KR102672578B1 - SNP marker composition for discriminating Cyperaceae plant 'Carex nervata' and uses thereof - Google Patents

Abstract

The present invention relates to a SNP marker composition and its use for identifying the Cyperaceae plant 'Sedge Sedge'. Since the SNP marker of the present invention can accurately identify Sedge Sedge, a Cyperaceae plant, it provides variety protection and seed management systems for Cyperaceae plants. It may be useful for establishment.

Description

SNP marker composition for discriminating Cyperaceae plant 'Carex nervata' and uses thereof}

The present invention relates to a SNP marker composition and its use for identifying the Cyperaceae plant 'Yangji Sedge'.

Yangji sedge ( Carex nervata ) is a perennial plant of the Cyperaceae family. It is distributed in Korea (Mt. Hallasan, Mt. Jiri, northern regions) and Japan, and mainly grows in dry grasslands. The rhizome extends sideways and is thin, and the stems are clustered and are 10 to 30 cm high. The leaves are flat, 2-3 mm wide, shorter than the flower stem, and the leaf sheath at the bottom is split like fibers and is light brown. Two to four spikelets hang on the upper part of the stem. The spikelet at the end of the stem has male flowers, and the spikelet hanging next to the stem has female flowers and has a short stalk. The lowest bract is needle-shaped and the bottom is barrel-shaped. The bracts surrounding the fruit run at an angle and are shaped like an upside-down triangular egg, 2 to 2.5 mm long, with thin veins and fine hairs. The fruit is an achene, shaped like an upside-down triangular egg with a length of 1.5 mm, a tray-shaped shape with a compressed end, and three stigmas.

Cyperaceae is a plant group belonging to the Poales family of monocots of the angiosperm group. It is widely distributed in temperate regions of the northern and southern hemispheres, and about 109 genera and 5,500 species have been reported worldwide. It is known that there are over 300 taxa in Korea, and the Cyperaceae plants, which were organized by Linne into the genus Scirpus sp., Cyperus sp., and Carex sp., were recognized by later scholars. Many classifications have been made at the genus level.

Cyperaceae is a plant that is treated as a weed along with plants of the Poaceae family, but it is one of the developed plant groups with very large species diversity in the history of plant evolution, and is a major plant group that makes up the grassland ecosystem. Cyperaceae and Poaceae have similar shapes of leaves, stems, etc., and the ripening period of the fruit, which is the most important characteristic in classifying the species, is only 2 to 3 weeks, and when the fruit ripens, it falls off, leaving only the leaves, making accurate identification difficult. Although Cyperaceae is widely used for medicinal and edible purposes, there is a lack of systematic research worldwide. Therefore, in order to utilize Cyperaceae plants as biological resources, it is urgent to reveal their taxonomic identity and identify their characteristics.

Meanwhile, Korean Patent No. 2086854 discloses 'antioxidant or anti-inflammatory cosmetic composition containing extract of Cyperaceae plants', but the SNP marker composition for identifying the Cyperaceae plant 'Yangji Cypress' of the present invention and its use are not described. There is no bar.

The present invention was derived from the above needs, and the present inventors assembled the chloroplast genome sequence of Carex nervata and then assembled the reference chloroplast genome sequence of Carex laevissima (GenBank accession No. MZ846224.1). ), a SNP (single nucleotide polymorphism) marker was discovered in the rpoB and atpB intergenic regions. In addition, a set of KASP (Kompetitive allele specific PCR) primers based on the SNP marker was prepared and PCR was performed on 10 species of Cyperaceae plants, including Sedge sedge. As a result, it was found that the KASP primer set of the present invention can accurately distinguish Sedge sedge. By confirming that this was possible, the present invention was completed.

In order to solve the above problem, the present invention is a polynucleotide consisting of 8 or more consecutive nucleotides containing a SNP (single nucleotide polymorphism) base located at the 301st position in the base sequence of SEQ ID NO: 1, or a complementary polynucleotide thereof. , Provides a SNP marker composition for identifying Yangji sedge ( Carex nervata ).

In addition, the present invention provides a microarray for discriminating onychophyllum, including a polynucleotide containing the above SNP base or cDNA thereof.

