KR20240033715A - KASP Primer Set Based on SNP for Discriminating Anser fabalis and Anser albifrons, and Uses thereof - Google Patents

Abstract

The present invention relates to a primer set for SNP-based KASP and its use for species identification of whooping geese and white geese.
The primer set for KASP of the present invention has the advantage of being able to quickly and accurately determine the species of common wild goose and white-fronted goose, and through this, the presence of common white-fronted and white-fronted geese can be confirmed by using samples of the common white-fronted and white-fronted geese, which are difficult to capture due to the nature of wild birds. The presence or absence can be easily confirmed and can be useful for avian influenza control and wild bird research.

Description

Primer set for SNP-based KASP for species discrimination of whooping goose and black goose and its uses {KASP Primer Set Based on SNP for Discriminating Anser fabalis and Anser albifrons, and Uses thereof}

The present invention relates to a primer set for SNP-based KASP and its use for species identification of whooping geese and white geese.

The base sequence of mtDNA is widely used in various birds as an important marker in molecular biological species identification, and it is known that species identification of approximately 16,684 species of birds is possible through this (Hebert et al., 2004).

Meanwhile, geese are highly pathogenic avian influenza-infected birds, and are a species in which the disease can be easily spread due to their ecological and behavioral characteristics, so it is very important to establish a molecular biological species identification technique. Avian influenza virus is a zoonotic disease that causes great damage to human health and livestock animals such as poultry, and also causes significant economic losses. In addition, it poses a great threat not only to breeding farms but also to wild birds, so research on wild birds in response to avian influenza is important for the conservation of wild birds.

In order to accurately predict avian influenza, it is recommended to check for the presence of the virus in individuals whose species has been identified after capture. However, due to the nature of wild birds, it is not easy to capture them, so virus detection in fecal samples and species identification using the mitochondrial COI (Cytochrome Oxidase subunit I) gene are recommended. proceeded.

However, in the case of species with close evolutionary history, such as the white-cheeked duck and the mallard, the degree of genetic differentiation between the two species is low, so when species identification is performed using mtDNA, species identification is impossible because the two species have identical base sequences. It is well known (Kulikova et al., 2003). This phenomenon is presumed to be caused by the slow rate of genetic evolution and frequent hybridization between bird species (Degnan & Rosenberg, 2009; Pamilo & Nei., 1988; Ruokonen, 2000). According to research results, Anser species They have a very close evolutionary relationship, and the degree of nucleotide sequence differentiation between species was found to be lower than that of other bird species, at about 0.9 to 5.5%.

Whooping geese and black geese are species that are relatively easy to identify due to their morphological characteristics, but there are two problems when identifying molecular biological species through samples such as feces. ) and NUMT (nuclear DNA of mitochondrial origin), it was confirmed that species identification was difficult using the COI (cytochrome c oxidase I) gene and control region of mtDNA.

Specifically, NUMT ("new might" nuclear mitochondrial DNA) is DNA introduced into nuclear DNA by duplicating the base sequence of mtDNA. Avian NUMT was first discovered in snow geese and occurs frequently within wild goose species. Caution is needed. Caution is also needed in the case of Whooper geese and White geese, as they may be misidentified as wild geese when amplifying their mitochondrial control region.

In addition, the commonly used barcode gene COI (mitochondrial gene cytochrome c oxidase I) is also not suitable for species identification of wild geese. According to the results of previous studies conducted by these researchers, the COI genes of common geese, common geese, and wild geese were used to identify species. As a result of comparing the relationships, it was confirmed that the COI barcode is not suitable for species identification within the geese genus.

Meanwhile, GBS (Genotyping-By-Sequencing) analysis is a method that allows the acquisition of many SNP loci from multiple individuals at once at a relatively low cost, unlike general base sequence acquisition technologies in the past. Because of these advantages, it was mainly used in early plant breeding and has recently been used in population genetics and phylogeny (Elshire et al., 2011). In addition, SNP markers can identify a large amount of nucleotide sequence variation in nuclear DNA, so they can be used as species identification markers for species with hybrids, slow evolutionary rates, and short differentiation histories (Kang et al., 2013).

Under this background, the present inventors performed GBS (Genotyping-By-Sequencing) analysis of greater white-fronted geese and white-fronted geese, which cannot be identified using mtDNA base sequences, and discovered 21 SNP markers. However, the base sequence analysis results and GBS (Genotyping by Sequencing) When comparing the Homo/Hetero results of the Sequencing map, it was confirmed that the SNP results did not match for 19 markers except for two markers, and the ability to distinguish the two species from each other was poor.

