KR20230165039A - Snp markers for discriminating italian ryegrass hs104 varieties and method for discriminating italian ryegrass hs104 varieties using the same - Google Patents

Abstract

The present invention relates to a composition for identification of Italian ryegrass HS104 variety, in which a polynucleotide consisting of the base sequences of SEQ ID NO: 1 or SEQ ID NO: 2, and SEQ ID NOs: 3 to 7, wherein the 301st base of SEQ ID NO among each base sequence It includes an agent that can detect single nucleotide polymorphism (SNP) markers.

Description

SNP marker for identification of Italian ryegrass HS104 variety and method for identifying Italian ryegrass HS104 variety using the same

The present invention relates to a SNP marker for identification of Italian Ryegrass Volcano No. 104 (HS104) variety and a method for identifying Italian Ryegrass Volcanic No. 104 (HS104) variety using the same.

In order to stabilize the unstable supply and demand of grain feed due to recent intensifying fluctuations in international grain prices and extreme weather events and to alleviate the burden of feed costs on cattle farms, the government is implementing various support policies along with measures to increase the production of high-quality forage to expand the forage production base. . As the domestic forage production base is expanding and the cultivation area is rapidly increasing, the amount of seed imports of feed crops from overseas is also increasing every year.

Since the optimal cultivation method for each variety is somewhat different, identifying the variety to produce high-quality forage can increase the productivity of the forage and ultimately increase the profits of farmers. However, Italian ryegrass seeds have similar morphological characteristics, so it is difficult to distinguish varieties with the naked eye, and varieties are distinguished using growth and morphological characteristics after emergence. Even if varieties are distinguished using post-emergence growth and morphological characteristics, it is difficult to clearly distinguish between extremely early-maturing, early-maturing, mid-maturing, late-maturing, and/or imported varieties of Italian ryegrass.

To solve these problems in breed identification, many researchers have recently attempted to approach breed identification using biochemical analysis methods.

The purpose of the present invention is to provide a SNP marker for identifying the Italian Ryegrass Volcano No. 104 (HS104) variety and a method for identifying the Italian Ryegrass Volcano No. 104 (HS104) variety using the same. It is provided.

The present invention relates to a composition for identification of Italian ryegrass HS104 variety, in which a polynucleotide consisting of the base sequences of SEQ ID NO: 1 or SEQ ID NO: 2, and SEQ ID NOs: 3 to 7, wherein the 301st base of SEQ ID NO among each base sequence It includes an agent that can detect single nucleotide polymorphism (SNP) markers.

In one embodiment of the present invention, the single nucleotide polymorphism marker is G or A at the 301st base in the polynucleotide represented by the nucleotide sequence of SEQ ID NO: 1, and 301 in the polynucleotide represented by the nucleotide sequence of SEQ ID NO: 2. The 301st base is C or G, the 301st base in the polynucleotide represented by the base sequence of SEQ ID NO: 3 is T or C, and the 301st base in the polynucleotide represented by the nucleotide sequence of SEQ ID NO: 4 is A or G, In the polynucleotide represented by the nucleotide sequence of SEQ ID NO: 5, the 301st base is T or C, and in the polynucleotide represented by the nucleotide sequence of SEQ ID NO: 6, the 301st base is A or G, represented by the nucleotide sequence of SEQ ID NO: 7 It may be a single nucleotide polymorphism marker where the 301st base in the polynucleotide is T or A.

In one embodiment of the present invention, the agent capable of detecting the single nucleotide polymorphism marker is a polynucleotide consisting of 10 to 400 consecutive DNA sequences, including a single nucleotide polymorphism marker site; Or it may be a polynucleotide that specifically hybridizes with its complementary polynucleotide.

In one embodiment of the present invention, the polynucleotide may be one or more selected from the group consisting of primers, probes, and combinations thereof capable of detecting a single nucleotide polymorphism marker.

The present invention relates to an Italian ryegrass HS104 variety identification kit, in which a polynucleotide consisting of the base sequences of SEQ ID NO: 1 or SEQ ID NO: 2, and SEQ ID NOs: 3 to 7, the 301st base of SEQ ID NO among each base sequence. It includes a composition for identification of Italian ryegrass HS104 variety, including an agent capable of detecting a single nucleotide polymorphism (SNP) marker.

The present invention relates to a method for identifying Italian ryegrass HS104 variety, comprising the steps of extracting DNA from Italian ryegrass; Using the extracted DNA as a template and performing polymerase chain reaction (PCR) using a composition for identifying Italian ryegrass HS104 varieties; And analyzing the product produced by the polymerase chain reaction to identify the variety of Italian ryegrass.

In one embodiment of the present invention, in the step of identifying the variety, the base of the DNA corresponding to the 301st base in the polynucleotide represented by SEQ ID NO: 1 among the DNA of the Italian ryegrass is G or sequence The base of the DNA corresponding to the position of the 301st base in the polynucleotide represented by number 2 is C, the base of the DNA corresponding to the position of the 301st base in the polynucleotide represented by SEQ ID No. 3 is T, and the sequence The base of the DNA corresponding to the position of the 301st base in the polynucleotide represented by number 4 is A, the base of the DNA corresponding to the position of the 301st base in the polynucleotide represented by SEQ ID No. 5 is T, and the sequence If the base of the DNA corresponding to the position of the 301st base in the polynucleotide represented by number 6 is A, and the base of the DNA corresponding to the position of the 301st base in the polynucleotide represented by SEQ ID NO: 7 is T, The Italian ryegrass can be identified as the HS104 variety.

In one embodiment of the present invention, the Italian ryegrass varieties are Kowinner, Kospeed, Green Call, Green Call No. 2, IR601, IR602, IR603, IR605, IR901, Green Farm, Green Farm No. 2, Green Farm No. 3, Kowinearly, AE, Hwasan No. 101, Hwasan No. 102, Hwasan 104 It may be any one of Ho, Hercules (HC), Kowinmaster, Kogreen, Florida, Billiken, and Rio.

The present invention relates to a composition for identification of the Italian ryegrass HS104 variety, and in the polynucleotide consisting of the base sequences of SEQ ID NO: 1 and SEQ ID NO: 3 to 7, a single nucleotide polymorphism at the 301st base of SEQ ID NO among each base sequence ( It contains an agent that can detect single nucleotide polymorphism (SNP) markers.

The present invention relates to a composition for identification of Italian ryegrass HS104 variety, and in the polynucleotide consisting of the base sequences of SEQ ID NOs: 2 to 7, a single nucleotide polymorphism (single nucleotide polymorphism) at the 301st base of the sequence number among each base sequence. Includes an agent capable of detecting SNP) markers.

By using the SNP marker for identification of the Italian ryegrass HS104 variety and the Italian ryegrass HS104 variety identification method using the same according to an embodiment of the present invention, the Italian ryegrass HS104 variety can be identified.

