KR102315977B1 - Molecular marker derived from complete sequencing of chloroplast genome for discrimination of Pinus species and uses thereof - Google Patents

Abstract

The present invention relates to a molecular marker for identifying pine species based on a chloroplast genome sequence and its use, and by using the molecular marker according to the present invention, it is possible to quickly and easily distinguish morphologically difficult pine related species. It will be useful for securing the genetic specificity of pine ( Pinus densiflora ) and developing pine varieties.

Description

Molecular marker derived from complete sequencing of chloroplast genome for discrimination of Pinus species and uses thereof

The present invention relates to a molecular marker for identifying a pine species based on the chloroplast genome sequence of five pine trees, and to a use thereof.

Pinaceae is the largest gymnosperm family, comprising 10 genera and more than 230 species. Most pine species are divided into forest species and wood species, mainly distributed in the northern hemisphere. Pinus densiflora siebold & zucc., also called Korean red pine, is widely distributed in East Asia including Korea, Japan and China. This species accounts for more than 23% of South Korea's forest land and is the most important and popular coniferous species in Korea.

The chloroplast genome is an important resource for phylogenetic studies at the molecular level, exhibits uniparental inheritance, and contains conserved sequences due to the slower rate of evolutionary change compared to the nuclear genome. Circular DNA (110-210 kb) exists in multiple copies in the chloroplast, and the chloroplast DNA of seed plants contains a large single copy (LSC) region, a small single copy (SSC) region, and a pair of inverted repeats (IRs). It has a quadripartite structure. Most land plants have between 110 and 130 genes in the chloroplast genome.

The IR structure of the chloroplast genome generally ranges from 15 to 30 Kb. Due to the expansion or rearrangement of the IR, the number and sequence of genes exhibits diversity within species. Large IRs play an important role in the stabilization of the chloroplast genome, and loss of large IRs leads to some variations in genome structure and gene content. The reduction in IR sequence has been confirmed in species of Pineaceae, Taxaceae, Cephalotaxaceae and Legumes.

The development of Next Generation Sequencing (NGS) technology has made it possible to obtain full-length chloroplast sequences easily and inexpensively. However, this technique has limitations in producing short reads of several hundred bases or less. Short reads cause mis-mappings and mis-alignments, and produce heterogeneous and repetitive regions of the genome that are difficult to access. In addition, when only short leads are used, it is difficult to identify structural variations or haplotype structures. Oxford Nanopore Technology (ONT), a new third-generation sequencing platform, offers a future-proof USB-powered sequencer with significantly longer read lengths than previous technologies. The use of long reads in ONT is efficient because there is no need to align short reads for complete sequence. To overcome the high error rate of ONT reads, high quality MiSeq short reads can be used for error correction.

Meanwhile, Korea Patent No. 1409012 discloses a 'SNP primer for distinguishing pine trees, a kit including the same and a method for distinguishing pine trees using the same' based on the trnT(UGU)-trnL(UAA)3' exon of the chloroplast. , There is no description of a molecular marker for identifying a pine species based on the chloroplast genome sequence of the present invention and its use.

The present invention was derived from the above needs, and the present inventors completely analyzed the chloroplast genome of Pinus densiflora, a Korean pine tree, through Oxford Nanopore MinION and Illumina MiSeq sequencing. Thereafter, the sequence of the chloroplast genome of the analyzed pine tree was compared with the chloroplast genome sequence of P. sylvestris , P. thunbergii , Chinese pine ( P. tabuliformis ) and Teda pine ( P. taeda ). After confirming the polymorphic site, four primer sets that can distinguish pine species based on the polymorphic site were prepared, and then the pine species discrimination ability of the primer set was evaluated for pine, spruce pine, gom pine, and teda pine. As a result of the assay, the present invention was completed by confirming that the four primer sets can effectively distinguish each pine species.

In order to solve the above problems, the present invention comprises one or more primer sets selected from the group consisting of oligonucleotide primer sets of SEQ ID NO: 5 and SEQ ID NO: 6 and oligonucleotide primer sets of SEQ ID NO: 7 and SEQ ID NO: 8, pine A primer set for species identification is provided.

In addition, the present invention provides a kit for identifying the species of pine, including the primer set and reagents for performing the amplification reaction.

In addition, the present invention comprises the steps of isolating genomic DNA from a pine tree sample; amplifying a target sequence by using the isolated genomic DNA as a template and performing an amplification reaction using the primer set of the present invention; and detecting the product of the amplification step; it provides a pine species identification method comprising a.

