A probe set, molecular marker, kit and identification method for identifying elliott pine and loblolly pine

Abstract

The present application relates to a kind of probe group for identifying Pinus elliottii and Pinus taeda, molecular marker, kit and its identification method, belong to biological molecular breeding technical field.The present application provides a kind of probe group for identifying Pinus elliottii and Pinus taeda, is by based on Pinus taeda reference genome, Pinus elliottii three generation full-length transcriptome and Pinus elliottii and Pinus taeda SNPs dataset, 26 liquid phase probes are developed to be composed.The liquid phase probe group of the application is used to obtain 55 SNPs markers, the minimum allele frequency of these markers is greater than 0.05, the detection rate is 100%, and the PCA and system evolution analysis based on 55 SNPs markers can successfully distinguish Pinus elliottii and Pinus taeda, and the test proves that the liquid phase probe group of the application can efficiently and quickly complete the identification of Pinus elliottii and Pinus taeda species.

Description

Technical Field

[0001] The present invention relates to the technical field of biomolecule breeding, in particular to a probe group, a molecular marker, a kit and an identification method for identifying elliott pine and loblolly pine. Background Art

[0002] The slash pine (Pinus elliottii Engelm. var. elliottii) and loblolly pine (Pinus taeda L.), naturally distributed in the southeastern United States, are important pine species worldwide. Due to their rapid growth, strong adaptability, high timber value, pest and disease resistance, and high resin yield, they are widely cultivated in subtropical and some tropical regions of the world. They serve as a major source of various industrial materials, construction materials, pulp materials, and resins, bringing substantial value to society.

[0003] In the 1830s, slash pine and loblolly pine were successfully introduced to my country and are now cultivated on a large scale. During the seedling stage, it is difficult to distinguish the two species through morphological observation. Furthermore, since there is no clear geographical boundary between the natural distribution areas of the two species, and their cultivation areas in China almost overlap, natural hybridization may occur. Therefore, the morphological variation between the species is continuous, making it difficult to accurately identify all individual trees through phenotypic analysis, even for mature trees. Therefore, it is necessary to develop markers that can distinguish the two pine species when it is difficult to distinguish them, and to identify hybrids between the two species. Summary of the Invention

[0004] The purpose of the present invention is to provide a liquid phase probe set for identifying elliot pine and loblolly pine. 55 SNPs were obtained using this set of liquid phase probes, which can efficiently and quickly complete the identification of elliot pine and loblolly pine species.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The present invention provides a liquid phase probe group for identifying elliott pine and loblolly pine. The liquid phase probe group comprises probes with sequences shown as SEQ ID NOs. 1 to 26.

[0007] The present invention provides a SNPs marker for distinguishing elliott pine and loblolly pine, wherein the SNPs marker includes the SNPs markers shown in Table 2.

[0008] The present invention also provides a method for distinguishing elliott pine and loblolly pine, the method comprising the following steps:

[0009] The liquid phase probe group is hybridized with the DNA library of the sample to be tested, and the DNA library hybridized with the capture probe and the target region is amplified by PCR, and then subjected to high-throughput sequencing, genotyping, and cluster analysis to determine the classification of the sample to be tested, thereby identifying the species of the sample to be tested.

[0010] Preferably, the DNA library of the sample to be tested is obtained by fragmenting and end-repairing the total DNA of the sample to be tested, connecting with adapters, amplifying the library and purifying it.

[0011] Preferably, the method further comprises a reference substance, and the reference substance is a standard species of Pinus elliottii and Pinus taeda.

[0012] Preferably, the samples to be tested that are clustered together with the standard species of Pinus elliottii are Pinus elliottii, and the samples to be tested that are clustered together with the standard species of Pinus taeda are Pinus taeda.

[0013] The present invention also provides a kit for identifying elliott pine and loblolly pine, wherein the kit comprises the above-mentioned liquid phase probe group and / or the above-mentioned SNPs marker.

[0014] The present invention also provides the use of the liquid phase probe, the SNPs marker, the method or the kit in species identification of Pinus elliottii and Pinus taeda.

[0015] The present invention provides a liquid phase probe set for distinguishing between longitudinal pine and loblolly pine. The probe set comprises 26 liquid phase probes developed based on a loblolly pine reference genome, a third-generation full-length transcriptome of longitudinal pine, and SNP datasets for longitudinal pine and loblolly pine. Using the liquid phase probe set, 55 SNP markers were identified, each with a minimum allele frequency greater than 0.05 and a detection rate of 100%. PCA and phylogenetic analysis based on the 55 SNP markers successfully distinguished longitudinal pine from loblolly pine. Experimental results confirm that the liquid phase probe set can efficiently and rapidly distinguish longitudinal pine and loblolly pine species.