Accordingly, the present inventors used KASP (Kompetitive Allele Specific PCR genotyping system) analysis technology on the additionally secured 30 SNP loci to develop a set of SNP-based KASP primers that can finally distinguish between white geese and white geese, and completed the present invention. I did it.

(0001) Hebert et al., 2004; PLoS Biology, Vol 2, Iss 10, p e312 (2004), Identification of Birds through DNA Barcodes (0002) Kulikova et al., 2003, Russian Journal of Genetics, 39(10), 1143-1151, Low genetic differentiation of and close evolutionary relationships between Anas platyrhynchos and Anas poecilorhyncha: RAPD-PCR evidence (0003) Degnan & Rosenberg, 2009, Trends in ecology & evolution, 24(6), 332-340, Gene tree discordance, phylogenetic inference and the multispecies coalescent (0004) Pamilo & Nei., 1988, Molecular biology and evolution, 5(5), 568-583, Relationships between gene trees and species trees (0005) Ruokonen, 2000, Journal of Evolutionary Biology, 13, 532-540, Close relatedness between mitochondrial DNA from seven Anser goose species (0006) Elshire et al., 2011, PloS one, 6(5), e19379, A robust, simple genotyping-by-sequencing (GBS) approach for high diversity species (0007) Kang et al., 2013, Molecular ecology resources, 13(3), 369-376, Identification of Culex complex species using SNP markers based on high resolution melting analysis (0008) Pritchard et al., 2000, Genetics, 155(2), 45-959, Inference of population structure using multilocus genotype data (0009) Tamura et al., 2013, Molecular biology and evolution, 30(12), 2725-2729

Therefore, the main purpose of the present invention is to provide a primer set for SNP-based KASP for species discrimination of Greater White Goose and White Goose and a method for species discrimination of White Goose and White Goose using the primer set.

Other objects and advantages of the present invention will become clearer from the following detailed description, claims, and drawings.

According to one aspect of the present invention, in the oligonucleotides shown in SEQ ID NO: 1 to 42, the oligonucleotides of SEQ ID NO: 3n, SEQ ID NO: 3n-1, and SEQ ID NO: 3n-2, where n has the same value, are one Provided is a primer set for KASP (kompetiive allele specific PCR) for species identification of greater white goose and white goose, which includes a primer set of 14 oligonucleotide primers (n is a natural number from 1 to 14).

The term "KASP (kompetitive allele specific PCR)" of the present invention is one of the PCR (polymerase chain reaction)-based analysis methods, and is a homogenous and fluorescence-based genotyping technology. KASP is a technology based on fluorescence resonance energy transfer for allele-specific oligo extension and signal generation.

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 allow for initiation of 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 invention, the oligonucleotide used as a primer may also include a nucleotide analogue, for example, phosphorothioate, alkylphosphorothioate or peptide nucleic acid, Alternatively, it may include an intercalating agent. Additionally, primers may incorporate additional features that do not change the basic nature of the primer, which serves 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.

The appropriate length of the primer is determined by the characteristics of the primer to be used, but is usually used at a length of 15 to 30 bp. 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 primer set of the present invention is a primer set that detects the base type of a single nucleotide polymorphism (SNP) marker that is specifically differentiated at the loci of greater white goose and black goose.

The term "Single Nucleotide Variation" used in the present invention is also called "SNP (single nucleotide polymorphism)", which means polymorphism at a single base. In other words, it refers to the case where some bases in the entire genome of a certain group are different for each chromosome. It is generally known to exist about one base in 300 to 1000 bases, but is not limited to this.

The term "polymorphism" used in the present invention refers to the presence of two or more alleles at one genetic locus, and among the polymorphic sites, only a single base differs depending on the individual. It is called a single nucleotide polymorphism. Preferred polymorphic markers have two or more alleles with an occurrence frequency of 1% or more, more preferably 5% or 10% or more in the selected population.

The term “allele” used in the present invention refers to several types of one gene that exist at the same genetic locus on a homologous chromosome. Alleles are also used to refer to polymorphisms.

The primer set of the present invention consists of three consecutive oligonucleotides with SEQ ID NOs, forming one primer set, and consists of two forward primers and one reverse primer. Specifically, a FAM or HEX fluorescent substance is attached to the 5' end of the forward primer, and a base representing the SNP is attached to the 3' end, and the reverse primer allows replication of the allele of the SNP portion of the forward primer. It can be designed (see [Table 7]).