By using the molecular marker based on the chloroplast genome sequence according to the present invention, it is possible to quickly and easily distinguish pine related species that are difficult to morphologically classify. In addition, it may be usefully utilized as a marker for securing the genetic specificity of Pinus densiflora , a Korean pine tree, and for developing pine varieties.

1 is a pine ( Pinus densiflora ) genetic map of the chloroplast genome.
2 is a pairwise alignment result of chloroplast genome sequences of five pine species. P. densiflora : Pine, P. sylvestris : Old pine, P. thunbergii : Gombri, P. tabuliformis : Chinese pine, P. taeda : Teda pine.
3 shows the distances between adjacent intergenic, SSC, LSC, and junction points of two IRs in the chloroplast genomes of five pine species. P. densiflora : Pine, P. sylvestris : Old pine, P. thunbergii : Gombri, P. tabuliformis : Chinese pine, P. taeda : Teda pine.
4 is a phylogenetic tree based on CDS of pine ( P. densiflora ) and 12 reference species.
5 is an analysis result of amplified DNA fragments of each pine tree sample using four primer sets (Marker 1 to Marker 4). P. densiflora : Pine, P. sylvestris : P. sylvestris: P. thunbergii : Gompine, P. taeda : Tedapine.

In order to achieve the object of the present invention, the present invention comprises one or more primer sets selected from the group consisting of oligonucleotide primer sets of SEQ ID NO: 5 and SEQ ID NO: 6 and oligonucleotide primer sets of SEQ ID NO: 7 and SEQ ID NO: 8 , provides a primer set for identification of pine species.

In the primer set for discrimination of pine species of the present invention, the pine species is pine ( Pinus densiflora , red pine), old pine tree ( P. sylvestris ), gomsol ( P. thunbergii ) and the pine tree ( P. taeda ) The group consisting of It may be one or more selected from, but is not limited thereto.

In the primer set according to an embodiment of the present invention, the primer set is based on a polymorphic marker based on the complete sequence of the chloroplast genome of 5 types of pine trees (pine pine, old pine, gom pine, Chinese pine, and teda pine), SEQ ID NO: The oligonucleotide primer set of 5 and SEQ ID NO: 6 (Marker 3) and the oligonucleotide primer set of SEQ ID NO: 7 and SEQ ID NO: 8 (Marker 4) are markers capable of distinguishing between pine, spruce pine, gomsol and teda pine, respectively. (See Table 5 and Figure 5).

The primer set of the present invention preferably comprises one or more primer sets selected from the group consisting of the two primer sets, and two primer sets, namely, the oligonucleotide primer sets of SEQ ID NO: 5 and SEQ ID NO: 6 and the sequences It may include both the oligonucleotide primer sets of No. 7 and SEQ ID No. 8. If two primer sets are used simultaneously, pine species identification can be performed more efficiently.

In addition, in the primer set according to the present invention, the primer set may further include an oligonucleotide primer set of SEQ ID NO: 3 and SEQ ID NO: 4, and the oligonucleotide primer set of SEQ ID NO: 3 and SEQ ID NO: 4 is a savior It is a marker that can specifically distinguish only Korean pine ( P. densiflora ) from pine, pine, and cedar pine.

The primer may include an oligonucleotide consisting of a fragment of 16 or more, 17 or more, 18 or more, 19 or more, 20 or more consecutive nucleotides in the sequence of SEQ ID NOs: 3 to 8, depending on the sequence length of each primer. have. For example, the primer (22 oligonucleotides) of SEQ ID NO: 3 is a fragment of 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more consecutive nucleotides in the sequence of SEQ ID NO: 3 It may comprise an oligonucleotide consisting of In addition, the primer may also include an addition, deletion or substitution of the nucleotide sequence of SEQ ID NOs: 3 to 8.

In the primer set of the present invention, the nucleotide sequences shown in SEQ ID NOs: 3, 5 and 7 represent forward primers, and the nucleotide sequences shown in SEQ ID NOs: 4, 6 and 8 represent reverse primers.

In the present invention, "primer" refers to a single-stranded oligonucleotide sequence complementary to a nucleic acid strand to be copied, and can serve as a starting point for synthesis of a primer extension product. The length and sequence of the primers should allow synthesis of the extension product to begin. The specific length and sequence of the primer will depend on the conditions of use of the primer, such as temperature and ionic strength, as well as the complexity of the DNA or RNA target required.