[0016] At the same time, the present invention also provides a method for efficiently identifying elliott pine and loblolly pine. Due to the use of high-throughput sequencing typing technology, the procedure is simple and standardized, which reduces the errors of individual operations, making the unit price of batch identification analysis extremely low, with the characteristics of economy and efficiency, especially suitable for large sample analysis and identification, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a phylogenetic analysis of the relationship between Pinus taeda and Pinus elliottii based on probe-captured SNPs markers, where Pta represents Pinus taeda and Pel represents Pinus elliottii.

[0018] Figure 2This is the PCA analysis of Pinus taeda and Pinus elliottii based on probe-capture SNPs markers, where Pta represents Pinus taeda and Pel represents Pinus elliottii.

[0019] Figure 3 This is a phylogenetic analysis of the relationship between Pinus taeda and Pinus elliottii based on probe-capture SNPs markers, where Pta represents Pinus taeda, Pel represents Pinus elliottii, and Pel / Pta represents samples of unknown species of Pinus taeda and Pinus elliottii.

[0020] Figure 4 This is the PCA analysis of Pinus taeda and Pinus elliottii based on probe-capture SNPs markers, where Pta represents Pinus taeda, Pel represents Pinus elliottii, and Pel / Pta represents samples of unknown species of Pinus taeda and Pinus elliottii. DETAILED DESCRIPTION

[0021] The present invention provides a liquid phase probe set for identifying elliott pine and loblolly pine, wherein the liquid phase probe set includes the probes shown in Table 1 below:

[0022] Table 1 Liquid phase probe information for identifying 26 species of Pinus elliottii and Pinus taeda

[0023]

[0024]

[0025]

[0026] Among them, the scaffolds are the scaffolds after the second edition of the reference genome of loblolly pine was assembled.

[0027] In the present invention, the liquid phase probe group is preferably designed based on the genome reference sequence of loblolly pine V2.0 version, the three-generation full-length transcriptome data of elliott pine, the loblolly pine SNPs data set and the elliott pine data set. In the present invention, the liquid phase probe group is preferably the smallest probe set that can distinguish between the two species of elliott pine and loblolly pine. In the present invention, the probe is preferably designed according to the design principles of liquid phase probes, and the design principles of the probe preferably include: 1) the GC content of the SNPs marker sequence and the sequence 100bp upstream and downstream (a total of 201bp) are both between 0.35 and 0.65%; 2) the copy number on the genome is low to ensure the efficiency of probe capture.

[0028] The present invention provides a SNPs marker for distinguishing elliot pine and loblolly pine, wherein the SNPs marker includes the SNP markers shown in Table 2 below:

[0029] Table 2 Relevant information of 55 SNPs markers

[0030]

[0031]

[0032]

[0033] In the present invention, the SNPs markers are obtained by targeted capture sequencing and typing of 37 elliottii pine and 5 loblolly pine species using liquid probes.

[0034] The present invention also provides a method for distinguishing elliott pine and loblolly pine, the method comprising the following steps:

[0035] The liquid phase probe group is hybridized with the DNA library of the sample to be tested, and the DNA library hybridized with the capture probe and the target region is amplified by PCR, and then subjected to high-throughput sequencing, genotyping, and cluster analysis to determine the classification of the sample to be tested, thereby identifying the species of the sample to be tested.

[0036] In the present invention, the DNA library of the sample to be tested is obtained by fragmenting and end-repairing the total DNA of the sample to be tested, connecting the adapter, amplifying the library, and purifying the total DNA. The present invention does not particularly limit the method for extracting the total DNA. In an embodiment of the present invention, the method for extracting the total DNA is preferably the CTAB method. In the present invention, the data obtained by the genotyping are preferably subjected to phylogenetic relationship analysis and PCA analysis of the sample to be tested. When the sample to be tested is clustered with the standard species of Pinus elliottii, it is Pinus elliottii. When the sample to be tested is clustered with the standard species of Pinus taeda, it is Pinus taeda.

[0037] In the present invention, a reference substance corresponding to the sample to be tested is also required when distinguishing elliot pine and loblolly pine; the reference substance is preferably a standard species of elliot pine and loblolly pine, more preferably genotyping data of the standard species of elliot pine and loblolly pine.

[0038] The present invention also provides a kit for identifying elliott pine and loblolly pine, wherein the kit comprises the above-mentioned liquid phase probe group and / or the above-mentioned SNPs marker.