The primer set of the present invention may preferably include one or more primer sets selected from the group consisting of the 14 primer sets, but at least 10 primer sets corresponding to 70% of the 14 primer sets are used simultaneously. By doing this, species discrimination between greater white geese and white geese can be performed more efficiently.

The primers of the present invention are 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more depending on the sequence length of each primer. It may include an oligonucleotide consisting of a segment of 25 or more, 26 or more consecutive nucleotides. For example, the primer (22 oligonucleotides) of SEQ ID NO: 1 is 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more nucleotides in the sequence of SEQ ID NO: 1. It may contain an oligonucleotide consisting of a fragment of. In addition, the primer may also include sequences in which the base sequences of SEQ ID NOs: 1 to 42 are added, deleted, or substituted. The oligonucleotide primers of SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, and 40 are forward primers to which the fluorescent substance FAM is attached, and SEQ ID NOs: 2 and 5 The oligonucleotide primers of 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, and 41 are HEX-attached forward primers, and have SEQ ID NOs: 3, 6, 9, 12, 15, Oligonucleotide primers 18, 21, 24, 27, 30, 33, 36, 39, and 42 are reverse primers.

The primer set according to one embodiment of the present invention is capable of distinguishing between the above-mentioned species of greater white goose and black goose by combining 14 primer sets represented by SEQ ID NOs: 1 to 42. For example, SEQ ID NOs: 1, 2, and 3 of [Table 7] below; SEQ ID NOS: 4, 5 and 6; SEQ ID NOs: 7, 8 and 9; SEQ ID NOS: 10, 11 and 12; SEQ ID NOS: 13, 14 and 15; SEQ ID NOS: 16, 17 and 18; SEQ ID NOS: 19, 20 and 21; SEQ ID NOS: 22, 23 and 24; SEQ ID NOS: 25, 26 and 27; SEQ ID NOs: 28, 29, and 30; SEQ ID NOs: 31, 32 and 33; SEQ ID NOS: 34, 35 and 36; SEQ ID NOS: 37, 38 and 39; And by combining the primer sets of SEQ ID NOs: 40, 41, and 42, it is possible to distinguish between white goose and white goose.

According to another aspect of the present invention, the present invention provides the primer set; and a kit for KASP for species identification of greater white-fronted geese and white-fronted geese, including reagents for performing an amplification reaction.

In the KASP kit for species identification of common white goose and white goose of the present invention, reagents for performing the amplification reaction may include DNA polymerase, dNTPs, and buffer, but are not limited thereto. The dNTPs include dATP, dCTP, dGTP, and dTTP. DNA polymerase is a heat-stable DNA polymerase, and commercially available polymerases such as Taq DNA polymerase and Tth DNA polymerase can be used. In addition, the kit of the present invention may additionally include user instructions describing optimal reaction performance conditions. The guide is a printed material that explains how to use the kit, for example, how to prepare reverse transcription buffer and 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.

According to another aspect of the present invention, the present invention includes the steps of isolating genomic DNA from a sample; Using the isolated genomic DNA as a template and performing KASP using the primer set of claim 1; and analyzing the amplification product of the KASP. It provides a method for discriminating between species of white goose and white goose, including the step of analyzing the amplification product of the KASP.

In the method for species identification of greater white goose and white goose of the present invention, the primer set for KASP is the same as described above.

The method of the present invention includes the step of isolating genomic DNA from a sample. Methods known in the art for isolating the genomic DNA may be used, for example, the CTAB method may be used, or the Wizard prep kit (Promega) may be used. The target sequence can be amplified by performing an amplification reaction using the isolated genomic DNA as a template and a primer set according to an embodiment of the present invention as primers. Methods for amplifying target nucleic acids include polymerase chain reaction (PCR), ligase chain reaction, nucleic acid sequence-based amplification, and transcription-based amplification system. 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 these, “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. These PCR methods are well known in the art, and commercially available kits can also be used.

In the method of the present invention, the amplified sequence can be labeled with a detectable label. In one embodiment, the labeling material may be a material that emits fluorescence, phosphorescence, or radioactivity, 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 set used to amplify the target sequence is as described in [Table 7] below.

Additionally, in the method according to one embodiment of the present invention, the step of analyzing the amplification product of the KASP may be performed through genotyping. Specifically, the genotype can be distinguished through the fluorescent substance FAM or HEX attached to the 5' end of the forward primer and the SNP attached to the 3' end, thereby distinguishing between whooping goose and white goose species.