As used herein, the oligonucleotide used as a primer may also contain a nucleotide analogue, for example, a phosphorothioate, an alkylphosphorothioate or a peptide nucleic acid or An intercalating agent may be included.

The present invention also provides a kit for identifying pine species, including the primer set and reagents for performing the amplification reaction.

In the kit of the present invention, the primer set is the same as described above, and the reagent for performing the amplification reaction may include DNA polymerase, dNTPs, a buffer, and the like. In addition, the kit of the present invention may further include a user's guide describing optimal conditions for performing the reaction. A handbook is a printout explaining how to use the kit, eg, how to prepare a PCR buffer, and suggested reaction conditions. Instructions include a brochure in the form of a pamphlet or leaflet, a label affixed to the kit, and instructions on the surface of the package containing the kit. In addition, the guide includes information published or provided through electronic media such as the Internet.

In the kit according to the present invention, the pine species is pine ( Pinus densiflora ), pine tree ( P. sylvestris ), gom pine ( P. thunbergii ) and cedar pine ( P. taeda ) It may be at least one selected from the group consisting of However, it is not limited thereto.

The present invention also provides a method for discriminating a pine species using the primer set of the present invention. The method is specifically,

isolating genomic DNA from a pine tree sample;

amplifying a target sequence by using the isolated genomic DNA as a template and performing an amplification reaction using the primer set of the present invention; and

It may include; detecting the product of the amplification step.

The method of the present invention includes isolating genomic DNA from a pine tree sample. A method for isolating genomic DNA from the sample may use a method known in the art, for example, a CTAB method may be used, or a Wizard prep kit (Promega) may be used. A target sequence may be amplified by using the isolated genomic DNA as a template and performing an amplification reaction using the primer set according to an embodiment of the present invention. Methods for amplifying a target nucleic acid include polymerase chain reaction (PCR), ligase chain reaction, nucleic acid sequence-based amplification, and transcription-based amplification systems. amplification system), strand displacement amplification or amplification via Qβ replicase, or any other suitable method for amplifying nucleic acid molecules known in the art. Among them, PCR is a method of amplifying a target nucleic acid from a primer pair that specifically binds to the target nucleic acid using a polymerase. Such PCR methods are well known in the art, and commercially available kits may be used.

In the method of the present invention, the amplified target sequence may be labeled with a detectable labeling substance. In one embodiment, the labeling material may be a material emitting fluorescence, phosphorescence, or radioactivity, preferably, FAM, HEX, VIC, JOE, ROX, TAMRA, Cy3 or Cy5, etc., but is not limited thereto. When PCR is performed by labeling the labeling material at the 5'-end of the primer during amplification of the target sequence, the target sequence may be labeled with a detectable fluorescent labeling material. ^{In addition, when a radioactive isotope such as 32} P or ³⁵ S is added to the PCR reaction solution for labeling using a radioactive material, the amplification product is synthesized and radioactivity is incorporated into the amplification product, so that the amplification product can be radioactively labeled. The primer sets used to amplify the target sequence are as described above.

In the method according to an embodiment of the present invention, the detection of the amplification step product may be performed through a DNA chip, gel electrophoresis, capillary electrophoresis, radiometric measurement, fluorescence measurement or phosphorescence measurement, but is not limited thereto. As one of the methods for detecting the amplification product, capillary electrophoresis may be performed. Capillary electrophoresis may use, for example, the ABI Sequencer. In addition, gel electrophoresis can be performed, and agarose gel electrophoresis or acrylamide gel electrophoresis can be used for gel electrophoresis depending on the size of the amplification product. In addition, in the fluorescence measurement method, when PCR is performed by labeling the 5'-end of the primer with Cy-5 or Cy-3, the target sequence is labeled with a detectable fluorescent labeling material, and the labeled fluorescence is measured using a fluorometer. can do. In addition, the radioactive measurement method is a ^{radioactive isotope such as 32} P or ³⁵ S is added to the PCR reaction solution during PCR to label the amplification product, and then a radioactive measuring instrument, for example, a Geiger counter (Geiger counter) or liquid scintillation The radioactivity can be measured using a liquid scintillation counter.

below. The present invention will be described in detail by way of Examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