In addition, the present invention provides a probe for discriminating between Sedge sedges, comprising a polynucleotide containing the above SNP base or cDNA thereof.

In addition, the present invention provides a primer set for discriminating Sedge sedge, consisting of oligonucleotide primers of SEQ ID NOs: 2 to 4 for amplifying polynucleotides containing the SNP base.

In addition, the present invention provides the primer set; And a reagent for performing an amplification reaction; It provides a kit for determining Yangji Sedge.

In addition, the present invention includes the steps of isolating genomic DNA from a Cyperaceae plant sample; Using the isolated genomic DNA as a template and performing an amplification reaction using the primer set according to the present invention to amplify the target sequence; and detecting the product of the amplification step. It provides a method for identifying Yangji Sedge, including a.

Since the SNP marker of the present invention can accurately identify Sedge sedge, a sedge plant, it can be usefully used to protect varieties of sedge plants and establish a seed management system.

Figure 1 is a chloroplast genetic map of Yangji sedge ( Carex nervata ).
Figure 2 shows the KASP (Kompetitive allele specific PCR) primer set of the present invention (Table 6) used to test 10 species of Cyperaceae plants (Sedge sedge, Sedge sedge, White sedge, Gaetcheong sedge, Mokpo sedge, Gagcheong sedge, This is the result of KASP analysis on fine white sedge, Jindo sedge, Yangji sedge, and wrinkled sedge).

In order to achieve the purpose of the present invention, the present invention provides a polynucleotide consisting of 8 or more consecutive nucleotides containing a SNP (single nucleotide polymorphism) base located at position 301 in the base sequence of SEQ ID NO: 1, or a complementary polynucleotide thereof. It provides a SNP marker composition for discrimination of Yangji sedge ( Carex nervata ), including:

In one embodiment of the present invention, the contiguous nucleotides may be 8 to 100 contiguous nucleotides, but are not limited thereto.

In this specification, the term 'nucleotide' refers to a deoxyribonucleotide or ribonucleotide that exists in single-stranded or double-stranded form, and includes analogs of natural nucleotides unless specifically stated otherwise.

In the SNP marker composition according to one embodiment of the present invention, the SNP position base is 301st in the base sequence of SEQ ID NO: 1, and the polymorphic base information is indicated as [/] in the SNP sequence information in Table 5. The base sequence of SEQ ID No. 1 of the invention refers to a sequence containing the base located before the slash (/) in Table 5.

The reason that the SNP marker of the present invention can be used to identify Sedge sedge is based on the fact that the 301st SNP mutation position in the nucleotide sequence shown in SEQ ID NO: 1 is expressed differently as T or C. In the base at the SNP position, if the 301st base in the base sequence of SEQ ID NO. 1 is T, it is C. brevispicula, and if the 301st base is C, it is C. brevispicula and Aster. ( C. tristachya ), White sedge ( C. mollicula ), C. breviculmis var. fibrillosa ), Mokpo sedge ( C. genkaiensis ), Branched sedge ( C. polyschoena ), White sedge ( C. alopecuroides ), C. taihokuensis , or C. rugata .

The present invention relates to a nucleotide variant of the SNP position in the nucleotide sequence of SEQ ID NO: 1, but when such SNP nucleotide mutation is found in double-stranded gDNA (genomic DNA), it also includes a polynucleotide sequence complementary to the nucleotide sequence. It is interpreted that Therefore, the base at the SNP position in the complementary polynucleotide sequence also becomes a complementary base. In this respect, all sequences presented herein are based on sequences in the sense strand of genomic DNA, unless otherwise specified.

The present invention also provides a microarray for discriminating between Sedge chinensis and its cDNA, including a polynucleotide containing the SNP base or its cDNA.

Preferably, the polynucleotide may be immobilized on a substrate coated with an active group of amino-silane, poly L-lysine, or aldehyde, but is not limited thereto. Additionally, preferably, the substrate may be a silicon wafer, glass, quartz, metal, or plastic, but is not limited thereto. Methods for immobilizing the polynucleotide on a substrate include micropipetting using a piezoelectric method, a method using a pin-shaped spotter, etc.