[0039] The present invention also provides the use of the liquid phase probe, the SNPs marker, the method or the kit in species identification of Pinus elliottii and Pinus taeda.

[0040] In the present invention, the raw materials, reagents and equipment used are all known products and can be conventionally commercially available products.

[0041] In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0042] Example 1

[0043] Development of liquid phase probe sets

[0044] (1) Download the data of 2,822,609 SNPs of Pinus taeda from the NCBI Single Nucleotide Polymorphism Database (dbSNP) (Accession numbers ss1995911273-ss1996900602);

[0045] (2) Transcriptome sequencing was performed on 240 domestic elliottii pine accessions at a sequencing depth of 10×, obtaining 974,733 original SNPs.

[0046] (3) Use vcftools software to filter the SNPs data of loblolly pine and elliott pine, retaining SNPs with a minimum allele frequency greater than 0.01 and a sample detection rate greater than 80%; extract the SNP site and the 100bp sequence upstream and downstream, and use bedtools software to align the extracted 201bp fragment to the loblolly pine V2.0 genome, retaining the successfully aligned fragments. Analyze the GC content and copy number of the aligned 201bp fragments on the genome, retaining fragments with a GC content between 0.35 and 0.65, fragments with a copy number of 1 to 3, and areas covered by both elliott pine and loblolly pine as candidate capture regions;

[0047] If the distance between two candidate capture regions was less than 200-300 bp, they were merged into the capture region of one probe, resulting in a total of more than 30,000 candidate probe capture regions screened;

[0048] (4) A self-written Perl script was used to analyze the location of the probe-captured candidate regions on the genome, and candidate regions located within the CDS region and within 300 bp upstream of the CDS were retained;

[0049] (5) Selecting 1000 candidate regions from step (4), designing and synthesizing 100 bp probes in suitable regions, and performing capture sequencing in 37 individuals of Pinus elliottii and 5 individuals of Pinus taeda;

[0050] (6) Selecting the smallest liquid phase probe set with a detection rate of 100% and capable of distinguishing between Pinus taeda and Pinus elliottii from the 1000 probes obtained in step (5) to obtain the liquid phase probe set of the present invention comprising 26 probes.

[0051] Example 2

[0052] Method for identifying Pinus taeda and Pinus taeda using the 26 liquid phase probes described in Example 1

[0053] (1) Genomic DNA extraction;

[0054] Young needles of elliot pine and loblolly pine were collected, including 37 elliot pine samples, 5 standard loblolly pine species control samples and 15 unknown test samples of loblolly pine and elliot pine. They were stored with ice packs and transported back to the laboratory in time. The total DNA of elliot pine and loblolly pine needles was extracted using the CTAB method.

[0055] (2) genomic DNA fragmentation and end repair;

[0056] 1. Prepare the reaction system shown in Table 3 in a sterile PCR tube placed on ice;

[0057] Table 3 Fragmentation reaction system

[0058] Reagent Volume Input DNA* X μL (250 ng) Buffer 4.5 μL Enzymes 5 μL 1 x TE Buffer Up to 30 μL Total Volume 30 μL

[0059] Where X represents any volume not exceeding 20 μL, which is added to 4.5 μL of buffer and 5 μL of enzymes. The portion less than 30 μL is made up with 1× TE buffer.

[0060] 2. Use a pipette to pipette up and down or shake to ensure the system is fully mixed, then centrifuge briefly;

[0061] 3. Immediately place the PCR tubes in a PCR instrument preheated to 32°C and run the reaction program shown in Table 4. Set the PCR instrument's heated lid to 75°C. Once the temperature drops to 4°C, immediately place the tubes on ice for the next step. Do not keep them at 4°C.

[0062] Table 4 Fragmentation and end-repair reaction procedures

[0063] Temperature Time 32℃ 5-25 min 65℃ 30 min 4℃ Drop to 4°C immediately for next step

[0064] The fragmentation time (i.e., the time spent at 32° C.) was adjusted according to the size of the target insert fragment in Table 5.

[0065] Table 5 Fragmentation time selection

[0066] Target insert size Fragmentation time Time optimization range 200 bp 20 min 18-25 min 250 bp 15 min 13-17 min 300 bp 10 min 8-12 min 550 bp 5 min 4-6 min

[0067] (3) Connector connection;

[0068] 1. Prepare the reaction system as shown in Table 6 below;

[0069] Table 6 Linker ligation reaction system

[0070] Reagent Volume End Prep Reaction Mix (Step (2) reaction product) 30 μL Ligation Buffer 15 μL <![CDATA[ddH2O]]> 2.5 μL Ligation Enzymes 5 μL Truncated Adaptor 2.5 μL Total Volume 55 μL

[0071] All operations were performed on ice, and ddH2O, Ligation Buffer, and Ligation Enzymes were used to prepare premixed solutions.