Materials and Methods

1. Sampling, DNA Extraction and Sequencing

Fresh leaves of pine ( Pinus densiflora ) (MK285358) were collected from designated cultural properties (333-360 Jungyeong-gil, Miro-myeon, Samcheok-si, Gangwon-do, Jungyeongmyo, N 37"22'2" E 129"3'32"), and total genomic DNA was ~100 mg of frozen leaves were extracted using the Exgene ^™ Plant SV kit (GeneAll Biotechnology Co., LTD, Korea) according to the manufacturer's instructions. To construct two types of DNA libraries, Illumina MiSeq and Oxford Nanopore libraries were constructed using the TruSeq Nano DNA kit having an average insert size of 670 bp and the Rapid sequencing kit (SQK-RAD004) having an average insert size of 55 kbp, respectively. prepared. The two genomic libraries were then sequenced on PHYZEN (http://phyzen.com) with Illumina MiSeq and Oxford Nanopore MinION platforms.

2. Assembling and Annotating Chloroplast Genome Sequences

ONM raw data are basecalled to the default option of the albacore program (https://github.com/JGI-Bioinformatics/albacore/blob/master/README.md), and then adapter and chimeric sequences are Removed as default option of porechop program (https://github.com/rrwick/Porechop). Thereafter, MinION leads were assembled de novo using smartdenovo (https://github.com/ruanjue/smartdenovo). Thereafter, the assembled contigs were compared to the NCBI (National Center for Biotechnology Information) base database with the BlastN program to select chloroplast contigs. The overlapping of unitigs was used to assemble the complete chloroplast genome sequence. Illumina MiSeq reads were mapped to the full-length chloroplast genome using the CLC_ref_assemble tool from the CLC assembly cell package (version 4.21, CLC Inc, Aarhus, Denmark). Error correction was performed by manual curation. Gene annotation is the default option of the GeSeq program (https://chlorobox.mpimp-golm.mpg.de/geseq-app.html), which uses Blast searches to identify protein-coding gene regions, ARAGORN (version 1.2. 3) and tRNAscan-SE (version 2.0.3) were used to determine the tRNA gene. The exact gene site was determined by manual curation using the Artemis annotation tool (https://www.sanger.ac.uk/science/tools/artemis). The circular gene map was prepared using Organellar Genome DRAW software (ORDRAW).

3. Sort and build phylogenetic trees

A BlastN search was performed in the NCBI database with the newly sequenced chloroplast genome sequence. Through this process, the entire chloroplast genome sequence of the species with the highest similarity was identified and downloaded. The assembled chloroplast DNA sequence was compared with the chloroplast genome sequence of the most similar species, P. sylvestris. The sequence of the protein coding gene (CDS) was aligned using mVISTA. For phylogenetic analysis, 12 Pineaceae (Pinus, Picea, Larix and Abies) plants and 1 yew (Taxus) plants were selected. The outgroup was set as Western attention ( Taxus baccata ). 59 commonly conserved protein-coding genes were extracted from a total of 13 plant species as follows: accD, atpA, atpB, atpE, atpF, atpH, atpI, chlB, chlL, chlN, infA, matK, petA, petB, petD, petG, psaA, psaB, psaC, psaI, psaJ, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, rpl16, psbT rpl2, rpl20, rpl22, rpl23, rpl32, rpl33, rpl36, rpoA, rpoB, rpoC1, rpoC2, rps11, rps12, rps14, rps15, rps18, rps19, rps3, rps4, rps7, rps4 . Multiple sequence alignment was performed using the MAFFT program (version 7), and then, the maximum likelihood phylogenetic tree was fabricated with a general time reversible (GTR) model using the MEGA6 program and 1,000 bootstraps.

Example 1. Korean pine ( P. densiflora ) chloroplast genome sequencing

The complete chloroplast genome of pine ( P. densiflora ) was sequenced using the ONM sequencer, and errors were corrected through short reads of Illumina MiSeq. After mapping the ONM reads to the complete chloroplast genome sequence, the sequencing error rate for each nucleotide position was calculated and confirmed to be 9.29%. The total amount of raw data production was 14.6 Gb for MiSeq and 2.1 Gb for ONM. The average coverage of the chloroplast genome was 303.27× for MiSeq and 28.46× for ONM. The complete chloroplast genome sequence of pine ( P. densiflora ) was submitted to GenBank (accession number MK285358).