As used herein, the term 'substrate' refers to any substrate that possesses hybridization properties and to which a marker can be attached under conditions that the background level of hybridization is kept low. Typically, the substrate may be a microtiter plate, membrane (eg, nylon or nitrocellulose), microspheres (beads), or a chip. Before application to or immobilization on a membrane, nucleic acid probes can be modified to facilitate immobilization or improve hybridization efficiency. The modifications may include homopolymer tailing, coupling with different reactive functional groups such as aliphatic groups, NH ₂ groups, SH groups and carboxyl groups, or coupling with biotin, haptens or proteins. .

The microarray according to the present invention can be prepared by conventional methods known to those skilled in the art using the polynucleotide according to the present invention or its complementary polynucleotide, the polypeptide encoded by it, or its cDNA.

The present invention also provides a probe for discriminating onychophyllum, comprising a polynucleotide containing the above SNP base or cDNA thereof.

As used herein, the term 'probe' refers to a hybridization probe, which refers to a linear oligomer having a natural or modified monomer or linkage containing deoxyribonucleotides and ribonucleotides capable of sequence-specific binding to the complementary strand of a nucleic acid. . The probe of the present invention is an allele-specific probe, in which a polymorphic site is present among nucleic acid fragments derived from two members of the same species, and hybridizes to the DNA fragment derived from one member, but does not hybridize to the fragment derived from the other member. . Preferably, the probe may be single stranded for maximum efficiency in hybridization, more preferably a deoxyribonucleotide, but is not limited thereto.

As used herein, the term 'hybridization' refers to complementary single-stranded nucleic acids forming double-stranded nucleic acids. Hybridization can occur between two nucleic acid strands that are either completely matched or substantially matched with some mismatches. Complementarity for hybridization may vary depending on hybridization conditions, especially temperature.

As a probe used in the present invention, a sequence that is perfectly complementary to the polynucleotide containing the SNP may be used, but a substantially complementary sequence may be used to the extent that it does not interfere with specific hybridization. It could be. Preferably, the probe used in the present invention contains a sequence capable of hybridizing to a sequence containing 8 to 100 consecutive nucleotides including the nucleotide at position 301 of SEQ ID NO: 1, which is a SNP nucleotide. More preferably, the 3'-end or 5'-end of the probe has a base complementary to the SNP base. In general, since the stability of the duplex formed by hybridization tends to be determined by the match of the terminal sequences, the terminal portion of the probe having a base complementary to the SNP base at the 3'-end or 5'-end Without hybridization, these duplexes can disassemble under stringent conditions. Suitable conditions for hybridization can be determined by referring to information commonly known in the art. The stringent conditions used for hybridization must be sufficiently stringent to ensure hybridization to only one of the alleles, and can be determined by controlling temperature, ionic strength (buffer concentration), and the presence of compounds such as organic solvents. These stringent conditions may vary depending on the sequence being hybridized.

The present invention also provides a primer set for discriminating between Sedge sedges, consisting of oligonucleotide primers of SEQ ID NOs: 2 to 4 for amplifying polynucleotides containing the SNP base.

In one embodiment of the present invention, the primer set may be a KASP (Kompetitive allele specific PCR) primer set capable of confirming the 301st SNP mutation position in the nucleotide sequence represented by SEQ ID NO: 1, but is not limited thereto.

In this specification, the term 'KASP' is one of the PCR (polymerase chain reaction)-based analysis methods, which is a homogenous and fluorescence-based genotyping analysis technology. KASP is a technology based on fluorescence resonance energy transfer for allele-specific oligo extension and signal generation.

The KASP primer set of the present invention consists of three oligonucleotides with SEQ ID NO: 3 oligonucleotides forming one primer set, and consists of two forward primers and one reverse primer. In the primer set of the present invention, the oligonucleotides of SEQ ID NOs: 2 to 4 are composed of a forward primer to which FAM is bound, a forward primer to which HEX is bound, and a reverse primer, in that order. Specifically, at the 5' end of the forward primer, FAM or HEX fluorescent material is attached, a SNP base is attached to the 3' end, and it consists of one common reverse primer.