Figure 1 shows the results of gDNA electrophoresis of an Italian ryegrass sample according to an embodiment of the present invention.
Figure 2 shows the results of electrophoresis on the GBS library according to an embodiment of the present invention.
Figure 3 shows an analysis schematic diagram according to an embodiment of the present invention.
Figure 4 shows a phylogenetic tree that is the result of analysis of inter-individual kinship relationships according to an embodiment of the present invention.
Figures 5a and 5b show the results of principal component analysis based on two factors according to an embodiment of the present invention.
Figure 6 shows the results of principal component analysis based on three factors according to an embodiment of the present invention.
Figure 7 is a graph showing the results of the delta-K method according to an embodiment of the present invention.
Figure 8 is a graph showing the results of population structure analysis at a peak with a K value of 2 according to an embodiment of the present invention.

Hereinafter, with reference to the attached drawings, a SNP marker for identifying Italian ryegrass HS104 variety according to the present invention and a method for identifying Italian ryegrass HS104 variety using the same will be described in detail.

Prior to this, the terms used in this specification and claims should not be construed limited to their dictionary meaning, and based on the principle that the inventor can appropriately define the concept of the term in order to explain his or her invention in the best way, , should be interpreted with meaning and concept consistent with the technical idea of the present invention.

The exemplary embodiments described above in the detailed description, drawings, and claims are not intended to be limiting, and other embodiments may be used, and other changes may be made without departing from the spirit or scope of the technology disclosed herein. .

Accordingly, the embodiments described in this specification and the configurations shown in the drawings are only preferred embodiments of the present invention and do not express the entire technical idea of the present invention, so various equivalents that can replace them at the time of filing the present application It should be understood that variations and variations may exist.

Those skilled in the art will be able to arrange, configure, combine, and design the components of the present disclosure, i.e., the components generally described herein and shown in the drawings, into a variety of different configurations, all of which are expressly contrived, and which are consistent with the present disclosure. It will be easy to understand that it forms part of .

The present invention is to identify the Italian Ryegrass Volcano No. 104 (HS104) variety using a SNP marker for identification of the Italian Ryegrass Volcano No. 104 (HS104) variety and a method for identifying the Italian Ryegrass Volcano No. 104 (HS104) variety using the same. .

Biological species can generally be identified through morphological and molecular biological methods, and in particular, molecular biological identification methods are helping to improve the identification accuracy of biological species by complementing the limitations of morphological classification.

Recently, as next-generation sequencing technology has developed rapidly and become more sophisticated, it has become possible to produce genomic information from living organisms more efficiently. In particular, GBS (Genotyping-By-Sequencing) is one of the newly developing analysis methods based on next-generation sequencing technology. The GBS (Genotyping-By-Sequencing) analysis uses restriction enzymes (RE) to sequence only the sequences surrounding the region cut out of the genome sequence, thus securing a wide range of genome information without performing full-length genome analysis. can do. Additionally, by selecting an appropriate restriction enzyme, repetitive regions in the genome sequence can be avoided and at the same time, it is possible to secure the base sequence of the desired region.

In this specification, 'Single Nucleotide Polymorphism (SNP)' refers to a change in one nucleotide sequence consisting of A (Adenine), T (Thymine), G (Guanine), and C (Cytosine) in the genome to a different nucleotide sequence. means that Polymorphism refers to the presence of two or more alleles at one genetic locus, and a polymorphic site in which only a single base differs depending on the individual is called a single nucleotide polymorphism (SNP). Preferred polymorphic markers have two or more alleles with an occurrence frequency of 1% or more in the selected population.

In this specification, 'allele' refers to several types of one gene that exist on the same locus of a homologous chromosome. The allele can be used to indicate polymorphism.

In this specification, 'marker' refers to a base sequence used as a reference point when identifying a genetically unspecified genetic locus, and the location of the marker on the genetic map can be indicated by a genetic locus.

The present invention is a composition for identifying Italian ryegrass Volcano No. 104 (HS104), a polynucleotide consisting of the base sequences of SEQ ID NO: 1 or SEQ ID NO: 2, and SEQ ID NOs: 3 to 7, wherein the sequence number of each base sequence is It may include an agent capable of detecting a single nucleotide polymorphism (SNP) marker at base 301.

For example, the composition for identifying the variety of Italian ryegrass Volcano No. 104 (HS104) is a polynucleotide consisting of the base sequences of SEQ ID NO: 1 and SEQ ID NO: 3 to 7, and the 301st base of SEQ ID NO among each base sequence. It may include an agent capable of detecting a single nucleotide polymorphism (SNP) marker.

In addition, for example, the composition for identifying the Italian ryegrass Volcanic No. 104 (HS104) variety is a polynucleotide consisting of the base sequences of SEQ ID NOs. 2 to 7, and a single base at the 301st base of SEQ ID NO. It may include an agent capable of detecting polymorphic (SNP) markers.

The single nucleotide polymorphism (SNP) marker according to an embodiment of the present invention is a polynucleotide represented by the nucleotide sequence of SEQ ID NO: 1, where the 301st base is G or A, and the 301st base is G or A, and the nucleotide sequence represented by SEQ ID NO: 2 is The 301st base in the polynucleotide is C or G, the 301st base in the polynucleotide represented by the base sequence of SEQ ID NO: 3 is T or C, and the 301st base in the polynucleotide represented by the base sequence of SEQ ID NO: 4 is A. or G, the 301st base in the polynucleotide represented by the base sequence of SEQ ID NO: 5 is T or C, the 301st base in the polynucleotide represented by the base sequence of SEQ ID NO: 6 is A or G, and the 301st base is A or G in the polynucleotide represented by the base sequence of SEQ ID NO: 7 In a polynucleotide expressed as a base sequence, the 301st base may be T or A.

If any of A, C, G, and T can be included at a specific point in the base sequence of SEQ ID NOs. 1 to 7 according to an embodiment of the present invention, it can be written as “n” according to the multiple base description method. .

Among the base sequences of SEQ ID NOs: 1 to 7 according to an embodiment of the present invention, the 301st base of SEQ ID NOs may be prepared according to the multiple base description method.

For example, if the allele is C/G, it can be either C or G, so it can be written as “s”. Also, for example, if the allele is T/C, it may be T or C, so it may be written as “y”. Also, for example, if the allele type is A/T, it may be A or T, so it may be written as “w”. Also, for example, if the allele is G/A, it may be G or A, so it may be written as “r”. Also, for example, if the allele type is A/C, it can be either A or C, so it can be written as “m”. Also, for example, if the allele is G/T, it may be G or T, so it may be written as “k”.

An agent capable of detecting a single nucleotide polymorphism (SNP) marker according to an embodiment of the present invention includes a polynucleotide consisting of 10 to 400 consecutive DNA sequences, including a single nucleotide polymorphism marker site; Or it is preferably a polynucleotide that specifically hybridizes with its complementary polynucleotide, but is not limited thereto. Preferably, the agent capable of detecting the single nucleotide polymorphism (SNP) marker may be a polynucleotide, which may be one or more selected from the group consisting of primers, probes, and combinations thereof capable of detecting the single nucleotide polymorphism marker. there is.