[0072] 2. Place the PCR tube on the PCR instrument and set the reaction program at 22°C for 15 minutes. Do not set the heat cover on the PCR instrument. Then store at 4°C (proceed to the next step immediately after cooling to 4°C).

[0073] 3. DNA Sample Purification

[0074] 3.1 Add 44 μL (0.8×) DNA Clean Beads to each sample and mix thoroughly; incubate at room temperature for 5 minutes;

[0075] 3.2 Place the PCR tube on a magnetic rack for 3 minutes. After the solution becomes clear, remove the supernatant.

[0076] 3.3 Add 180 μL 80% ethanol to rinse the beads, incubate for 30 seconds, remove the supernatant, and repeat the operation once;

[0077] 3.4 Keep the PCR tube on the magnetic rack and use a 10 μL pipette to remove the ethanol remaining at the bottom of the tube. Dry until no ethanol remains.

[0078] 3.5 Resuspend the magnetic beads in 21 μL ddH2O and let it stand at room temperature for 1 min to fully release the DNA from the beads.

[0079] 3.6 Place the PCR tube on a magnetic stand for 2 minutes and transfer 20 μL of supernatant to a new PCR tube for library amplification.

[0080] (4) Library amplification and purification;

[0081] 1. Prepare the PCR reaction system as shown in Table 7 below, mix thoroughly by pipetting, and centrifuge briefly;

[0082] Table 7 PCR reaction system

[0083] Reagent Volume Ligated DNA 20 μL 2 x PCR Mix 12.5 μL PCR Index Primer 2.5 μL Universal PCR Primer 2.5 μL Total Volume 35 μL

[0084] 2. The prepared reaction system was subjected to the PCR amplification program shown in Table 8 below for amplification.

[0085] Table 8 PCR amplification program

[0086]

[0087] 3. Add 35 μL (1×) DNA Clean Beads to the amplified product, mix well, and incubate at room temperature for 5 minutes;

[0088] 4. Place the sample on a magnetic rack for 2 minutes. Once the solution is clear, remove the supernatant.

[0089] 5. Add 200 μL of 80% ethanol to rinse the magnetic beads, incubate for 30 seconds, remove the supernatant, and repeat this step once;

[0090] 6. Keep the PCR tube on the magnetic rack, use a 10μL pipette to remove the ethanol remaining at the bottom of the tube, open the tube cap and dry until no ethanol remains;

[0091] 7. Resuspend the magnetic beads in 31 μL ddH2O and let it stand at room temperature for 1 minute to fully release the DNA from the beads;

[0092] 8. Place the sample on a magnetic stand for 2 minutes, transfer 30 μL of the supernatant to a new PCR tube, and store the library at -20°C for subsequent library quality testing and sequencing.

[0093] (5) Hybridization of library and probe;

[0094] 1) Take 750 ng of the library constructed in step (4) and add it to a PCR tube and mark it;

[0095] 2) Add purified magnetic beads to the library and gently pipette to mix;

[0096] 3) Incubate at room temperature for 5 minutes and place the PCR tube on a magnetic rack for 3 minutes to clarify the solution.

[0097] 4) Remove the supernatant, place the PCR tube on the magnetic stand, add 180 μL of 80% ethanol, and let it stand for 30 seconds;

[0098] 5) Remove the supernatant and add 180 μL of 80% ethanol to the PCR tube. Let it stand for 30 seconds and then completely remove the supernatant.

[0099] 6) Leave at room temperature for 5 minutes to allow the residual ethanol to evaporate completely;

[0100] 7) Add the hybridization reaction system to the PCR tube according to Table 9 below;

[0101] Table 9 Hybridization reaction system

[0102] Reagent Volume Hyb Buffer 13 μL Hyb Human Block 5 μL Adapter Blocker 2 μL Rnase Block 5 μL Nuclease Free Water 3 μL Total Volume 28 μL

[0103] 8) Gently pipette to mix, let stand at room temperature for 3 minutes, centrifuge briefly, place the PCR tube on a magnetic rack, and let stand for 3 minutes;

[0104] 9) Pipette 28 μL of supernatant into a new PCR tube, add 2 μL of Target Probe, gently pipette to mix, and centrifuge briefly;

[0105] 10) Set the PCR instrument parameters as follows:

[0106] Heating cover temperature: 85℃; 80℃ for 5min; 50℃ hold;

[0107] Place the PCR tube on the PCR instrument, run the above program, and incubate overnight.