The primer set may include oligonucleotides consisting of segments of 20 or more, 21 or more, 22 or more, 23 or more, 24 or more consecutive nucleotides in the sequences of SEQ ID NOs. 2 to 4, depending on the sequence length of each primer set. You can. For example, the primer (24 oligonucleotides) of SEQ ID NO: 2 may include an oligonucleotide consisting of a segment of 20 or more, 21 or more, 22 or more, or 23 or more consecutive nucleotides within the sequence of SEQ ID NO. . In addition, the primer may also include sequences in which the base sequences of SEQ ID NOs: 2 to 4 are added, deleted, or substituted.

As used herein, the term 'primer' refers to a single-stranded oligonucleotide sequence complementary to the nucleic acid strand to be copied, and can serve as a starting point for the synthesis of a primer extension product. The length and sequence of the primers should be used to initiate synthesis of the extension product. The specific length and sequence of the primer will depend on the complexity of the DNA or RNA target desired as well as the conditions under which the primer is used, such as temperature and ionic strength.

In the present specification, oligonucleotides used as primers may also include nucleotide analogues, such as phosphorothioate, alkylphosphorothioate, or peptide nucleic acid, or May contain an intercalating agent. Additionally, primers may incorporate additional features that do not change the basic nature of the primer serving as the starting point for DNA synthesis. The primer nucleic acid sequence of the present invention may, if necessary, contain a label detectable directly or indirectly by spectroscopic, photochemical, biochemical, immunochemical or chemical means. Examples of labels include enzymes (e.g., horse radish peroxidase (HRP), alkaline phosphatase), radioisotopes (e.g., ³² P), fluorescent molecules, chemical groups (e.g., biotin), etc. there is.

The appropriate length of the primer is determined by the characteristics of the primer to be used. Primers do not have to be exactly complementary to the sequence of the template, but must be complementary enough to form a hybrid-complex with the template.

The present invention also provides the primer set; And a reagent for performing an amplification reaction; It provides a kit for determining Yangji Sedge.

In the kit of the present invention, the primer set is as described above.

In the kit of the present invention, reagents for performing the amplification reaction may include, but are not limited to, DNA polymerase, dNTPs, and buffer.

The kit for identifying Sedge officinalis of the present invention may additionally include a user guide describing optimal reaction performance conditions. The guide is a printed material that explains how to use the kit, for example, how to prepare PCR buffer, and the suggested reaction conditions. Instructions include information leaflets in the form of pamphlets or leaflets, labels affixed to the kit, and instructions on the surface of the package containing the kit. Additionally, the guide includes information disclosed or provided through electronic media such as the Internet.

The present invention also,

Isolating genomic DNA from a Cyperaceae plant sample;

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

It provides a method for identifying Yangji Sedge, including the step of detecting the product of the amplification step.

In the method according to one embodiment of the present invention, the primer set is the same as described above.

In the method according to one embodiment of the present invention, the Cyperaceae plants include Carex brevispicula , C. tristachya , C. mollicula , and C. breviculmis var. fibrillosa , C. genkaiensis , C. polyschoena , C. alopecuroides , Jindo sedge ( C. taihokuensis ), C. nervata , or wrinkled sedge (. C. rugata ), but is not limited thereto.

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

In the method according to one embodiment of the present invention, the sample of the Cyperaceae plant may be a plant seed, leaf, fruit, root, or stem, but is not limited thereto.

In the method according to one embodiment of the present invention, the amplified target sequence may be labeled with a detectable labeling substance. The labeling material may be a fluorescent, phosphorescent, or radioactive material, but is not limited thereto. Preferably, the labeling substance may be FAM, HEX, VIC, JOE, ROX, TAMRA, Cy3 or Cy5. When amplifying a target sequence and performing PCR by labeling the 5' end of the primer with the labeling material, the target sequence can be labeled with a detectable fluorescent labeling material. The oligonucleotide primer sets used to amplify the target sequence are listed in Table 6 below.

In the method according to one embodiment of the present invention, the method of identifying Yangji Sedge includes analyzing the product of the amplification step, and the analysis of the product of the amplification step is performed using a SNP genotyping (single nucleotide polymorphism genotyping) method. may be used, and may preferably be performed through HRM (High resolution melting) analysis or sequencing, but is not limited thereto.