In this specification, 'primer' is a base sequence with a short free 3' terminal hydroxyl group, which can form a base pair with a complementary template and is used for copying the template strand. A short sequence that serves as a starting point. The primer can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization at an appropriate buffer solution and temperature. When designing the primers, there are various restrictions, such as the A, G, C, and T content ratio of the primers, prevention of primer complex (dimer) formation, and prohibition of repeating the same base sequence more than three times. In addition, in the single PCR reaction conditions, the template (template) Conditions such as the amount of DNA, concentration of primers, concentration of dNTP, concentration of Mg ²⁺ , reaction temperature, and reaction time must be appropriate.

The above primers may incorporate additional features without changing the basic properties. That is, nucleic acid sequences can be modified using many means known in the art. Examples of such modifications include methylation, capping, substitution of a nucleotide with one or more homologs, and phosphonate, phosphotriester, phosphoroamidate, or carbamate ( It is possible to modify nucleotides into uncharged linkages such as carbamate or charged linkages such as phosphorothioate or phosphorodithioate. In addition, nucleic acids include nucleases, toxins, antibodies, signal peptides, proteins such as poly L-lysine, inserts such as acridine or psoralen, metals, and radioactive substances. , may have one or more additional covalently bonded moieties such as chelants and alkylating agents such as iron oxidizing metals.

Additionally, the primer sequence of the present invention can be modified using a label that can directly or indirectly provide a detectable signal. The primer may contain a label that can be detected using spectroscopy, photochemistry, biochemistry, immunochemistry, or chemical means.

The present invention is capable of detecting a single nucleotide polymorphism (SNP) marker at the 301st base of SEQ ID NO: 1 or SEQ ID NO: 2, and SEQ ID NO: 3 to 7 in the polynucleotide. Provided is an Italian Ryegrass Volcano No. 104 (HS104) variety identification kit containing a composition for identifying the Italian Ryegrass Volcano No. 104 (HS104) variety containing an agent.

The Italian ryegrass Volcano No. 104 (HS104) variety identification kit according to an embodiment of the present invention can identify whether Italian ryegrass is the Volcano No. 104 (HS104) variety by detecting a single nucleotide polymorphism (SNP) marker. The Italian ryegrass HS104 variety identification kit may include polynucleotides, primers, and probes for detecting single nucleotide polymorphism markers, and may include one or more other components, solutions, or devices suitable for the identification method. You can.

For example, the Italian ryegrass HS104 variety identification kit of the present invention may be a kit containing essential elements for performing allele-specific polymerase chain reaction (PCR). The allele-specific polymerase chain reaction (PCR) kit includes a test tube or other appropriate container, reaction buffer, and deoxyribonucleotide triphophate (dNTP) in addition to a specific polynucleotide, primer, or probe for the single nucleotide polymorphism (SNP) marker of the present invention. , Taq-polymerase, and reverse transcriptase, DNase, RNAse inhibitor, DEPC (DiEthyl PyroCarbonate) water, and sterile water.

The present invention extracts the DNA of Italian ryegrass, performs polymerase chain reaction (PCR) using the extracted DNA as a template and a composition for identifying the Italian ryegrass HS104 variety, and performs the polymerase chain reaction (PCR) Provides an Italian ryegrass HS104 variety identification method for identifying the Italian ryegrass variety by analyzing the product produced by.

The Italian ryegrass varieties include Kowinner, Kospeed, Green Call, Green Call No. 2, IR601, IR602, IR603, IR605, IR901, and Green Farm. ), Green Farm No. 2, Green Farm No. 3, Kowinearly, AE, Hwasan No. 101 (HS101), Hwasan No. 102 (HS102), Hwasan No. 104 (HS104) ), Hercules (HC), Kowinmaster, Kogreen, Florida, Billiken, and Rio, but other varieties can also be called Italian. The ryegrass HS104 variety identification method can be applied, but is not limited thereto.

The Kowinner is a tetraploid species that was bred by creating a crossbreeding combination with a total of 5 superior nutritional lines, X8907, X9517, X9518, X9519, and As a variety, the color of the leaves is dark green, the first form before winter is a semi-prostrate type, and the first form in spring is an intermediate type. The Cowwinner is a mid-late ripening variety that is planted around May 19, and has the characteristics of longer plant height and longest stem compared to Hwasan No. 101, similar leaf lengths, and thick stems.

The above-mentioned Kospeed is a species bred by creating a crossbreeding combination with a total of 5 superior nutritional lines 00CR10, 00CR15, 00CR18, 00CR19, and 00CR41 through research by the Rural Development Administration to cultivate Italian ryegrass early-maturing varieties that are hardy and hardy. As a diploid variety, the color of the leaves is green, and the plant form before winter is semi-erect, and the plant form in spring is erect. The Kospid is an early-maturing variety that is harvested around May 1, and has wider leaf width, longer leaf length, and shorter plant height than Florida 80. In addition, the above-mentioned Cospid has a medium stem thickness and is a variety with stronger cold resistance than Florida 80.

The Green Call was developed by creating a crossbreeding combination with a total of 5 early-maturing lines, 09CR02, 09CR04, 09CR06, 09CR09, and 09CRR11, through research by the Rural Development Administration to cultivate Italian ryegrass varieties that ripen quickly and produce high production. It is a diploid variety with green leaf color, semi-erect form before winter, and erect form in spring. The Green Call is an extremely early maturing variety that goes out around April 25th, and has shorter leaf width, leaf length, and plant height, thicker stems, and comparable ear lengths compared to Florida 80.

The Green Call No. 2 was crossed with a total of five early-maturing lines, 09CR01, 09CR05, 09CR07, 09CR10, and 09CRR12 (K191022), through research by the Rural Development Administration to cultivate Italian ryegrass varieties with rapid ripening and high production. It is a species bred by creating a combination. It is a diploid variety with green leaf color, semi-erect form before winter, and erect form in spring. The Green Call No. 2 is an extremely early maturing variety that is harvested around April 24, and has longer leaf length, shorter leaf width and plant height, thicker stems, and equal ear length compared to Florida 80.

The IR601 is a species bred by creating a crossbreeding combination of a total of 5 09CR17, 09CR19, 09CR20, 09CR22, and 09CR18 through research by the Rural Development Administration. It is a tetraploid variety with green leaf color and pre-wintering plant type. Semi-erect type, spring's first type is an erect type. The IR 601 is a late-ripening variety that is harvested on May 18, and has longer leaf and ear lengths than Corwinery, a large number of spikelets, similar leaf widths, thick stems, and the longest liver life. It has the characteristic of being short.

The IR602 is a species bred by creating a crossbreeding combination of a total of 5 09CR15, 09CR16, 09CR21, 09CR13, and 09CR14 through research by the Rural Development Administration. It is a diploid variety with green leaf color and pre-wintering plant type. Upright, spring's first type is an upright individual. IR602 is a medium-growing variety that is harvested on May 10, and has slightly longer leaf lengths than Corwinery, similar leaf width, ear length, number of spikes, and stem thickness, and the longest stem is short. It has characteristics.