[0108] (6) Capturing the target region DNA library;

[0109] 1. Magnetic bead pretreatment:

[0110] 1) Remove capture beads from 4°C, vortex to resuspend, and equilibrate at room temperature for 30 minutes;

[0111] 2) Add 50 μL of magnetic beads to a new PCR tube, place on a magnetic rack for 1 minute to allow the solution to clear, and remove the supernatant.

[0112] 3) Remove the PCR tube from the magnetic rack, add 180 μL Binding Buffer, gently pipette several times to mix, and resuspend the magnetic beads; place on the magnetic rack for 1 minute, remove the supernatant, and repeat this step once;

[0113] 4) Remove the PCR tube from the magnetic stand, add 180 μL Binding Buffer, and gently pipette to resuspend the magnetic beads.

[0114] 2. Capture target region DNA library:

[0115] 1) Keeping the hybridization product on the PCR instrument, add 180 μL of capture magnetic beads resuspended in step 1 to the hybridization product, mix by pipetting, and place on a rotary mixer at room temperature for 30 minutes;

[0116] 2) Place the PCR tube on a magnetic rack for 2 minutes to clarify the solution and remove the supernatant;

[0117] 3) Add 150 μL of 50°C preheated Wash Buffer, gently pipette to mix, then briefly centrifuge and incubate on a constant temperature shaker at 50°C for 10 min.

[0118] 4) Briefly centrifuge and place the PCR tube on a magnetic rack for 2 minutes to clarify the solution. Remove the supernatant. Repeat this step twice, washing the beads a total of three times.

[0119] 5) Keeping the sample on the magnetic rack, add 150 μL of 80% ethanol to the PCR tube. Let it stand for 30 seconds, then completely remove the ethanol solution and air dry at room temperature.

[0120] 6) Add 24 μL of Nuclease-free Water to the PCR tube, remove the PCR tube from the magnetic stand, and gently pipette to resuspend the magnetic beads for later use.

[0121] (7) PCR amplification after capture;

[0122] 1) Remove PostPCR MasterMix and PostPCR Primer from the -20°C freezer and place them on an ice box to thaw. After thawing, mix well and place on ice or at 4°C until ready to use.

[0123] 2) After capture, the DNA library needs to be amplified by PCR. The reaction system is prepared according to Table 10 below:

[0124] Table 10 Reaction system

[0125] Reagent Volume Step (6) Capture DNA library of target region 24 μL Post PCR Master Mix 25 μL Post PCR Primer (select according to library type) 1 μL Total Volume 50 μL

[0126] 3) Adjust the pipette to 40 μL, gently pipette up and down 6 times to mix, and then immediately place the tube in the PCR instrument;

[0127] 4) Run the PCR program:

[0128] Heating cover temperature: 105℃

[0129] Program: 95°C for 1 min; 98°C for 20 s; 60°C for 30 s (N cycles) (referring to the probe tube wall label parameter); 72°C for 30 s; 72°C for 5 min; hold at 4°C.

[0130] 5) After PCR, add 55 μL of purified magnetic beads to the sample, vortex or pipette to mix, and let stand at room temperature for 5 minutes;

[0131] 6) Briefly centrifuge and place the PCR tube on a magnetic rack for 3 minutes to allow the solution to clear.

[0132] 7) Keep the PCR tube on the magnetic rack, remove the supernatant, add 180 μL of 80% ethanol solution to the PCR tube, and let it stand for 30 seconds;

[0133] 8) Keep the PCR tube on the magnetic rack, remove the supernatant, add 180 μL of 80% ethanol solution to the PCR tube again, let it stand for 30 seconds, and then completely remove the supernatant. Let it stand at room temperature for 5 minutes to allow the residual ethanol to evaporate completely.

[0134] 9) Add 25 μL of nuclease-free water, remove the PCR tube from the magnetic stand, shake or pipette to mix 10 times, and let it stand at room temperature for 2 minutes;

[0135] 10) Briefly centrifuge and place the PCR tube on a magnetic rack for 2 minutes to allow the solution to clear.

[0136] 11) Use a pipette to transfer 23 μL of supernatant to a 1.5 mL centrifuge tube and label the sample information;

[0137] 12) Take 1 μL of library and quantify using the Qubit dsDNA HS Assay Kit. Record the library concentration, which is approximately 1-20 ng / μL.

[0138] 13) Take 1 μL of sample and use Agilent 2100 Bioanalyzer system (Agilent DNA 1000 Kit) to measure the fragment length.