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.

As described above, the primer set for KASP of the present invention can quickly and accurately determine the species of Greater White Goose and White Goose.

Accordingly, it is possible to easily confirm the presence or absence of Greater Geese and Lesser Geese, which are difficult to capture due to the nature of wild birds, using samples of Lesser Geese and Lesser Geese, which can be useful for avian influenza control and wild bird research.

Figure 1 is a photo of a common goose (left) and a common goose (right).
Figure 2 is a photograph of the electrophoresis results of the Greater Goose and Lesser Goose samples (the individual NO, marked in red, was excluded from the analysis due to low DNA quality).
Figure 3 is a graph showing the results of in silico digestion of Great White Goose and White Goose DNA using two restriction enzymes, ApeKI and PstI-MspI.
Figure 4 is an explanatory diagram explaining the production of GBS (Genotyping by Sequencing) library.
Figure 5 is a graph showing the results of PCA analysis using 1,322 SNP loci of Greater Wild Goose (red) and White Goose (blue).
Figure 6 is a graph showing the results of population STRUCTURE structure analysis using 1,322 SNP loci of whooping geese and white geese.
Figure 7 is a phylogenetic tree diagram of the greater white goose (red) and the lesser goose (blue) using the MEGA6 program (Tamura et al., 2013).
Figure 8 is a graph showing the results of PCA analysis using 21 SNP loci of the greater white goose (red) and the lesser goose (blue).
Figure 9 is a graph showing the results of PCA analysis of the genotypes of the 14 SNP loci of the selected Greater Goose (red) and White Goose (blue).
Figure 10 is an explanatory diagram summarizing the amplification conditions of Real-Time PCR.
Figure 11 is a graph summarizing Delta K values by examining the population structure of greater white-fronted geese and white-fronted geese.
Figure 12 is a graph analyzing the population STRUCTURE structure using the genotypes of the 14 SNP loci of the Whooper Goose (Pop1, yellow) and White Goose (Pop2, red).
Figure 13 is a graph showing the results of 2D PCA analysis of the genotypes of the 14 SNP loci of the Great White Goose (red) and White Goose (blue).
Figure 14 is a graph showing the results of 3D PCA analysis of the genotypes of the 14 SNP loci of the Great White Goose (red) and White Goose (blue).

Hereinafter, the present invention will be described in more detail through examples. Since these examples are merely for illustrating the present invention, the scope of the present invention is not to be construed as limited by these examples.

Example 1. Research samples and DNA extraction

1-1. research sample

The research samples were used for analysis using 56 white-fronted geese obtained from Gyeonggi-do, Gangwon-do, and Chungcheongnam-do, and samples collected from 36 individuals from Gyeonggi-do, Gyeongsangnam-do, and Busan Metropolitan City. The list of Greater White Goose and White Goose samples is as shown in [Table 1] below.

[Table 1]

1-2. DNA extraction and quality check

DNA was extracted from the geese muscle and blood samples collected from the samples in [Table 1] using the DNeasy® Blood & Tissue Kit (Qiagen) according to the manufacturer's manual.

Qualitative analysis of DNA from extracted samples was based on electrophoresis results and absorbance (260/280 ratio). For electrophoresis, 2 μl of each sample was loaded onto a 1% agarose gel (Nucleic Acid Gel Stain-GelRed®) and the results were confirmed using Gel Doc.

As a result of electrophoresis [Figure 2], samples with clear and clearly visible bands were selected and used in the next step of analysis. Individuals that did not meet the criteria were excluded from the analysis, and a total of 76 individuals were used for GBS analysis.

Example 2. Restriction enzyme selection and GBS library generation

Prior to creating a GBS (Genotyping by Sequencing) library, an in silico digestion process was performed with two restriction enzymes, ApeKI and PstI-MspI, to select enzymes suitable for cutting the DNA of the Great White Goose and White Goose. As a result, the ApeKI enzyme showed a higher fragment size ratio of 200 to 500 bp compared to the PstI-MspI enzyme, and was selected as an appropriate enzyme for cleavage of white goose and white goose DNA (Figure 3), and the selected restriction enzyme A GBS library was constructed using ApeKI enzyme as described in [Figure 4].