The IR603 is a species bred by creating a crossbreeding combination of a total of 5 15CR19, 15CR18, 15CR17, 15CR13, and 15CR21 through research by the Rural Development Administration. It is a tetraploid variety with green leaf color and pre-wintering plant type. Semi-erect type, spring's first type is an erect type. The IR 603 is a late-ripening variety that is harvested on May 16, and has significantly longer leaf and ear lengths than Corwinery, a significantly higher number of sugar spikes, similar leaf widths, and a significantly thicker stem. It has the characteristic of having a slightly shorter longest soy sauce.

The IR605 is a species bred through research by the Rural Development Administration by creating a crossbreeding combination with a total of 5 12EtYG2N18, 12EtYG2N18, 12EtYG2N27, ARIKG07, and ARIXRO18. It is a diploid variety with green leaf color and pre-wintering plant type. Semi-erect type, spring's first type is an erect type. The IR 605 is a medium-growing variety that is harvested on May 15, and has the characteristics of slightly longer leaf length, plant height, and ear length, slightly more number of spikelets, and similar leaf width and stem thickness compared to Corwinery. have

The IR901 is a species bred through research by the Rural Development Administration by creating a crossbreeding combination of a total of 5 12EtHN4NN14, 12EtHN4NN23, ARIXJN09, ARIXJN14, and ARIXJN18. It is a diploid variety with green leaf color and pre-wintering plant type. Semi-erect type, spring's first type is an erect type. The IR 901 is a late-ripening variety that is harvested on May 22, and has the characteristics of having a slightly longer ear length, a slightly larger number of spikelets, and similar leaf length, leaf width, and stem thickness compared to Corwinery.

The Green Farm is a species bred through research by the Rural Development Administration by creating a crossbreeding combination of a total of 5 06CR47, 06CR16, 06CR80, 06CR11, and 06CR26. It is a diploid variety with green leaf color and pre-wintering plant type. is an erect type, and Bom's initial type is a semi-erect type. The Green Farm is an extremely early maturing variety that is harvested on April 28. Compared to Florida 80, the stem thickness is thicker, the longest stem is similar, and the leaf length, leaf width, and ear length are shorter, It has the characteristic of having a small number of allowances.

The Green Farm No. 2 is a species bred by creating a crossbreeding combination of a total of 5 06CR33, 06CR52, 06CR05, 06CR38, and 06CR79 through research by the Rural Development Administration. It is a diploid variety with green leaf color and winter. The former first type is a semi-erect type, and the spring type is an erect type. The Green Farm No. 2 is an extremely early maturing variety that is harvested on May 4th. Compared to Florida 80, the stem thickness is thicker, the longest stem and leaf width are similar, and the leaf length and ear length are shorter. , it has the characteristic that the number of permissible persons is significantly small.

The above-mentioned Green Farm No. 3 is a species bred by creating a crossbreeding combination of a total of 5 08CR01, 08CR06, 08CR07, 08CR11, and 08CR16 through research by the Rural Development Administration. It is a diploid variety with green leaf color and winter. The former first type is a semi-erect type, and the spring type is an erect type. The Green Farm No. 3 is an extremely early maturing variety that is harvested on April 27th. Compared to Green Farm, the leaf length, ear length, and maximum liver length are longer, and the stem thickness and number of sorghum plants are similar. , it has the characteristic of narrow leaf width.

The Kowinearly is a species bred through research by the Rural Development Administration by creating a crossbreeding combination of a total of 5 00CR04, 00CR32, 00CR40, 00CR48, and 00CR58. It is a diploid variety with green leaf color and pre-wintering plant type. It is a semi-prone type, and the first type in spring is a semi-erect type. The above-mentioned Kowinearly is an early-maturing variety that is harvested on May 3, and has longer leaf and ear lengths, wider leaf width, and similar stem thickness and number of spikelets than Florida 80, and has the longest soy sauce. It has the characteristic of being short.

The above-mentioned Hwasan No. 101 (HS101) is a species bred through seed synthesis with a total of 5 94CR01, 94CR02, 94CR03, 94CR10, and 93CR06 through research by the Rural Development Administration. It is a tetraploid variety with dark green leaf color and a reddish-green pre-wintering initial type. The double type, Bom's first type, is an intermediate type individual. The volcano No. 101 is a mid-late ripening variety that is planted on May 20, and has the characteristics of having wider leaf widths than Marshall, similar ear length and number of spikes per seed, and low plant height at the time of planting.

The above-mentioned Hwasan No. 102 (HS102) is a species bred through seed synthesis with a total of 5 93CR04, 93CR05, 93CR07, 93CR08, and 93CR09 through research by the Rural Development Administration. It is a tetraploid variety with dark green leaf color and a reddish-green pre-wintering initial type. The double type, Bom's first type, is an intermediate type individual. The volcano No. 102 is a mid-late ripening variety that is planted on May 19, and has the characteristics of having a wider leaf width than Marshall, similar ear length and number of spikes per seed, and low plant height at the time of planting.

The above-mentioned Hwasan No. 104 (HS104) is a species bred by creating a cross combination with a total of 5 P8702, P8705, is a semi-prone type, and Bom's supertype is an intermediate type. The Hwasan No. 104 is a late-ripe variety that is planted on May 19, and has wider leaf width, longer leaf length and plant height, and thicker stems than Hwasan No. 101.

The Kowinmaster is a species bred by seed synthesis with a total of 5 00CR22, 00CR35, 00CR31, 00CR46, and 00CR55 through research by the Rural Development Administration to cultivate cold-resistant Italian ryegrass varieties. It is a diploid variety and has different leaf colors. The species is light green, the first form before winter is a medium type, and the first form in spring is a semi-erect type. The above-mentioned Kowinmaster is a medium-growing variety that is harvested on May 10, and has the characteristics of longer leaf length and ear length compared to Florida 80, and similar leaf width, number of sorghum sorghums, longest stem, and stem thickness. has

The Kogreen is a species bred by seed synthesis with a total of 5 00CR02, 00CR05, 00CR24, 00CR28, and 00CR43 through research by the Rural Development Administration to cultivate cold-resistant Italian ryegrass varieties. It is a diploid variety and has different leaf colors. is green, the first form before wintering is semi-erect, and the first form in spring is an erect form. The above-mentioned Kogreen is an early-maturing variety that is harvested on May 4, and has the characteristics of wider leaf width, thicker stem, and similar leaf length, ear length, and number of spikelets compared to Florida 80. have

In the step of identifying the variety, the DNA of the Italian ryegrass can be identified by analyzing the product produced by the polymerase chain reaction (PCR), and the nucleotide sequence of the identified DNA can be confirmed to determine the variety of the Italian ryegrass. can be identified.

