (6) The probes do not match each other and form dimers; and

[0026] (7) The Tm values ​​of the probes are uniform.

[0027] Through the inventors' research and experience, the present invention has obtained a combination of SNP loci associated with Chinese fir growth traits (tree height, diameter at breast height, individual volume) and wood material traits (wood basic density, heartwood ratio), comprising a total of 13,530 SNP loci. Based on these SNP loci, detection probes for the SNP loci combination associated with Chinese fir growth and wood material traits were obtained, and the detection probe liquid phase chip was loaded with the detection probes. The SNP loci combination associated with Chinese fir growth and wood material traits, the detection probes, and the corresponding liquid phase chip can be used for early selection of Chinese fir growth and wood material traits, kinship analysis, and genetic structure analysis. The obtained prediction information on Chinese fir growth and wood material traits, kinship information, and genetic structure can be used for Chinese fir breeding needs and improve breeding efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Venn diagram of SNPs associated with growth and wood traits of Chinese fir with a threshold of -log10(p)>2.

[0029] Figure 2 Distribution map of predicted and measured trait values ​​based on 13,530 SNP loci (Longshan Forest Farm).

[0030] Figure 3 Distribution map of predicted and measured trait values ​​based on 13,530 SNP loci (Xiaokeng Forest Farm).

[0031] Figure 4Distribution diagram between predicted and measured values ​​of traits based on 13,530 SNP loci (mixed samples from two forest farms).

[0032] Figure 5 Distribution diagram of predicted and measured growth material properties of 20 Chinese fir samples based on the liquid phase chip in Example 3.

[0033] Figure 6 The phylogenetic tree of Chinese fir parents based on liquid phase chip in Example 4.

[0034] Figure 7 The structure diagram of the Chinese fir parent population based on liquid phase chip in Example 4. DETAILED DESCRIPTION

[0035] To facilitate 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 present disclosure more thorough and comprehensive.

[0036] Experimental procedures in the following examples, where specific conditions are not specified, generally followed conventional conditions, such as those in Molecular Cloning: A Laboratory Manual (4th edition, edited by Green and Sambrook, published in 2013), or according to manufacturer recommendations. All commonly used chemical reagents used in the examples were commercially available.

[0037] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as those belonging to the art and

[0038] The terms "and / or" and "and / or" used in the present invention are intended to be used only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" as used in the present invention includes any and all combinations of one or more of the related listed items.

[0039] The present invention is further described in detail below with reference to specific embodiments.

[0040] Example 1 Development of SNP sites related to Chinese fir growth and material traits, probe design, and liquid phase chip preparation

[0041] 1.1 Determination of growth and wood properties of Chinese fir parent populations

[0042] The present invention uses 57 core breeding parents from the third generation of Chinese fir breeding garden parent populations in Longshan Forest Farm, Lechang City, Guangdong Province and Xiaokeng Forest Farm, Qujiang District, Shaoguan City as test objects. These parents come from major Chinese fir producing areas including Guangdong, Fujian, Guangxi, Hunan, Jiangxi, Guizhou and other provinces. The height and diameter at breast height of each tree were measured (tree age was 6 years old). Then, according to the Chinese fir tree volume formula V = 0.00005877042 × H 0.89646157 ×D 1.9699831 At the same time, the growth cone of the tree was used to drill the wood core at the diameter of each tree at breast height (1.3m high) to determine the heartwood ratio and basic density of the wood. The heartwood ratio was calculated using the following formula: Heartwood ratio = (r 2 ×π) / (R 2 ×π), where r is the heartwood radius and R is the whole plant radius; the basic density of wood is determined using the saturated moisture content method, and the specific formula is: basic density of wood = 1 / ((w1-w2) / w2+1 / ρ cw ), where w1 is the saturated weight of the wood core, w2 is the oven-dried weight of the wood core, and ρ cw is the density of wood cell wall material (1.53 g / cm 3 The actual test results of the growth and wood properties of the parents are shown in Table 1.

[0043] Table 1 Results of 6-year-old growth and material traits of 57 core breeding parents

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[0046] Note: The growth and material data on the left of the slash ( / ) are measured data from Longshan Forest Farm, and on the right are measured data from Xiaokeng Forest Farm. - indicates missing data.