Example 3. GBS Data Generation and SNP Analysis

3-1. SNP matrix creation and SNP selection

A total of 124,184,189,048 bp of nucleotide sequence was obtained from DNA samples of Greater Wild Goose and White Goose, and SNP selection was performed based on the obtained nucleotide sequences. To perform comparative analysis of SNPs between the Whooper Goose and White Goose groups, an integrated SNP matrix between individuals was created and SNPs were selected according to the criteria below.

① Homozygotes: read rate ≥ 90%

② Heterozygous: heterozygous 40%≤read rate≤60%

③ Other SNPs: 20%＜read rate≤40%, 60<read rate<90%

SNP filtering items and steps are summarized in Table 2 below.

[Table 2]

Referring to [Table 2] above, the nucleotide sequences of 72 individuals of Whooper Goose and White Goose were analyzed and approximately 2,729,302 SNP loci were selected.

The raw SNPs obtained in this way were used to secure 716,702 SNP loci through the SNP matrix, and biallelic SNPs were selected to remove individuals and SNP loci containing more than 30% deletion, a total of 12 individuals were removed, and 206,526 SNP loci were selected. did.

Next, MAF, Hardy-Weinberg equilibrium, Linkage disequilibrium, and Fst values corresponding to steps 4 to 7 in [Table 2] were calculated to select 1,322 SNP loci from 60 individuals.

3-2. SNP analysis

Based on 1,322 SNP loci generated from 60 individuals of Greater White Goose and White Goose, the analysis was conducted by dividing the Great White Goose into two groups: Greater White Goose and White Goose. To confirm the correlation between the Whooping Goose and White Goose groups, Principal Component Analysis (PCA) was performed using the SNP Relate R package program, and DAPC (Discriminant analysis of principal components) analysis was performed using the adegent R package program. proceeded. Additionally, STRUCTURE v2.3.4 (Pritchard et al., 2000, Genetics, 155(2), 45-959, Inference of population structure using multilocus) was used to confirm the genetic population structure between the two species of whooping goose and white goose for the selected SNP. Using the genotype data) program, analysis was conducted under the following conditions: Length of burnin period=10,000, Number of MCMC reps after Burnin=100,000, and K=1∼10.

In addition, a phylogenetic tree was created using the MEGA6 program (Tamura et al., 2013, Molecular biology and evolution, 30(12), 2725-2729, MEGA6: molecular evolutionary genetics analysis version 6.0).

Referring to [Figure 5], which summarizes the results of the principal component analysis, and [Figure 6], which summarizes the results of the STRUCTURE analysis, it was confirmed that the white geese and the white geese were clearly separated into two groups.

Also, refer to [Figure 7] which summarizes the phylogenetic tree analysis results. It was confirmed that the groups of Greater Goose and Lesser Goose were clearly divided.

3-3. Exploration of polymorphic SNPs between species of Whooper Goose and White Goose

Common SNPs were selected for each group of Lesser Goose and Lesser Goose from 1,322 SNP loci in 60 individuals, and polymorphic SNP selection was attempted by comparing common SNPs between the two groups.

[Table 3]

Referring to [Table 3], common SNPs were selected for both the Whooper Goose and White Goose groups, but polymorphic SNPs between the two groups were not selected, so polymorphic SNP selection was attempted again by adjusting the common SNP ratio for each group, and the common SNP ratio was set to 70. By adjusting the percentage, 21 SNP loci were finally selected.

[Table 4]

PCA analysis was performed again using data from 21 polymorphic SNP loci of selected whooping geese and white geese.

Referring to [Figure 8], it was confirmed that the genotypes of greater white goose and white goose were clearly divided into two groups.

Example 4. SNP locus verification

4-1. Verification of 21 SNP loci

To verify the 21 SNP loci selected in Example 3, primers were designed after confirming the surrounding sequences centered on the SNP. The DNA of two large wild geese and two common white geese were amplified using the designed primers, and the base sequences were analyzed using Sanger sequencing.

When comparing the sequence analysis results and the Homo/Hetero results of the GBS (Genotyping by Sequencing) map, the SNP results did not match for 19 markers except for two markers, and the ability to distinguish the two species from each other was poor. It has been confirmed.

4-2. Additional SNP loci verification

In order to discover SNP loci that can distinguish the two species from each other, the 1,322 SNP loci discovered in Example 3-1 above were reconfirmed through GBS analysis results and additional markers that could distinguish the two species were selected.

The Fst value between the two species was large, the SNP was located in the center of the read, and the SNP was selected based on the criteria of having less overall nucleotide sequence variation. In addition, loci with many repeats in the sequence were not selected even if the Fst value was large.