[0139] (8) High-throughput sequencing;

[0140] The library obtained after capture and amplification in step (7) was subjected to high-throughput sequencing using the MGI DNBSEQ-T7 sequencing platform to obtain the sequencing results of the tree species genomic DNA, and the obtained data was basically cleaned.

[0141] (9) Genotyping analysis;

[0142] The cleaned sequencing data were aligned to the loblolly pine reference sequence using bwa software, and the SNP genotyping data of elliott pine and loblolly pine were obtained using GATK software.

[0143] (10) Species identification

[0144] Phylogenetic analysis and PCA analysis were performed using SNP genotyping data for the standard species, Pinus elliottii, and Pinus taeda, as well as for the unknown samples. Species identification was based on cladistics and PCA clustering. Species clustered with the standard species Pinus elliottii were identified as Pinus elliottii, while those clustered with the standard species Pinus taeda were identified as Pinus taeda.

[0145] Note: When using the present invention for species identification, the genotyping data of the 42 samples in the present invention can be directly used as a reference standard.

[0146] Example 3

[0147] Identification of known elliott pine and loblolly pine samples

[0148] (1) Test materials

[0149] In August 2021, needles from 37 known slash pine and 5 known loblolly pine species were collected from the seed orchard of Wuchan Zhongda Changle Forest Farm Co., Ltd. in Yuhang District, Hangzhou. After refrigeration, these needles were promptly transported to the laboratory for total DNA extraction. The sample species and numbers are shown in Table 11.

[0150] Table 11 Sample tree species type and number

[0151]

[0152] (2) Acquisition of genotyping data and species identification

[0153] Hybridization and genotyping of 26 liquid probes were performed on each single strain in Table 11 using the method described in Example 2, and the genotyping data of each single strain were obtained, a total of 55 SNP markers were obtained, as shown in Table 2, and the detection rate was 100%. Among them, the genotyping results of 26 liquid probes in 37 slash pine samples and 5 standard samples of loblolly pine in this example are as shown in Table 12.

[0154] Table 12 Genotyping results of 37 slash pine samples and 5 standard samples of loblolly pine

[0155]

[0156]

[0157]

[0158] Based on the 55 SNP marker genotyping results of the standard species loblolly pine sample detected by 26 probes, phylogenetic analysis and PCA analysis were performed, and the classification results are shown in Figure 1 and Figure 2 .

[0159] From the classification results, it can be seen that slash pine and loblolly pine are clearly classified into two categories, and the identification rate reaches 100%.

[0160] Example 4

[0161] Identification of unknown tree species samples

[0162] (1) Test materials

[0163] In October 2021, 15 slash pine and loblolly pine seedlings of unknown species were selected from the experimental forest farm nursery of the Institute of Subtropical Forestry, Chinese Academy of Forestry, and leaf tissues were collected. After cold storage, the total DNA of each single needle was extracted in the laboratory. Among them, the unknown species codes are as shown in Table 13.

[0164] Table 13 Unknown species code

[0165] Pel / Pta1 Pel / Pta2 Pel / Pta3 Pel / Pta4 Pel / Pta5 Pel / Pta6 Pel / Pta7 Pel / Pta8 Pel / Pta9 Pel / Pta10 Pel / Pta11 Pel / Pta12 Pel / Pta13 Pel / Pta14 Pel / Pta15

[0166] (2) Acquisition of genotyping data and species identification

[0167] According to the steps described in Example 2, the hybridization and genotyping of 26 liquid probes were performed on the total DNA of each single strain, and the 55 SNP genotyping data of each single strain were obtained, and the detection rate was 100%. The genotyping data of 15 unknown slash pine and loblolly pine species are as shown in Table 14.

[0168]

[0169] The 55 SNPs marker typing results of Table 14 detected by the 26 probes of the liquid phase probe set of the present invention were subjected to phylogenetic and PCA analysis with the known slash pine and loblolly pine genotyping results in Example 3 to obtain the following: Figure 3 、 Figure 4 The classification results are shown.

[0170] The results showed that 12 of the 15 unknown samples clustered with Pel and 3 with Pta, concluding that 12 of the unknown samples were Pinus elliottii and 3 were Pinus taeda. Observation of the dormancy characteristics of these 15 identification samples during winter 2021 and spring 2022 revealed that the probe identification results fully conformed to the biological learning characteristics of dormancy and shoot development for both species, thus confirming the reliability of this identification method. This demonstrates that the liquid-phase probe set of the present invention can efficiently and rapidly identify Pinus elliottii and Pinus taeda species.