Hereinafter, the present invention will be described in detail by examples. However, the following examples only illustrate the present invention, and the content of the present invention is not limited to the following examples.

Materials and Methods

1. Plant material and DNA extraction

10 species of Cyperaceae collected and held at the National Baekdudaegan Arboretum (Sedge sedge, White sedge, Mokpo sedge, Yangji sedge, Jindo sedge, White sedge, Wrinkled sedge, Fine white sedge, Branched sedge, and Gaetcheong sedge ) Genomic DNA was extracted from the seeds using the APrep ^TM Total DNA kit (APBIO, Korea). Specifically, 200 μl of DNA extraction buffer containing 20 μl of proteinase K (100 μg/ml) and 4 μl of RNase (10 mg/μl) was added to a small amount of seed sample and pulverized by bead beating. Next, the culture was incubated for 10 minutes in a constant temperature container adjusted to 65°C, the reaction solution was centrifuged at about 13,000 rpm for 1 minute to obtain a supernatant, and the supernatant was transferred to a new 1.5 ml micro tube. An equal amount of 100% ethanol was added, the reaction solution was placed in a spin column, centrifuged, and washed twice using a washing buffer. DNA was extracted by adding 30 μl of sterilized triple distilled water to the spin column, which was transferred to a new tube.

2. Library production and data production for whole genome sequencing analysis

Quantification and quality of the extracted genomic DNA was confirmed using electrophoresis on a 1.5% agarose gel, nanodrop, and Qubit. The recommended concentration of genomic DNA for library production for WGS analysis is more than 20 ng/μl based on nanodrop measurement, more than 10 ng/μl based on measurement using Qubit, and more than 30 μl in total volume.

The library for WGS analysis was prepared using the QIAseq FX DNA Library Kit (Qiagen, Germany) according to the manufacturer's instructions. The produced library was quality inspected for the size of the template inserted into the library using TapeStation HS D1000Screen Tape (Agilent, USA) equipment, and the average inserted size was 330 to 365 bp. Using the NovaSeq6000 equipment (Illumina Inc, USA), 150 bp was read in both directions (2 × 150 bp paired-end), producing 10 Gb of data for each Cyperaceae sample.

3. Chloroplast genome assembly of Yangji sedge

Sequence pre-processing of short reads removes PCR duplicate reads and adapter sequences with Trimmomatic (v. 0.39) (Anthony M. Bolger et al ., Bioinformatics, 2014, 30(15), 2114-2120). This was performed after removing. Trimming and QC (quality control) were performed using the SLIDINGWINDOW, LEADING, and TRAILING options according to the following conditions. 1) window size=4, mean quality≥15; 2) LEADING, TRAILING≥3; 3) minimum length of reads≥36 bp.

For chloroplast genome assembly, Getorganelle is evaluated as having high accuracy among current assembly tools. The first assembly was performed without a reference genome sequence using the program, and when the long contigs generated during the WGS analysis were BLASTed to NCBI, the Cyperaceae chloroplast genome (Carex breviculmis chloroplast) of NCBI GenBank NC_072263.1 with high accuracy was obtained. , complete genome) was selected as the reference genome for assembling the chloroplast genome. Using the reference genome, the NOVOPlasty program (https://github.com/ndierckx/NOVOPlasty), the Fast-Plast program (https://github.com/mrmckain/Fast-Plast), and GetOrganelle Chloroplast genome assembly was performed using the program (https://github.com/Kinggerm/GetOrganelle). Several generated contig fragments were combined based on the reference genome to create one contig sequence, and the GeSeq program (https://chlorobox.mpimp-golm.mpg.de/geseq.html) was used with reference to the reference genome sequence. The gene region of the chloroplast genome sequence was determined through chloroplast genome annotation (Table 1). Chloroplast genome annotation results were visualized using ODGRAW tools and created as a chloroplast genome map (Figure 1).

Chloroplast genome assembly results of Yangji sedge Sample Total size (bp) GC(%) Total genes protein-coding genes tRNA genes rRNA genes Yangji Sedge 206,470 33.58 155 104 44 9

4. WGS analysis and SNP exploration

4-1. Sequence pre-processing

Preprocessing of short reads was performed after removing PCR duplicate reads and adapter sequences with Trimmomatic (v. 0.39). Pretreatment conditions are the same as above.