In addition, the step of identifying the variety is when the base of the DNA corresponding to the position of the 301st base in the polynucleotide represented by SEQ ID NO: 1 among the DNA of the Italian ryegrass is G or in the polynucleotide represented by SEQ ID NO: 2 The base of the DNA corresponding to the position of the 301st base is C, and in the polynucleotide represented by SEQ ID NO: 3, the base of the DNA corresponding to the position of the 301st base is T, and in the polynucleotide represented by SEQ ID NO: 4 The base of the DNA corresponding to the position of the 301st base is A, and the base of the DNA corresponding to the position of the 301st base in the polynucleotide represented by SEQ ID NO: 5 is T, and in the polynucleotide represented by SEQ ID NO: 6 If the base of the DNA corresponding to the position of the 301st base is A and the base of the DNA corresponding to the position of the 301st base in the polynucleotide represented by SEQ ID NO: 7 is T, the Italian ryegrass variety is HS104 It can be identified by breed.

Example 1: GBS library sequence analysis using Italian ryegrass samples

Example 1-1: Italian ryegrass sample preparation

Table 1 below shows a list of 96 Italian ryegrass samples used to create a GBS (Genotyping-By-Sequencing) library.

Referring to Table 1 above, you can see a list of 96 Italian ryegrass samples used to produce the GBS library. KW refers to the Kowinearly variety, KS refers to the Kospeed variety, GC refers to the Green Call variety, and GC2 refers to the Green Call No. 2 variety. And 601, 602, 603, 605, and 901 refer to the varieties IR601 (IR601), IR602 (IR602), IR603 (IR603), IR605 (IR605), and IR901 (IR901), and GF is It refers to the Green farm variety, GF2 refers to the Green Farm variety No. 2, GF3 refers to the Green Farm variety No. 3, and KWR refers to the Kowinner variety. AE refers to a line selected from the ACE variety with an early seeding period, HS101, HS102, and HS104 refer to Hwasan 101, Hwasan 102, and Hwasan 104 varieties, and HC refers to Hercules. In the variety, it refers to a line selected with a fast heading period, KM refers to the Kowinmaster variety, KG refers to the Kogreen variety, FLO refers to the Florida80 variety, and BK refers to the variety. It refers to the Billiken variety, and Rio refers to the Rio variety. Since 4 samples each were prepared for a total of 24 varieties, a total of 96 Italian ryegrass samples were prepared.

Example 1-2: gDNA QC (Quality Check) of Italian ryegrass samples

Figure 1 shows the results of gDNA electrophoresis of an Italian ryegrass sample according to an embodiment of the present invention.

Referring to Figure 1, it can be seen that electrophoresis was performed on gDNA of a total of 96 samples previously prepared in Example 1-1. Through the above process, gDNA of 96 samples suitable for GBS (Genotyping-By-Sequencing) library production was confirmed.

Example 1-3: Production of GBS (Genotyping-By-Sequencing) library of Italian ryegrass samples

GBS (Genotyping-By-Sequencing) library according to an embodiment of the present invention includes the steps of annealing oligonucleotides containing the top and bottom strands of each barcode adapter and a common adapter, and preparing Italian Digesting the DNA of the ryegrass sample with Apek I, a restriction enzyme (RE), connecting an adapter to the end of the gDNA fragment, heating to inactivate the ligase, and pooling each sample. Purifying to remove unreacted adapters, adding an appropriate primer having a binding site to the ligated adapter and performing multiplex PCR, and cleaning up the PCR products and performing agarose gel running. ) was produced through a verification process.

In more detail, the GBS library was constructed using the restriction enzyme Apek I. Oligonucleotides containing each barcode adapter and the common adapter were diluted in 50 uM of TE buffer (10 mM Tris, 0.1 mM EDTA), and annealed in a thermocycler. Afterwards, the barcode and general adapter were diluted to 10 uM using 10x adapter buffer (500 mM NaCl, 100 mM Tris-Cl) and mixed together at a 1:1 ratio, and then 2.4 ㎕ of the mix was added to a 96-well PCR plate. . Sample DNA (100 ng/μl) was added to the wells containing individual adapters, and samples (DNA + adapter) were reacted at 75°C in a 20 μl volume containing 1x NEB Buffer 3 and 3.6 U Apek I. Samples were incubated at 22°C and heated at 65°C for 20 min to inactivate T4 DNA ligase. Then, a set of 96 DNA samples (5 μl each) with different barcode adapters were combined and purified using a kit (QIAquick PCR Purification Kit, Qiagen, CA). Using 2 ㎕ of pooled DNA fragments, DNA polymerase (Agilent), and 25 pmol of primers, PCR was performed using a Life ECO Thermal Cycler (Bioer Technology Co.) to amplify the restriction fragments of each library to construct the library. did.

Example 1-4: Purification of the produced GBS library

Two purification processes were performed on the GBS (Genotyping-By-Sequencing) library produced in Examples 1-3, and the results are shown in Table 2 below.

No Sample 1 ^st Purification ^2nd Purification One Italian ryegrass GBS library set-1(IRG-1) O O 2 Italian ryegrass GBS library set-2(IRG-2) O O 3 Italian ryegrass GBS library pooling X O 4 Positive control O O 5 Negative control X O

Figure 2 shows the results of electrophoresis on the GBS library according to an embodiment of the present invention. The electrophoresis was performed for 30 minutes at 200v using a 1.5% agarose gel, and 5 ㎕ of each 100bp marker (M, Marker) and library were used.

Referring to Figure 2, it can be seen that the GBS (Genotyping-By-Sequencing) library according to an embodiment of the present invention is formed in a smear-like manner. In particular, the concentration was high at 250bp to 350bp.

Example 1-5: GBS library QC (Quality Check)

Previously, a quality check (QC) was performed on the GBS (Genotyping-By-Sequencing) library purified in Examples 1-4.

Table 3 below shows the results of quality check (QC) conducted on the GBS (Genotyping-By-Sequencing) library.

Library Name Library Type Conc.
(ng/㎕) Conc.
(nM) Size
(bp) Result One IRG-1 GBS library (single enzyme) 11.62 39.62 451 Pass 2 IRG-2 GBS library (single enzyme) 11.68 35.64 504 pass

Referring to Table 3 above, it can be seen that the concentration value in nM units of IRG-1 is higher. Accordingly, it was decided to use the IRG-1 library in the following examples.

Figure 3 shows an analysis schematic diagram according to an embodiment of the present invention.

Referring to FIG. 3, the order of analysis to be performed in subsequent examples can be confirmed. In subsequent examples, GBS (Genotyping-By-Sequencing) data is extracted, sequence demultiplexing and pre-processing are performed, and mapping is performed to a reference genome. Detect raw SNPs (In/Del), extract consensus sequences, create SNP matrices, perform SNP filtering and selection, and select SNPs. You can apply or select polymorphic SNPs from a group. More details will be disclosed through the examples below.

Example 2: GBS data

Example 2-1: Sequencing raw data

Using the IRG-1 library prepared in Example 1, 1 lane (approximately 100 Gbp) sequencing was performed using an Illumine HiSeq

Table 4 below shows the results of sequencing conducted using the IRG-1 library.