[0171] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention. Sequence Listing <110> Research Institute of Subtropical Forestry, Chinese Academy of Forestry <120> A probe set, molecular marker, kit and identification method for identifying elliott pine and loblolly pine <160> 26 <170> SIPOSequenceListing 1.0 <210> 1 <211> 100 <212> DNA <213> Artificial Sequence <400> 1 gggagggcat ccatgtcggc acgaagtgca acgactgggg cagtgcccaa tccgatggtg 60 gcaaccacac ccgtctgtgc aaagggccat tcgaaatgca 100 <210> 2 <211> 100 <212> DNA <213> Artificial Sequence <400> 2 gtttttgggt tcaacacaac ctgaacatgc tcagttttct tctctggcaa tcaattagct 60 gtatttgact ggaatataaa tattcatggc cacaggtatg 100 <210> 3 <211> 100 <212> DNA <213> Artificial Sequence <400> 3 aggccgggca ccaggaagca gaacggccgc atagcttcgc cttgggcgac ccatcggctg 60 cggccccgct gagcaggagc aacagaagaa aggggaacat 100 <210> 4 <211> 100 <212> DNA <213> Artificial Sequence <400> 4 aagtactgta tatcagagac gaaaataacc catttgtttc tggaaacagg atgatcaata 60 tcgtaatcta ctttaatttc cgggtcctgc gatcgatctc 100 <210> 5 <211> 100 <212> DNA <213> Artificial Sequence <400> 5 cgtctgggaa ttgggtaggg caatcgggtc tgaagggctg gttgcaggac aggttgtgga 60 tctttccagt ggtgggttat ccaatgtggg tttggatcta 100 <210> 6 <211> 100 <212> DNA <213>人工序列(Artificial Sequence) <400> 6 taagggcatt ggttcgtaac cacgaacgtc gccccttcag cacctgcact tgaccaaaca 60 atatttatgg catatacagt tgaatttaac tcgatggtat 100 <210> 7 <211> 100 <212> DNA <213>人工序列(Artificial Sequence) <400> 7 tggctttctt ccaaatggca atgatctatt actctgcggc actggttatt tgcttagcaa 60 gtatggcggt gctgtgtcag gggcaatacc agtatagcag 100 <210> 8 <211> 100 <212> DNA <213>人工序列(Artificial Sequence) <400> 8 gtgaatgttg ggcaggactc ctccattagc aatggttaca gcacccagca acttgctcag 60 ctcctcatca ttgcgaacgg caagctgaat atgtcttgga 100 <210> 9 <211> 100 <212> DNA <213>人工序列(Artificial Sequence) <400> 9 aattgtaagg aggtgagcag aggtgctttc tgtacctgta cttgacgttg gaagaggctg 60 gtaatatcgg gttcatatcg gaagagcgtg ctcagatctt 100 <210> 10 <211> 100 <212> DNA <213>人工序列(Artificial Sequence) <400> 10 ttcccctgtc gatggattgc cataaaact tgttcagcac gacgaaggga ggatacccgt 60 ttagaaggaa ccgtgaccaa ccctaataat gcatttcaaa 100 <210> 11 <211> 100 <212> DNA <213>人工序列(Artificial Sequence) <400> 11 tctagttcta gtgattttag cacacagtaa aacacagcag ctttgaatgt atagataaga 60 agatcaaggg aagtgagcag atttggggca atcctctctt 100 <210> 12 <211> 100 <212> DNA <213>人工序列(Artificial Sequence) <400> 12 agaggaacag gaatggggcc ggcgtacttc cttacgagtt attgctgcca acatcagttc 60 caggcgtaac tggtcgtggt atccccaaca gtatttcaat 100 <210> 13 <211> 100 <212> DNA <213>人工序列(Artificial Sequence) <400> 13 atacccatcc tctgcaggat cgtgatgcct ctggggtttc ctgtgtatct tcgaaaaatc 60 cttactaaca ctttgtcgta gaccgccgtg gataccacca 100 <210> 14 <211> 100 <212> DNA <213>人工序列(Artificial Sequence) <400> 14 aaactaaagc catagacagc ataaggacca agaattcaac atctatatga ctatacctgt 60 agagcttgag atgtttggcg agaagcatgt tcctttggtg 100 <210> 15 <211> 100 <212> DNA <213>人工序列(Artificial Sequence) <400> 15 ttacatatcg aattgataga gagaagttcg acttctcacc cgttaaactt ctccagcttt 60 agcccactta ttcctgcaat agtagagaga gcagtcctcc 100 <210> 16 <211> 100 <212> DNA <213>人工序列(Artificial Sequence) <400> 16 atctgattca ttccagtggc ttaagaactt tttggtgtcg tttaggtgtg cttttaactt 60 gagcaatgat atccctgcat tgtcccaaaa tagggaaaa 100 <210> 17 <211> 100 <212> DNA <213> Artificial Sequence <400> 17 agcaacatcg acatcacgcc caccacctgc gtcacgacgg gagtcgatat tctgcaatca 60 cagagtgtca cgcagctcaa caaatgggat tgcctgtacg 100 <210> 18 <211> 100 <212> DNA <213> Artificial Sequence <400> 18 aagctgctga ttcagtcagg tgcagatgtt aatagcttgg atgtttttgg aaggaggcct 60 gctgatttga tcattgttcc tcctaaatta gcccatgcta 100 <210> 19 <211> 100 <212> DNA <213> Artificial Sequence <400> 19 ccgaattggg gctggagtcg gcctgaggag ccgggatttg aacttaggac gtggcaatcg 60 tctgtctgaa gaattgttag gcgattcgga tggccgtgca 100 <210> 20 <211> 100 <212> DNA <213> Artificial Sequence <400> 20 ctccatgatc tcgtcgtcct gttcctgcgc gaatggcaat agttttatat ggttgccttt 60 tccatagcag ctcctgatac tgaacacaaa tctgaagtct 100 <210> twenty one <211> 100 <212> DNA <213> Artificial Sequence <400> twenty one ggtgatgctt ttcttctgag gagtctaagt tgccataact catgtgccca cccgtgtact 60 ccgggctgat cttgtaattg cccataacaa catttttctt 100 <210> twenty two <211> 100 <212> DNA <213> Artificial Sequence <400> twenty two ccattgaaaa attgattgag tccatgaaag taggttgcaa atatgcacat aatggatgta 60 gagagttggt taggtattcc caaatgactg ctcatgagtc 100 <210> twenty three <211> 100 <212> DNA <213> Artificial Sequence <400> twenty three ttcgaccaat attttcggaa ccgtcaaatt gtgagcactt tggggtttcc agccaggggg 60 cttgttgatg gttttgattt aacacttgtt atttgcatgc 100 <210> twenty four <211> 100 <212> DNA <213> Artificial Sequence <400> twenty four ctcgtagtgc ttgagatcgt ggtgtcacag catgaccaga accgaagtca acacgttgaa 60 atggtggcaa gtttgactat gctagcgcat actaacggaa 100 <210> 25 <211> 100 <212> DNA <213>人工序列(Artificial Sequence) <400> 25 aacgtgaaat caattcgaga tttagtagga aagaacagtt tgattgctgt acatggagac 60 ctgcacaaga agctttacgg catagcggcc agtcagctga 100 <210> 26 <211> 100 <212> DNA <213>人工序列(Artificial Sequence) <400> 26 gcctctgcat ccacccctgc caatcatgac ctcggccgcc aagattggtg tgccagagat 60 aaagctcaag agcagacaac gaacgaagga atgaagggtt 100