NGS result information of red pine sequencing platform Input reads Trimmed reads raw bases trimmed bases MiSeq 49,013,296 40,786,223 (83.21%) 14,563,262,097 10,101,761,675 (69.36%) ONM 305,965 306,493 (100.21%) 2,116,530,768 2,089,930,503 (98.74%)

The chloroplast of pine ( P. densiflora ) has a circular DNA of 119,875 bp in size and is a typical quadripartite containing a pair of large single copy (LSC), small single copy (SSC) and inverted repeats (IRs). had a structure. LSC of P. densiflora was identified as 65,654 bp, SSC as 53,231 bp, and each IR as 495 bp. The chloroplast genome of newly assembled pine ( P. densiflora ) was found to be somewhat larger than that of other reported pine species except for teda pine (Table 2).

Features of the chloroplast genome in the genus Pine Genome size (bp) LSC length (bp) SSC length (bp) IR length (bp) Number of genes P. densiflora (MK285358) 119,875 65,654 53,231 495 113 P. sylvestris (KR476379) 119,758 65,559 53,209 495 112 P. thunbergii (D17510) 119,707 65,696 53,021 495 113 P. tabuliformis (KT740995) 119,646 65,618 53,038 495 114 P. taeda (KC427273) 121,530 66,272 54,288 485 110

The chloroplast genome of Korean pine was confirmed to encode 108 unique genes, including 72 protein-coding genes, 32 tRNA genes, and 4 rRNA genes (Table 3). Among these genes, it was confirmed that trnI-CAU was repeated in the IR region, two copies of psaM and trnS-GCU were present in the LSC region, and one copy of trnH-GUG and trnT-GGU was present in the LSC and SSC regions, respectively. Duplicated genes were equally repeated, with the exception of trnT-GGU , four pairs of reversed orientations. A total of 113 genes encoding 73 proteins, 36 tRNAs and 4 rRNAs were found in the chloroplast genome. Of the 108 unique genes, 12 genes contained one intron and two genes contained two introns. The intron ranged from 479 bp of trnL-UAA to 2,501 bp of trnK-UUU. The rps12 gene has two exons, the first exon was identified at the LSC site, and the second and third exons were identified at the SSC site, suggesting that trans-splicing would be necessary. Five ndh genes ( ndhB, ndhD, ndhE, ndhH and ndhI ) were pseudogenerated. A genetic map of the newly produced pine ( P. densiflora ) chloroplast genome is shown in FIG. 1 .

List of Annotated Genes in the Chloroplast Genome of Pine ( P. densiflora ) Function Genes RNAs, ribosomal rrn4.5, rrn5, rrn16, rrn23 RNAs, transfer trnA-UGC ^* , trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnG-GCC, trnG-UCC ^* , trnH-GUG , trnI-CAU , trnI-GAU ^* , trnK-UUU ^*,T , trnL-CAA, trnL-UAA ^* , trnL-UAG, trnM-CAU, trnfM-CAU, trnN-GUU, trnP-UGG, trnP-GGG, trnQ-UUG, trnR-ACG, trnR-CCG, trnR-UCU, trnS-GCU , trnS-GGA, trnS-UGA, trnT-GGU , trnT-UGU, trnV-GAC, trnV-UAC ^* , trnW-CCA, trnY-GUA Transcription and splicing rpoA, rpoB, rpoC1 ^* , rpoC2, matK Translation, ribosomal proteins Small subunit rps2, rps3, rps4, rps7, rps8, rps11, rps12 ^**,T , rps14, rps15, rps18, rps19 Large subunit rpl2 ^* , rpl14, rpl16 ^* , rpl20, rpl22, rpl23, rpl32, rpl33, rpl36 Photosynthesis ATP synthase atpA, atpB, atpE, atpF ^* , atpH, atpI Photosystem I psaA, psaB, psaC, psaI, psaJ, psaM , ycf3 ^** , ycf4 Photosystem II psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ Calvin cycle rbcL Cytochrome complex petA, petB ^* , petD ^* , petG, petL, petN Chlorophyll biosynthesis chlB, chlL, chlN Others clpP, accD, cemA, ccsA, infA, ycf1, ycf2, ycf12

^* : gene including one intron, ^** : gene including two introns, ^T : trans-splicing of related genes, bold: 2 gene copies.