Plate No. of barcode No. of sample No. of reads Avg. length (bp) Total length (bp) GC
(%) Q30
(%) No. of
demultiplexed reads (%) 1st production 1 96 96 391,722,338 151 59,150,073,038 48.36 93.46 675,379,548 (86.21%) 1st production 2 391,722,338 151 59,150,073,038 Additional production 1 96 96 375,155,903 151 56,648,541,353 48.39 92.69 646,988,004 (86.23%) Additional production 2 375,155,903 151 56,648,541,353 Total 192 96 1,533,756,482 231,597,228,782

Referring to Table 4 above, it can be seen that sequencing law data for 96 samples of Italian ryegrass was secured using the previously produced IRG-1 library.

Example 2-2: GBS sequencing pre-processing

Demultiplexing (demultiplexing) was performed to separate sequences for each sample using the barcode sequence of the IRG-1 library previously produced in Example 1.

Table 5 below shows raw data statistics for each sample through demultiplexing according to an embodiment of the present invention.

BarCode Sample name Sum of raw reads Total length of raw reads CTCC KW_01 27,984,674 4,225,685,774 TGCA KW_02 20,508,824 3,096,832,424 ACTA KW_03 10,094,474 1,524,265,574 CAGA KW_04 11,023,322 1,664,521,622 AACT KS_01 24,973,212 3,770,955,012 GCGT KS_02 20,586,020 3,108,489,020 CGAT KS_03 16,037,266 2,421,627,166 GTAA KS_04 5,343,352 806,846,152 AGGC GC2_01 43,252,098 6,531,066,798 GATC GC2_02 28,706,912 4,334,743,712 TCAC GC2_03 18,224,244 2,751,860,844 TGCGA GC2_04 10,023,360 1,513,527,360 CGCTT GC_01 23,261,356 3,512,464,756 TCACC GC_02 26,581,886 4,013,864,786 CTAGC GC_03 19,102,102 2,884,417,402 ACAAA GC_04 13,195,778 1,992,562,478 TTCTC 901_01 56,763,672 8,571,314,472 AGCCC 901_02 24,411,272 3,686,102,072 GTATT 901_03 24,158,770 3,647,974,270 CTGTA 901_04 7,742,580 1,169,129,580 ACCGT 605_01 29,695,634 4,484,040,734 GCTTA 605_02 10,394,096 1,569,508,496 GGTGT 605_03 7,667,484 1,157,790,084 AGGAT 605_04 13,233,298 1,998,227,998 ATTGA 604_01 4,852,450 732,719,950 CATCT 604_02 25,011,476 3,776,732,876 CCTAC 604_03 34,245,834 5,171,120,934 GAGGA 604_04 12,545,584 1,894,383,184 GGAAC 603_01 22,336,060 3,372,745,060 GTCAA 603_02 16,499,444 2,491,416,044 TAATA 603_03 11,454,554 1,729,637,654 TACAT 603_04 15,046,218 2,271,978,918 TCGTT 602_01 10,197,584 1,539,835,184 GGTTGT 602_02 11,741,306 1,772,937,206 CCAGCT 602_03 10,880,226 1,642,914,126 TTCAGA 602_04 3,457,092 522,020,892 TAGGAA 601_01 5,693,774 859,759,874 GCTCTA 601_02 3,661,468 552,881,668 CCACAA 601_03 6,965,674 1,051,816,774 CTTCCA 601_04 7,619,840 1,150,595,840 GAGATA GF3_01 16,284,028 2,458,888,228 ATGCCT GF3_02 36,885,684 5,569,738,284 AGTGGA GF3_03 2,874,510 434,051,010 ACCTAA GF3_04 7,997,846 1,207,674,746 ATATGT GF2_01 11,568,158 1,746,791,858 ATCGTA GF2_02 6,219,668 939,169,868 CATCGT GF2_03 17,454,040 2,635,560,040 CGCGGT GF2_04 18,283,940 2,760,874,940 CTATTA GF_01 11,180,026 1,688,183,926 GCCAGT GF_02 16,376,054 2,472,784,154 GGAAGA GF_03 4,017,876 606,699,276 GTACTT GF_04 26,145,584 3,947,983,184 GTTGAA KWR_01 8,605,762 1,299,470,062 TAACGA KWR_02 5,258,768 794,073,968 TGGCTA KWR_03 7,583,138 1,145,053,838 TATTTTTT KWR_04 30,645,534 4,627,475,634 CTTGCTT AE_01 12,043,426 1,818,557,326 ATGAAC AE_02 17,350,990 2,619,999,490 AAAAGTT AE_03 15,840,586 2,391,928,486 GAATTCA AE_04 14,806,830 2,235,831,330 GAACTTC HS104_01 21,381,014 3,228,533,114 GGACCTA HS104_02 4,089,342 617,490,642 GTCGATT HS104_03 12,723,770 1,921,289,270 AACGCCT HS104_04 14,317,194 2,161,896,294 AATATGC HC_01 20,170,526 3,045,749,426 ACGTGTT HC_02 14,699,634 2,219,644,734 ATTAATT HC_03 8,487,060 1,281,546,060 ATTGGAT HC_04 11,535,506 1,741,861,406 CATAAGT HS102_01 7,520,160 1,135,544,160 CGCTGAT HS102_02 7,965,080 1,202,727,080 CGGTAGA HS102_03 2,530,602 382,120,902 CTACGGA HS102_04 8,076,456 1,219,544,856 GCGGAAT HS101_01 21,054,910 3,179,291,410 TAGCGGA HS101_02 13,352,672 2,016,253,472 TCGAAGA HS101_03 4,452,216 672,284,616 TCTGTGA HS101_04 8,781,464 1,326,001,064 TGCTGGA KM_01 8,381,220 1,265,564,220 ACGACTAC KM_02 15,430,376 2,329,986,776 TAGCATGC KM_03 10,553,204 1,593,533,804 TAGGCCAT KM_04 11,838,336 1,787,588,736 TGCAAGGA KG_01 6,387,922 964,576,222 TGGTACGT KG_02 13,455,236 2,031,740,636 TCTCAGTC KG_03 20,725,562 3,129,559,862 CCGGATAT KG_04 9,328,880 1,408,660,880 CGCCTTAT FLO_01 8,434,712 1,273,641,512 AACCGAGA FLO_02 3,945,090 595,708,590 ACAGGGAA FLO_03 3,202,022 483,505,322 ACGTGGTA FLO_04 5,163,006 779,613,906 CCATGGGT BK_01 7,995,282 1,207,287,582 CGCGGAGA BK_02 3,333,304 503,328,904 CGTGTGGT BK_03 4,666,524 704,645,124 GCTGTGGA BK_04 2,680,124 404,698,724 GGATTGGT Rio_01 8,503,012 1,283,954,812 GTGAGGGT Rio_02 5,582,316 842,929,716 TATCGGGA Rio_03 7,055,828 1,065,430,028 TTCCTGGA Rio_04 1,971,272 297,662,072 Average (AVG) 13,774,662 13,429,185 SUM 1,322,367,552 1,289,201,798

In addition, barcodes and adapter sequences were removed from raw data for each sample through demultiplexing, and sequence quality trimming was performed.