Claims

1. A liquid phase probe set for identifying Pinus elliottii and Pinus taeda, characterized in that: The liquid phase probe group is a probe having a sequence as shown in SEQ ID NO. 1 to 26.

2. A method for distinguishing Pinus elliottii and Pinus taeda, characterized in that: The method comprises the following steps: The liquid phase probe set according to claim 1 is hybridized with the DNA library of the sample to be tested, and the DNA library hybridized with the capture probe and the target region is amplified by PCR, and subjected to high-throughput sequencing, genotyping, and cluster analysis to determine the classification of the sample to be tested, thereby identifying the species of the sample to be tested.

3. The method according to claim 2, characterized in that The DNA library of the sample to be tested is obtained by fragmenting and end-repairing the total DNA of the sample to be tested, connecting with adapters, amplifying the library and purifying it.

4. The method according to claim 2, characterized in that The method further comprises a reference substance, which is a standard species of elliott pine and loblolly pine.

5. The method according to claim 2, characterized in that The samples to be tested that are clustered together with the standard species of Pinus elliottii are Pinus elliottii, and the samples to be tested that are clustered together with the standard species of Pinus taeda are Pinus taeda.

6. A kit for distinguishing Pinus elliottii and Pinus taeda, characterized in that: The kit comprises the liquid phase probe set according to claim 1.

7. Use of the liquid phase probe according to claim 1, the method according to any one of claims 2 to 5, or the kit according to claim 6 in the identification of elliottii and loblolly pine species.

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