Example 2. Comparative analysis of chloroplast genomes between species of pine trees to confirm DNA mutations

The genetic composition and sequence of the chloroplast genome of pine ( P. densiflora ) has been previously published chloroplast genomes of four species of pine ( P. sylvestris ): KR476379, P. thunbergii ): D17510, Chinese pine ( P. tabuliformis ): KT740995, P. taeda : KC427273) and ycf gene and psaM gene were almost similar except for duplication. The pine species SNP (single nucleotide polymorphisms) or InDel (insertion/deletions) polymorphisms were found in the inter-genic region ( FIG. 2 ). However, the ycf1 and ycf2 regions showed low sequence similarity between species. In addition, a huge base insertion was observed between the trnE- UUC and clpP genes in the LSC region of pine (P. densiflora).

The border structures were compared in detail in the chloroplast genomes of 5 species of pine trees (FIG. 3). Consistent with the pairwise sorting results, pine was most similar to old pine, and Teda pine showed a significant difference from the other 4 types of pines in the IR area. In the four types of pine except Teda pine, the gene positioning of the IRs region was stable, whereas the SSC and LSC regions were found to be mutated regions. The IR region had trnI-CAU and was identified as having a portion of psbA on the IRa/LSC border (border), and the gene size and location were well conserved. Genes adjacent to the border of the LSC and SSC regions were observed to have 1 to 11 bp mutations. The trnK-UUU gene was located in the LSC region 1,500-1,511 bp away from the SSC/IRa border. The first exon of the rpl2 gene was located 1,463~1,468 bp upstream of the LSC/IRb border, and the trnF-GAA and trnH-GUG genes were located in the SSC region of 1,530~1,537 bp and 144~169 bp from the border, respectively. .

A phylogenetic tree was prepared based on the chloroplast genome sequence alignment of 12 Pineaceae plant species and 1 Yum family (Taxaceae) plant species, and Pine ( P. densiflora ) was confirmed to be the most closely related to the Pine tree (Fig. 4 and Table 4). ).

Comparison of chloroplast genomes of 12 coniferous species Species Accession No. No. of protein coding genes No. of common protein coding genes with P. densiflora Pinus sylvestris KR476379 73 73 Pinus tabuliformis KT740995 74 73 Pinus thunbergii D17510 69 69 pinus taeda KC427273 71 71 Pinus strobus KP099650 - - Pinus koraiensis AY228468 74 72 Pinus sibirica KT723438 77 73 Picea abies HF937082 74 72 Larix Decidua AB501189 72 71 Abies koreana KP742350 74 73 Abies sibirica KR476376 74 73 Taxus baccata KR476375 81 70

Example 3. Molecular markers for identification of pine species

A primer set was prepared to amplify the PCR product including the target site with a gap of 6 bp or more in the pairwise alignment of Gomsol and spruce. The primer set was prepared using the Primer3 tool (v.0.4.0, http://bioinfo.ut.ee/primer3-0.4.0/), and the condition of the oligonucleotide during the preparation of the primer set was 18-27 bp in length. , Tm (dissolution temperature) 57-63°C, GC content ranged from 20-80%. The oligonucleotide primer set of SEQ ID NO: 1 and SEQ ID NO: 2 is a part of gene aptB, the oligonucleotide primer set of SEQ ID NO: 3 and SEQ ID NO: 4 is a part of gene chlN , and the oligonucleotide primer set of SEQ ID NO: 5 and SEQ ID NO: 6 is a gene A part of trnL-UAA, the oligonucleotide primer sets of SEQ ID NO: 7 and SEQ ID NO: 8 include a partial sequence of gene ycf2. Table 5 below summarizes the molecular markers for identifying pine species produced by the above method.