[0047] 1.2 RNA extraction from Chinese fir parent populations, RNA library construction, and high-throughput sequencing

[0048] Total RNA was extracted from the 57 Chinese fir samples using an RNA extraction kit (model: RN53, Beijing Aidelai Biotechnology Co., Ltd.). Total RNA was initially tested for concentration and purity using a Nanodro 2000, and RNA integrity and concentration were accurately quantified using an Agilent 4200 Bioanalyzer (test results are shown in Table 2). After testing, RNA samples were stored at -80°C. Total RNA of acceptable quality was used for transcriptome sequencing library construction.

[0049] Table 2 Information of Chinese fir samples and RNA concentration and integrity

[0050]

[0051] Note: RIN stands for RNA Integrity Number (RIN) counted by the Agilent 4200 instrument. It can accurately and intuitively assess the quality of RNA samples and screen out high-quality samples.

[0052] Transcriptome library using Ultra TM RNA Library Prep Kit for The DNA library was constructed using a kit from NEB (NEB, USA). The process involves enriching eukaryotic mRNA using magnetic beads with oligo(dT). Under high temperature and metal ion conditions, the RNA is fragmented and the first cDNA strand is synthesized using random hexamers. Subsequently, enzymes, buffer, and dNTPs (dATP, dTTP, dGTP, and dCTP) are added to synthesize the second cDNA strand. The synthesized double-stranded cDNA is purified using magnetic beads, followed by end-repair and A spiking, ligation with sequencing adapters, and size sorting using magnetic beads. The sorted fragments are enriched using PCR, and the PCR products are purified to obtain the final library. Sequencing was performed using the Illumina novaseq sequencing platform using a paired-end 150bp sequencing strategy. The sequencing capacity per sample was no less than 6 Gb, with a Q20 of no less than 90 and a Q30 of no less than 85. The sample clean read output and quality statistics are shown in Table 3. The original sequencing sequence was processed using Fastp (version 0.20.1) software for data quality control to obtain high-quality Clean Reads. Fastp quality control used default parameters.

[0053] Table 3 Statistics of sequencing data of Chinese fir samples

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[0057] Note: GC ratio (%): the ratio of G bases and C bases to the total number of bases; Q20: the ratio of bases with a quality value greater than 20 (error rate less than 1%) to the total number of bases; Q30: the ratio of bases with a quality value greater than 30 (error rate less than 0.1%) to the total number of bases.

[0058] 1.3 Development of SNP loci associated with growth and material traits of Chinese fir

[0059] Sequencing data from 57 Chinese fir parental lines were aligned to the Chinese fir reference genome (NCBI data ID: GCA_027924645.1, data source: https: / / www.ncbi.nlm.nih.gov / data-hub / genome / GCA_027924645.1 / ) using STAR software (version 2.7.10b), with default alignment parameters. After alignment, the sequences were sorted using the sort function in samtools (version 1.14). SNP detection was performed using deepvariant software (version 1.8.0), which utilizes an RNA deep learning model, with default parameters used for all other detections. After deepvariant detection, SNP genotyping, sequencing quality, and read alignment quality were rechecked using samtools mpileup and Python programs to comprehensively identify SNP polymorphic sites. A total of 207,859 SNPs were identified.

[0060] Subsequently, GWAS analysis was performed using GAPIT3 (version 3.5.0) software based on the SNP and growth and material data to identify SNP sets associated with growth and material traits. Before analysis, SNPs with a minimum allele frequency (MAF) < 0.05 were removed, as were sites with a detection efficiency less than 80% (i.e., at least 46 of the 57 samples required to be sequenced). After site selection, GWAS analysis of genotypes and phenotypes was performed using GAPIT3. Based on the statistical power of five models (MLM, CMLM, ECMLM, SUPER, and FramCPU), the FarmCPU model was selected for GWAS analysis of three growth traits (tree height, DBH, and individual volume) and two material traits (wood basic density and heartwood ratio). SNPs with a -log10(p) > 2 were selected as trait-associated sites for growth and material trait prediction model selection. The sites obtained in this step are SNP sites related to growth and material traits, and a total of 13,530 SNP sites were obtained ( Figure 1 After repeated screening, we found that 1,839 SNPs were associated only with tree height (Table 4), 1,197 SNPs were associated only with DBH (Table 5), and 1,174 SNPs were associated only with individual tree volume (Table 6). There were also 3,393 SNPs associated only with basic wood density (Table 7), and 3,265 SNPs associated only with heartwood ratio (Table 8). Furthermore, we found 2,662 SNPs associated with two or more of the five traits mentioned above (Table 9).