4-2. Alignment to reference genome

Cleaned reads that passed the preprocessing process were mapped to the reference genome sequence using the BWA (0.7.17-r1188) program. The chloroplast genome sequence of Carex laevissima (GenBank accession No. MZ846224.1) was used as a reference genome for SNP analysis. The mapping process is a prerequisite for detecting raw SNPs between the reference genome sequence and sequencing samples, and a file in BAM format was created.

4-3. Raw SNP detection and consensus sequence extraction

Raw SNPs (In/Del) were detected in BAM format files using the DeepVariant program, and common sequences were extracted. At this time, before the process of detecting SNP (In/Del), raw SNP (In/Del) was detected after SNP validation using SEEDERS in-house script.

4-4. Securing SNP marker candidates

Secured through WGS analysis of 10 species of Cyperaceae (Sedge sedge, White sedge, Mokpo sedge, Yangji sedge, Jindo sedge, Trifolium sedge, Wrinkled sedge, Slender white sedge, Branched sedge, and Gaetcheong sedge) From the results of one genome analysis, 37 locus mutations corresponding to 12 combinations were obtained in SNP marker set 1, which is a marker candidate that can distinguish 10 species of Cyperaceae (Table 2), and 17 combinations in SNP marker set 2. 1,168 locus mutations were obtained (Table 3).

4-5. SNP marker selection for identification of Yangji sedge

Among a total of 29 candidate SNP markers included in SNP marker set 1 and SNP marker set 2, markers that were amplified only in Cyperaceae and could distinguish each of 10 species of Cyperaceae were selected. Among the above SNP markers, SNP markers that can specifically distinguish Yangji Sedge were finally selected, and the final selected SNP markers are located in the rpoB and atpB intergenic regions based on the reference chloroplast genome sequence (GenBank accession No. MZ846224.1). . The SNP-specific SNP marker information, SNP flanking sequence information, and KASP primer set information produced based on the SNP of the present invention are shown in Tables 4 to 6, respectively.

SNP marker information for identification of Yangji Sedge Marker SNP marker set Position(bp) ref. wrinkled green sedge Other Cyperaceae Allele SA16 aaaaaaaaab 29,684 C T C T/C

SNP flanking sequence information - SNP T: Yangji Sedge
- SNP C: Reference genome and other Cyperaceae plants CACTGGAAACACCACTTCCCGTCCGACTTGCATGTGTTAAGCATGCCGCCAGCGTTCATCCTGAGCCAGGATCAAACTCTCCATGAGATTCCTAGTTCCATTACTTATAGCTTCCTTCTTCGTAGACAAAGCTGATTCGGACTTGTCTTTCACTCCAAGGCATCACTTGGATTTGTGTGAGCCTGGAGTTTTGCTTCCAACAATATAATAGATATTTCTTCTCTATGATGCATATCCCACAAGCCTCTTTTTCATTCTAGTTC GATCACGGCGGGGGGCTCAGCCCAAATAGAACAACT[ T/C ]AGATTGGCTTTTAGGTTAGGGATAATCAGGTTCGAACTGATGACTTCACCACGTCAAGGTGACACTCTACCGCTGAGTTATATCCCTTCCCTGCCTAATATACTAACTAATAGGGTCGATAAATTCAAGGCCACTACTCCTAAACAACTCGGAGTCGGGCCTTCTTTTCGTACTTTTTATTATGGATAACTCAGGAATGGGACCACTCCTATCGGAAATGGGATTGACCAAACCATCGCACATGCTCTCATCAAATCCGT ACTTGAGTCGGCTCAATCCTTTTTTTAGGAAAAGATTGGG (SEQ ID NO: 1)

Underlined and bold: SNP location base

KASP primer set information Marker Primer name Sequence(5'-3') (SEQ ID NO) Conjugated fluorescent dye SA16 SA16_FAM GGCTCAGCCCAAATAGAACAACT C (2) 5'-FAM SA16_HEX GGCTCAGCCCAAATAGAACAACT T (3) 5'-HEX SA16_common CAGCGGTAGAGTGCTCACCTTGACGT (4)

Underline: SNP location base

5. KASP analysis

PCR reaction and analysis were performed using a reaction solution containing 5 μl of template DNA, 5 μl of KASP-TF V4.0 2X Master Mix/Standard ROX, and 0.14 μl of KASP Assay mix (KASP primer set mix). Gene amplification conditions were the same as those in the 'Guide to running KASP genotyping on the ABI 7900 instrument' provided by LGC Biosearch Technologies, and the 61-55℃ touchdown protocol was used for amplification conditions.