Molecular markers for identification of pine species Marker 1 Forward 5'-TCTGATCGACCCAATATGGA-3' (SEQ ID NO: 1) Reverse 5'-TTTGAGCAATACGTCCCACA-3' (SEQ ID NO: 2) Species GenBank ID. start end PCR product (bp) P. densiflora MK285358 45028 45197 170 P. sylvestris KR476379 45011 45192 182 P. thunbergii D17510 45048 45216 169 P. taeda KC427273 45745 45907 163 Marker 2 Forward 5'-CAATTTCTCCACTTCTCCACAA-3' (SEQ ID NO: 3) Reverse 5'-TGATAATTGCCCGTTTCCACA-3' (SEQ ID NO: 4) Species GenBank ID. start end PCR product (bp) P. densiflora MK285358 93785 93992 208 P. sylvestris KR476379 93765 93961 197 P. thunbergii D17510 93805 94001 197 P. taeda KC427273 94604 94800 197 Marker 3 Forward 5'-CCGTAGCGTCTACCAATTCC-3' (SEQ ID NO: 5) Reverse 5'-CCAGTCATATTCGATTGGGGTA-3' (SEQ ID NO: 6) Species GenBank ID. start end PCR product (bp) P. densiflora MK285358 68671 68894 224 P. sylvestris KR476379 68664 68877 214 P. thunbergii D17510 68719 68914 196 P. taeda KC427273 69385 69602 218 Marker 4 Forward 5'-AGCACAATCCGTTCAACTCTC-3' (SEQ ID NO: 7) Reverse 5'-ATCCTCCCGTGCCTAATAGC-3' (SEQ ID NO: 8) Species GenBank ID. start end PCR product (bp) P. densiflora MK285358 115145 115345 201 P. sylvestris KR476379 115120 115314 195 P. thunbergii D17510 115016 115201 186 P. taeda KC427273 116977 117144 168

Example 4. Verification of molecular markers for identifying pine species

Using the molecular markers in Table 5, PCR was performed on DNA samples of pine, spruce pine, gom pine, and teda pine. The product amplified by PCR was analyzed using a fragment analyzer. For PCR, BioFACT™ A-Star Taq DNA Polymerase kit was used and a reaction solution was prepared according to the manufacturer's instructions. As primers, an oligo labeled with a fluorescent substance FAM in the forward sequence of each marker and an oligo having a reverse sequence of each marker were used as a set. The PCR step was repeated 25 times for 20 seconds at 95°C, 20 seconds at 58°C, and 20 seconds at 72°C after heat denaturation at 95°C for 2 minutes, followed by a final extension reaction at 72°C for 5 minutes. Fragment size was measured using ThermoFisher's GeneScan™ 500ROX™ Size Standard reagent, followed by reaction at 95°C for 5 minutes and ice for 15 minutes according to the manufacturer's instructions, and then analyzed with a fragment analyzer.

As a result of the analysis, it was confirmed that Marker 1 was able to distinguish the cedar pine from the pines, spruce pines, and pines, and Marker 2 specifically distinguished only Korean pines (P. It could be confirmed that this could be done, and it was found that Marker 3 and Marker 4 were markers that could distinguish pine, spruce pine, gom pine, and teda pine, respectively. In particular, it was found that Marker 2 could specifically distinguish Korean pines from Korean pine trees, which are the most closely related species.

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Claims (8)

A primer set composition for discrimination of pine species comprising at least one primer set selected from the group consisting of oligonucleotide primer sets of SEQ ID NO: 5 and SEQ ID NO: 6 and oligonucleotide primer sets of SEQ ID NO: 7 and SEQ ID NO: 8,
The pine species is pine ( Pinus densiflora ), old jujus tree ( P. sylvestris ), gomsol ( P. thunbergii ) and cedar pine ( P. taeda ) Primer for identifying pine species, characterized in that at least one selected from the group consisting of set composition. According to claim 1, wherein the primer set composition further comprises an oligonucleotide primer set of SEQ ID NO: 3 and SEQ ID NO: 4, the primer set composition for discrimination of pine species. delete A kit for identifying pine species, comprising the primer set composition of claim 1 or 2 and a reagent for performing an amplification reaction,
The pine species is a pine species ( Pinus densiflora ), old jujus tree (P. sylvestris ), gomsol ( P. thunbergii ) and pine tree ( P. taeda ) Kit for identifying pine species, characterized in that at least one selected from the group consisting of . The kit according to claim 4, wherein the reagents for performing the amplification reaction include DNA polymerase, dNTPs, and a buffer. isolating genomic DNA from a pine tree sample;
amplifying the target sequence by using the isolated genomic DNA as a template and performing an amplification reaction using the primer set composition of claim 1 or 2; and
As a pine species identification method comprising; detecting the product of the amplification step,
The pine species is a pine species ( Pinus densiflora ), old Jujus tree (P. sylvestris ), gomsol ( P. thunbergii ) and pine tree (P. taeda ) Pine species identification method, characterized in that at least one selected from the group consisting of. delete The method according to claim 6, wherein the detection of the product of the amplification step is performed through a DNA chip, gel electrophoresis, capillary electrophoresis, radiometric measurement, fluorescence measurement or phosphorescence measurement.

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