[0061] Table 4 SNP locus location and variation information related to tree height

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[0069] Table 5 SNP loci location and variation information related to DBH

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[0075] Table 6 SNP loci location and variation information related to single tree volume

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[0081] Table 7 SNP loci location and variation information related to wood basic density

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[0095] Table 8 SNP loci location and variation information related to heartwood ratio

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[0108] Table 9 SNP site locations and variation information associated with two or more phenotypes

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[0119] Example 2: Accuracy test of SNP loci predicting traits related to Chinese fir growth material traits

[0120] The present invention adopts a 5-fold cross-validation method to evaluate the trait prediction accuracy of SNP sites related to Chinese fir growth and material traits under models such as BRR, BayesB, RR, RRBLUP, BayesC, BL, BayesA, LASSO, SPLS, RFC, BRNN, RFR, and SVC. Specifically, the population is randomly divided into two subsets, and then one subset is used as the training population and the other subset is used as the validation population. Multiple random sampling iterations are performed. Assuming that the phenotype of the validation population is unknown, the training population is used to predict the phenotypic value of the validation population. The phenotypic value predicted by the validation population is then correlated with the actual measured value, and the accuracy of the prediction is evaluated using the correlation coefficient. The present invention uses single-site (Xiaokeng Forest Farm, Longshan Forest Farm) and cross-regional (two forest farms mixed) parent populations as objects, and adopts the above-mentioned 5-fold cross-validation method to verify the accuracy of the 13,530 SNP sites in Example 1 of the present invention for predicting Chinese fir growth and material traits. In the single-site (Xiaokeng Forest Farm and Longshan Forest Farm) validation, the correlation coefficients between the predicted and measured values ​​of all traits were above 0.8, and the correlations were extremely significant (P<0.001) ( Figure 2-Figure 3 ); In cross-regional prediction, the correlation coefficients between the predicted values ​​and the actual measured values ​​of all traits were above 0.8, and the correlations were extremely significant (P<0.001) ( Figure 4 The results showed that the 13,530 SNPs of the present invention can effectively predict the tree height, diameter at breast height, individual tree volume, wood basic density and heartwood ratio of Chinese fir, and can be used for the early selection of parents with excellent growth and wood properties of Chinese fir, shortening the breeding cycle of Chinese fir and accelerating the breeding process of Chinese fir.

[0121] Example 3

[0122] 1. Probe design and liquid chip preparation:

[0123] Liquid phase chip probes were designed based on the upstream and downstream sequences of the 13,530 SNP sites described in Example 1 of the present invention and the following principles: (1) the probe position was within 150 bp upstream and downstream of the target SNP site; (2) the probe length range was 80-120 bp; (3) the GC ratio of the probe was 40%-60%; (4) the probes were designed on repetitive structures; (5) the probes were prevented from generating secondary structures; (6) the probes did not match each other and form dimers; and (7) the Tm values ​​of the probes were uniform. Using the primer and probe design software Primer 3, according to the above requirements, the probes were designed by conventional methods (the specific sequences are not listed due to space limitations; according to the above design principles, they can be obtained conventionally through the design software) and synthesized. The probes were commissioned to the Guangzhou Branch of Beijing Liuhe BGI Genomics Technology Co., Ltd. to prepare a liquid phase chip containing the detection of the 13,530 SNP sites according to conventional methods.