For adapter trimming, use the cutadapt (version 1.8.3) program, and for sequence quality trimming, use DynamicTrim and Length Salt (in the SolexaQA package). The LengthSort) program was used.

The DynamicTrim performs a process of cutting out bad quality bases at both ends of short reads according to the phred score and refining them into good quality clean reads. In addition, LengthSort performed the process of removing reads with too many bases cut off in DynamicTrim. In DynamicTrim, a phred score of 20 or more was used, and in LengthSort, a short read length of 25 bp (base pair) or more was used.

Table 6 below shows the accuracy according to the score of the Fred quality score.

Phred Quality Score Probability of Incorrect Base Call Base call Accuracy 10 1 in 10 90% 20 1 in 100 99% 30 1 in 1,000 99.9% 40 1 in 10,000 99.99%

Referring to Table 6, it can be seen that the Dynamic Trim according to an embodiment of the present invention used a base call accuracy of 99% or more.

Table 7 below shows data statistics trimmed for each sample through demultiplexing according to an embodiment of the present invention.

BarCode Sample name Sum of trimmed reads Total length of trimmed reads (bp) Avg. length of trimmed reads (bp) Trimmed/Raw (%) CTCC KW_01 27,275,748 3,451,335,884 126.53 97.47% TGCA KW_02 19,994,044 2,535,790,267 126.83 97.49% ACTA KW_03 9,839,642 1,252,579,236 127.30 97.48% CAGA KW_04 10,751,044 1,363,582,041 126.83 97.53% AACT KS_01 24,367,894 3,098,411,409 127.15 97.58% GCGT KS_02 20,110,828 2,560,487,624 127.32 97.69% CGAT KS_03 15,657,066 1,989,405,726 127.06 97.63% GTAA KS_04 5,212,994 661,714,594 126.94 97.56% AGGC GC2_01 42,251,322 5,383,515,857 127.42 97.69% GATC GC2_02 28,055,162 3,561,867,436 126.96 97.73% TCAC GC2_03 17,775,900 2,256,018,257 126.91 97.54% TGCGA GC2_04 9,775,488 1,239,906,948 126.84 97.53% CGCTT GC_01 22,661,814 2,868,957,974 126.60 97.42% TCACC GC_02 25,919,568 3,292,060,831 127.01 97.51% CTAGC GC_03 18,598,778 2,342,989,872 125.98 97.37% ACAAA GC_04 12,855,874 1,630,601,653 126.84 97.42% TTCTC 901_01 55,333,290 7,009,107,424 126.67 97.48% AGCCC 901_02 23,805,438 3,011,204,578 126.49 97.52% GTATT 901_03 23,583,014 2,994,447,575 126.97 97.62% CTGTA 901_04 7,546,312 955,762,430 126.65 97.47% ACCGT 605_01 28,963,328 3,677,531,171 126.97 97.53% GCTTA 605_02 10,131,974 1,287,061,981 127.03 97.48% GGTGT 605_03 7,498,770 951,053,597 126.83 97.80% AGGAT 605_04 12,928,504 1,637,232,763 126.64 97.70% ATTGA 604_01 4,735,144 601,324,788 126.99 97.58% CATCT 604_02 24,394,414 3,088,358,722 126.60 97.53% CCTAC 604_03 33,350,966 4,230,887,688 126.86 97.39% GAGGA 604_04 12,262,020 1,559,723,150 127.20 97.74% GGAAC 603_01 21,797,130 2,766,281,029 126.91 97.59% GTCAA 603_02 16,119,982 2,052,084,130 127.30 97.70% TAATA 603_03 11,158,524 1,415,077,014 126.82 97.42% TACAT 603_04 14,663,850 1,859,221,925 126.79 97.46% TCGTT 602_01 9,957,556 1,264,468,759 126.99 97.65% GGTTGT 602_02 11,476,614 1,455,393,357 126.81 97.75% CCAGCT 602_03 10,596,762 1,336,288,603 126.10 97.39% TTCAGA 602_04 3,369,536 426,129,681 126.47 97.47% TAGGAA 601_01 5,553,860 703,419,411 126.65 97.54% GCTCTA 601_02 3,566,280 451,100,513 126.49 97.40% CCACAA 601_03 6,771,856 855,511,063 126.33 97.22% CTTCCA 601_04 7,412,280 934,676,535 126.10 97.28% GAGATA GF3_01 15,870,268 1,998,349,954 125.92 97.46% ATGCCT GF3_02 35,908,458 4,494,133,934 125.16 97.35% AGTGGA GF3_03 2,807,100 355,144,515 126.52 97.65% ACCTAA GF3_04 7,782,006 984,545,413 126.52 97.30% ATATGT GF2_01 11,281,864 1,426,204,139 126.42 97.53% ATCGTA GF2_02 6,056,750 765,613,900 126.41 97.38% CATCGT GF2_03 17,007,396 2,149,951,332 126.41 97.44% CGCGGT GF2_04 17,821,792 2,249,742,665 126.24 97.47% CTATTA GF_01 10,876,724 1,369,550,932 125.92 97.29% GCCAGT GF_02 15,966,054 2,020,329,611 126.54 97.50% GGAAGA GF_03 3,921,630 496,165,910 126.52 97.60% GTACTT GF_04 25,478,418 3,222,074,462 126.46 97.45% GTTGAA KWR_01 8,398,816 1,062,557,308 126.51 97.60% TAACGA KWR_02 5,116,732 645,289,422 126.11 97.30% TGGCTA KWR_03 7,387,354 934,175,295 126.46 97.42% TATTTTTT KWR_04 29,789,550 3,735,900,055 125.41 97.21% CTTGCTT AE_01 11,719,510 1,474,675,787 125.83 97.31% ATGAAC AE_02 16,896,068 2,132,239,778 126.20 97.38% AAAAGTT AE_03 15,459,336 1,950,733,471 126.18 97.59% GAATTCA AE_04 14,427,082 1,820,502,390 126.19 97.44% GAACTTC HS104_01 20,803,738 2,616,639,232 125.78 97.30% GGACCTA HS104_02 3,984,684 501,708,101 125.91 97.44% GTCGATT HS104_03 12,428,134 1,568,292,657 126.19 97.68% AACGCCT HS104_04 13,928,584 1,751,567,681 125.75 97.29% AATATGC HC_01 19,623,186 2,464,758,296 125.60 97.29% ACGTGTT HC_02 14,341,476 1,811,119,778 126.29 97.56% ATTAATT HC_03 8,275,758 1,044,415,849 126.20 97.51% ATTGGAT HC_04 11,271,380 1,424,145,107 126.35 97.71% CATAAGT HS102_01 7,325,992 923,497,075 126.06 97.42% CGCTGAT HS102_02 7,764,184 979,000,772 126.09 97.48% CGGTAGA HS102_03 2,465,222 310,685,306 126.03 97.42% CTACGGA HS102_04 7,856,212 986,175,040 125.53 97.27% GCGGAAT HS101_01 20,510,334 2,586,720,662 126.12 97.41% TAGCGGA HS101_02 13,010,938 1,638,915,759 125.96 97.44% TCGAAGA HS101_03 4,340,348 548,188,024 126.30 97.49% TCTGTGA HS101_04 8,559,258 1,079,498,526 126.12 97.47% TGCTGGA KM_01 8,166,996 1,029,238,008 126.02 97.44% ACGACTAC KM_02 15,009,954 1,887,498,346 125.75 97.28% TAGCATGC KM_03 10,264,012 1,286,776,171 125.37 97.26% TAGGCCAT KM_04 11,542,352 1,453,301,548 125.91 97.50% TGCAAGGA KG_01 6,224,482 783,442,672 125.86 97.44% TGGTACGT KG_02 13,112,664 1,650,397,076 125.86 97.45% TCTCAGTC KG_03 20,232,582 2,549,553,994 126.01 97.62% CCGGATAT KG_04 9,085,132 1,143,313,688 125.84 97.39% CGCCTTAT FLO_01 8,200,218 1,027,827,512 125.34 97.22% AACCGAGA FLO_02 3,844,176 484,104,712 125.93 97.44% ACAGGGAA FLO_03 3,123,198 393,699,697 126.06 97.54% ACGTGGTA FLO_04 5,028,826 629,143,384 125.11 97.40% CCATGGGT BK_01 7,806,476 984,852,104 126.16 97.64% CGCGGAGA BK_02 3,249,990 408,441,100 125.67 97.50% CGTGTGGT BK_03 4,559,274 574,531,180 126.01 97.70% GCTGTGGA BK_04 2,613,926 328,857,180 125.81 97.53% GGATTGGT Rio_01 8,318,062 1,048,888,019 126.10 97.82% GTGAGGGT Rio_02 5,468,334 690,763,234 126.32 97.96% TATCGGGA Rio_03 6,886,696 863,563,540 125.40 97.60% TTCCTGGA Rio_04 1,921,542 241,788,575 125.83 97.48% Average (AVG) 13,429,185 1,697,872,535 126.34 97.49% SUM 1,289,201,798 162,995,763,374