As shown in Table 7 below, after hot-start activation at 94°C for 15 minutes, amplification was performed for 10 cycles at 94°C for 20 seconds and 61-55°C touchdown, followed by 26 cycles of 94°C for 20 seconds and 55°C for 60 seconds. After repeating, one cycle was performed for 60 seconds at 30°C. The negative control group was treated with distilled water, and each sample was tested 3 to 4 times. When the amount of amplification was small, recycling was performed. As shown in Table 8 below, 3 cycles of 20 seconds at 94°C and 60 seconds at 57°C were repeated, followed by one cycle of 60 seconds at 30°C.

KASP standard conditions Protocol stage temperature hour cycle (number of times) <Stage 1>
Hot-start activation
94℃
15 minutes
One <Stage 2>
Touchdown 94℃ 20 seconds 10 61℃
(61℃ decreasing 0.6℃ per cycle to activate a final annealing/extension temperature of 55℃) 60 seconds <Stage 3>
Amplification 94℃ 20 seconds 26 55℃ 60 seconds <Optional Stage 4>
(Read stage for qPCR instruments only) 30℃ 60 seconds One

KASP recycling conditions Protocol stage temperature hour cycle (number of times) <Stage 1>
Amplification 94℃ 20 seconds 3 57℃ 60 seconds <Optional Stage 4>
(Read stage for qPCR instruments only) 30℃
(any temperature below 40℃ is suitable for the read stage) 60 seconds One

Example 1. Verification of the KASP primer set of the present invention for identification of Yangji Sedge

The KASP primer set of the present invention (Table 6) was applied to 10 types of Cyperaceae plants (Sedge sedge, White sedge, White sedge, Gaetcheong sedge, Mokpo sedge, Branch sedge, Fine white sedge, Jindo sedge, Yangji sedge, Wrinkle sedge KASP analysis was performed by applying it to Cheongsacho).

As a result, Yangji sedge appeared close to the y-axis due to the fluorescent substance HEX, and 9 other species of Cyperaceae plants appeared close to the x-axis due to the fluorescent substance FAM (Figure 2). Through this, it can be seen that the SNP marker of the present invention and the KASP primer set produced based on it can accurately distinguish only Yangji sedge among various sedge plants.

Claims (7)

A SNP for discrimination of Carex nervata, comprising a polynucleotide consisting of 8 or more consecutive nucleotides including the SNP (single nucleotide polymorphism) base located at position 301 in SEQ ID NO: 1, or a complementary polynucleotide thereof. Marker composition. The SNP marker composition according to claim 1, wherein the contiguous nucleotides are 8 to 100 contiguous nucleotides. A microarray for discriminating between Sedge sedges, comprising a polynucleotide or cDNA thereof consisting of 8 or more consecutive nucleotides including the SNP base located at position 301 in the base sequence of SEQ ID NO: 1. A probe for discriminating Sedge sedge, comprising a polynucleotide or cDNA thereof consisting of 8 or more consecutive nucleotides including the SNP base located at position 301 in the base sequence of SEQ ID NO: 1. A primer set for discriminating between the oligonucleotide primers of SEQ ID NO: 2 to 4 to amplify a polynucleotide consisting of 8 or more consecutive nucleotides containing the SNP base located at position 301 of SEQ ID NO: 1. The primer set of claim 5; And a reagent for performing an amplification reaction; a kit for determining Yangji Sedge. Isolating genomic DNA from a Cyperaceae plant sample;
Using the isolated genomic DNA as a template and performing an amplification reaction using the primer set of claim 5 to amplify the target sequence; and
Detecting the product of the amplification step; A method of identifying Yangji Sedge, including.