[0124] 2. Application of liquid phase chip in prediction of growth material properties of Chinese fir

[0125] In this example, 20 parents from the third generation breeding population in Guangdong were used as samples to be tested, including cx826, cx831, cx817, cx836, cx837, cx838, cx839, cx840, cx841, cx846, cx861, cx863, cx865, cx866, cx867, cx868, cx871, cx873, cx874, and cx876. Actual growth and texture property measurements of the samples to be tested were obtained according to the growth and texture property determination method in Example 1. The growth and texture property predictions of the samples to be tested were then performed using the aforementioned liquid phase chip, specifically comprising the following steps:

[0126] (1) DNA extraction: The genomic DNA of the sample to be tested was extracted using a plant genomic DNA extraction kit (kit manufacturer: Jiangsu Kangwei Century Biotechnology Co., Ltd.; kit model: CW0553). The extracted genomic DNA was subjected to 0.8% agarose gel electrophoresis to detect whether the DNA sample was degraded and had impurities, and the DNA concentration was estimated. At the same time, a Nanodrop spectrophotometer (ThermoFisher Scientific, USA) and The concentration and purity of the samples were detected using a 2.0 Fluorometer (Life Technologies, USA) and stored at -20°C after detection.

[0127] (2) DNA capture and high-throughput sequencing: According to the NGS high-throughput library construction and sequencing process, a double-end DNA sequencing library was constructed, and the library insert size was 350 bp. After fragment size selection and sequencing adapter ligation, the sequence fragments were amplified by PCR according to the following method: 98℃ polymerase activation for 2 min; 98℃ heat denaturation for 30 s, 65℃ annealing for 30 s, 72℃ extension for 60 s, and this process was repeated for 10 cycles; finally, extension at 72℃ for 4 min. The PCR product was purified by magnetic beads and added to the biotin-labeled probe sequence of the liquid chip to capture the specific DNA sequence in the library. After capture and purification, the DNA sequencing library of the target SNP site was obtained. The DNA library that passed the quality inspection was sequenced using the Illumina Novaseq6000 (Illumina, USA) high-throughput sequencing platform. The sequencing strategy was PE150 (Pair-End 150), and the sequencing amount was approximately 500 Mb / sample.

[0128] (3) SNP detection: Pair-end sequencing data were aligned to the reference genome using BWA software and the MEM alignment strategy. Default parameters were used for alignment, and only sequencing reads with unique pair-end alignments were retained. The samtoolsmpileup method was used to detect the coverage of target SNP sites (SNP sites in Tables 4 + 9, Tables 5 + 9, Tables 6 + 9, Tables 7 + 9, and Tables 8 + 9 were analyzed separately), as well as the base sequencing quality and sequence alignment quality of the site. The corresponding SNP genotype was detected using a Python program.

[0129] (4) Phenotypic prediction of the samples to be tested: Based on the BRR, BayesC, BL, BayesA, LASSO, RFR and other models trained by G2P software, the tree height, diameter at breast height, volume per tree, basic density of wood, heartwood ratio and other traits of the samples to be tested were predicted, and the predicted values ​​were correlated with the actual measured values. The results showed that the correlation coefficient between the predicted values ​​and the actual measured values ​​was between 0.605 and 0.823, and the correlation reached an extremely significant level (P < 0.001) ( Figure 5 ), from which it can be seen that the predicted value and the actual measured value are highly positively correlated, indicating that the liquid phase chip of the present invention can effectively predict the tree height, diameter at breast height, single tree volume, wood basic density and heartwood ratio of Chinese fir. The prediction results can provide reliable data support for the precise selection of Chinese fir breeding parents with excellent growth and material traits in the early stage, thereby quickly selecting excellent breeding parents for the next round of genetic improvement, shortening the Chinese fir breeding cycle, and accelerating the Chinese fir breeding process.

[0130] Example 4. Application of Liquid Microarray in Analysis of Phylogenetic Relationship and Population Genetic Structure of Chinese Fir