Through this process, the top 30 markers were selected, and a test was performed on 26 SNPs, excluding the 4 markers showing multibands, to determine whether they were SNPs capable of discriminating between species in the same manner as Example 4-1.

[Table 5] below shows the results of nucleotide sequence analysis using the Sanger sequencing method for the SNP locus named [Scf7180002171524] (analysis results for the remaining 25 SNPs are not shown).

[Table 5]

During verification, SNP markers that were capable of discriminating between the Whooper Goose and White Goose species were selected even if the Sanger sequencing and GBS (Genotyping by Sequencing) results did not match the matching marker. As a result of SNP verification, 14 SNP loci were finally selected.

No. SNP ID PCA by marker 1-7 scf7180002449464 1-14 scf7180002363362 2-3 scf7180002137055 2-4 scf7180002180573 2-5 scf7180002170975 2-6 scf7180002197706 2-8 scf7180002165023 2-10 scf7180002317930 2-14 scf7180002138002 2-19 scf7180002217963 2-21 scf7180002139145 2-22 scf7180002365952 2-23 scf7180002170219 2-24 scf7180002220589

As a result of PCA analysis of the genotypes of the 14 selected SNP loci, it was confirmed that there was a clear division into two groups, Greater Goose and Lesser Goose (Figure 9), and KASP primers were produced for the corresponding SNP loci.

Example 5. Production of SNP-based KASP primers

The KASP (Kompetitive allele specific PCR) primer set produced based on the 21 SNP markers selected above consists of two forward primers and one reverse primer. The forward primer was designed to have a FAM or HEX fluorescent substance attached to the 5' end and to distinguish differences in base sequence such as SNPs at the 3' end. And the reverse primer was designed to work with the two forward primers to allow the PCR reaction to proceed.

When performing PCR, the 5' ends of the two forward primers are amplified with different fluorescent substances attached, so the genotype was analyzed according to the difference in the wavelength of the fluorescent substances.

KASP marker set 1 was performed including 22 pieces of Greater White Goose and White Goose DNA and 2 Negative Control Samples (NTC). A master mix containing a total of 3 primers, including 2 forward primers and 1 common reverse primer, was used, and the primer sequences and amplified DNA sequences are summarized in [Table 7] below.

[Table 7]

The PCR mixture composition consisted of 5ul of sample DNA (6ng/ul) diluted to 30ng, 5ul of Master mix, and 0.14ul of assay mix, and the experiment was conducted with a total volume of 10ul. The basic amplification conditions for Real-Time PCR were 61-55°C and the Touch Down method was used (Figure 10).

As a result of investigating the population structure based on the analyzed genotype results, the Delta K value of the population was 2 (Figure 11), and it was confirmed that the population's STRUCTURE structure was clearly divided into white geese and white geese (Figure 12). In addition, in the PCA analysis, the 14 selected KASP markers were judged to be suitable for distinguishing between the two species, as they were divided into two groups: greater geese and lesser geese (Figures 13 and 14).

Among the 92 wild geese used in the KASP analysis, 5 large wild geese with low DNA quality had 40-50% missing data as a result of real-time PCR, and PCA analysis showed that they were distributed somewhat close to the white geese group ( (see red dotted line in Figure 13). Based on this, it was analyzed that reliable species identification is possible when more than 10 SNPs, which account for 70% of the total markers, are secured.

These results mean that the 14 types of KASP markers of the present invention can be applied to identify whooping geese and white geese.

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention.

Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (4)

In the oligonucleotides shown in SEQ ID NOs: 1 to 42, the oligonucleotides of SEQ ID NO: 3n, SEQ ID NO: 3n-1, and SEQ ID NO: 3n-2, where n has the same value, are one primer set (n is a natural number from 1 to 14) A primer set for KASP (kompetiive allele specific PCR) for species identification of greater white goose and white goose, including a set of 14 oligonucleotide primers, characterized in that: The primer set of claim 1; and a kit for KASP for species identification of Greater White Goose and White Goose, including reagents for performing an amplification reaction. According to paragraph 2,
The reagent for performing the amplification reaction is a kit for KASP for species identification of greater white goose and white goose, characterized in that it includes DNA polymerase, dNTPs, and buffer. isolating genomic DNA from the sample;
Using the isolated genomic DNA as a template and performing KASP using the primer set of claim 1; and analyzing the amplification product of the KASP.