[0131] This example used 57 samples from Example 1, 20 from Example 3, and 11 new third-generation Chinese fir breeding parents (cx66, cx232, cx233, cx240, cx242, cx245, cx250, cx251, cx269, cx420, and cx845), totaling 88 samples. DNA extraction, DNA capture and high-throughput sequencing, and SNP detection were performed on these 88 samples according to the methods of Example 3. Ultimately, the 13,530 SNPs described in Example 1 were detected in these 88 samples, achieving 100% coverage. These SNPs were then used for kinship analysis and population genetic structure analysis of the 88 samples. IQ-TREE 2.0.5 (http: / / www.iqtree.org / ) software was used to determine the optimal base substitution model of the SNP site as GTR+I+G based on the BIC (Bayesian Information Criterion scores) method. The phylogenetic tree was constructed based on the optimal model using the maximum likelihood method (ML method). The calculation was repeated 1000 times, and finally 88 sample phylogenetic trees were successfully constructed ( Figure 6 ) to detect the genetic relationship between 88 samples. Based on the 13,530 SNPs, admixture (version 1.3.0) was used to analyze the population structure of the 88 samples. Assuming that the number of clusters (K) of the samples was 1-10 for clustering, the optimal cluster was determined to be 3 based on the cross-validation (CV) error rate, that is, the genetic background of the 88 samples came from three different genetic components (corresponding to red, green and blue, respectively). Samples with the same color indicate that their genetic grouping is consistent and their genetic relationship is close. Results ( Figure 7) showed that the genetic components of cx100, cx552, cx554, cx569, cx571, cx861, and cx873 were consistent (blue), and they belonged to the same genetic grouping group, while the genetic components of cx180, cx232, cx241, cx242, cx246, cx263, cx310, cx538, cx262, cx251, cx269, and cx271 were consistent (green), and they belonged to another genetic grouping group. The genetic components of cx556, cx859, and cx863 were red, and they belonged to another genetic grouping group different from the first two genetic groups. The remaining 66 parents showed a mixture of the three genetic components, indicating that they had genetic components from the above three different genetic grouping groups at the same time. The parental relationship and population genetic structure information obtained based on the liquid phase chip of this invention can provide a basis for the configuration of Chinese fir hybrid parents. For example, in the process of Chinese fir hybrid breeding, mating of parents from the same genetic components or with close genetic relationships can be avoided, thereby effectively avoiding the occurrence of inbreeding depression. At the same time, considering the growth and material trait performance of the parents, the parents with the best growth and material performance and distant genetic relationship can be strongly combined to fully utilize hybrid advantages, thereby accelerating the genetic improvement process of Chinese fir.

[0132] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A SNP locus combination related to growth and material traits of Chinese fir, characterized in that: The SNP site combination includes a combination of SNP sites shown in at least one table selected from Table 4 to Table 8, and a combination of SNP sites shown in Table 9.

2. The SNP site combination according to claim 1, characterized in that: The SNP site combinations include the combination of SNP sites shown in Table 5 and the combination of SNP sites shown in Table 9.

3. The SNP site combination according to claim 1 or 2, characterized in that: The SNP site combinations include the combination of SNP sites shown in Table 7 and the combination of SNP sites shown in Table 9.

4. The SNP site combination according to any one of claims 2 to 3, characterized in that: The SNP site combinations include the combination of SNP sites shown in Table 8 and the combination of SNP sites shown in Table 9.

5. The SNP site combination according to any one of claims 2 to 4, characterized in that: Also included are combinations of SNP sites shown in at least one group in Table 4 or Table 6.

6. The SNP site combination according to claim 1, characterized in that: The SNP site combination includes the combination of all SNP sites shown in Tables 4 to 9.

7. Use of the SNP locus combination according to any one of claims 1 to 6 in detecting growth and material properties of Chinese fir, or use of the SNP locus combination according to any one of claims 1 to 6 in analyzing kinship and / or population genetic structure of Chinese fir.

8. Use of the SNP locus combination according to any one of claims 1 to 6 in Chinese fir breeding; or use of a reagent for detecting the SNP locus combination according to any one of claims 1 to 6 in Chinese fir breeding.

9. A kit for detecting the growth and material properties of Chinese fir, characterized in that: Comprising a reagent for detecting the SNP site combination according to any one of claims 1-6.

10. The kit according to claim 9, characterized in that The kit is a liquid phase chip loaded with detection probes for detecting the corresponding SNP sites in any one of the SNP site combinations according to claims 1-6; Preferably, the detection probe for the SNP site has the following characteristics: (1) The probe is located within 150 bp upstream and downstream of the target SNP site; (2) Probe length range is 80–120 bp; (3) The GC ratio of the probe is 40%-60%; (4) Avoid designing probes on repetitive structures; (5) Avoid the formation of secondary structures within the probe; (6) The probes do not match each other and form dimers; and (7) The Tm values ​​of the probes are uniform.

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