Example 3: Alignment to reference genome

Previously, clean reads of each sample obtained through demultiplexing and sequence quality trimming in Example 2 were mapped to a standard genome (reference genome) and statistics were extracted.

The reference genome is 'First assembly of the gene-space of Lolium multiflorum and comparison to other Poaceae genomes' disclosed on pages 125 to 134 of 'GRASSLAND SCIENCE' Volume 65, Issue 2, published on December 9, 2018. The genome of Lolium multiflorum included in was used.

Table 8 below shows statistical values of the standard genome.

No. of scaffolds Total length (bp) Min. length (bp) Max. length (bp) Avg. length (bp) N50 length (bp) 129,579 585,844,536 2,000 43,903 4,521 5,036

In extracting statistical values of the mapping, in more detail, clean reads that have passed the preprocessing process are compared to a standard genome (reference genome, Mapping was performed on the reference genome. Mapping is a prerequisite to finding raw SNPs (In/Del) between the standard genome and sequenced samples, and creates a file in BAM (Binary alignment Map) format, with options set to default.

Tables 9 and 10 below show the results of extracting statistical values by matching the clean reads of each sample to the standard genome.

Example 4: Genome-wide SNP

Example 4-1: Raw SNP detection and consensus sequence extraction

The BAM format file generated by mapping the clean reads extracted from a total of 96 previously disclosed Italian ryegrass samples to the reference genome was generated using the SAM tool (0.1.16) program. Using this method, raw SNPs (In/Del) were detected and a consensus sequence was extracted. At this time, before the SNP detection process, SNP validation is performed using the SEEDERS in-house script, and then raw SNP (In/Del) detection is performed. carried out. Except for the options disclosed in Table 11 below, default values were applied.

Minimum mapping quality for SNPs (-Q) = 30 Minimum mapping quality for gaps (-q) = 15 Minimum read depth (-d) = 3 Maximum read depth (-D) = 611 Min In/Del score for nearby SNP filtering (-G) = 30 SNP within INT bp around a gap to be filtered (-w) = 15 Window size for filtering dense SNPs (-W) = 15

Example 4-2: Generate SNP matrix

To perform comparative analysis of SNPs between analysis subjects, an integrated SNP matrix was created between a total of 96 Italian ryegrass samples. A list of unions was constructed using the raw SNP positions obtained by comparing each sample with the reference genome as candidates. At this time, a matrix was created through a filling process in which empty regions (non-SNP loci) were filled from the consensus sequence of the sample. Afterwards, through SNP comparison between samples, miss-calling SNP (In/Del) loci were filtered to create a final SNP matrix. Based on the corresponding locus, SNPs (In/Del) were classified according to the type classification criteria disclosed in Table 12 below.

Homozygous: read rate ≥ 90%
Heterozygous: 40% ≤ read rate ≤ 60%
Etc.: 20% ≤ read rate ≤ 40%, and 60% ≤ read rate ≤ 90% Read rate: Ratio of the number of mutated reads to the total number of mapped reads

Table 13 below shows the number of SNPs classified according to the above type classification criteria.

Sample No. of Total SNPs No. of Homozygous SNPs
(read rate ≥ 90%) No. of Heterozygous SNPs
(40% ≤ read rate ≤ 60%) No. of etc. SNP
(20% ≤ read rate < 40%,
60% < read rate < 90%) KW_01 334,041 258,794 27,911 47,336 KW_02 370,155 270,313 38,515 61,327 KW_03 198,035 156,595 16,702 24,738 KW_04 226,906 172,438 22,037 32,431 KS_01 380,929 281,305 37,616 62,008 KS_02 326,216 243,515 31,626 51,075 KS_03 281,094 212,041 27,242 41,811 KS_04 99,880 83,523 6,682 9,675 GC2_01 484,751 344,905 51,501 88,345 GC2_02 364,455 274,761 33,799 55,895 GC2_03 250,107 196,642 20,596 32,869 GC2_04 172,185 136,339 14,449 21,397 GC_01 334,595 244,670 34,585 55,340 GC_02 334,687 257,613 28,671 48,403 GC_03 260,707 200,099 23,525 37,083 GC_04 214,168 164,867 19,847 29,454 901_01 593,127 414,218 63,530 115,379 901_02 313,508 239,767 28,025 45,716 901_03 336,473 244,224 35,631 56,618 901_04 141,322 112,549 11,773 17,000 605_01 393,642 282,746 42,090 68,806 605_02 173,941 138,049 14,607 21,285 605_03 135,036 110,228 10,187 14,621 605_04 209,477 162,759 19,131 27,587 604_01 100,458 83,326 7,119 10,013 604_02 359,679 258,410 39,494 61,775 604_03 412,954 302,930 40,232 69,792 604_04 217,390 165,108 20,912 31,370 603_01 298,378 228,396 26,801 43,181 603_02 254,674 190,503 25,401 38,770 603_03 198,842 152,766 18,685 27,391 603_04 246,443 186,899 23,935 35,609 602_01 154,886 126,361 11,438 17,087 602_02 187,921 150,479 14,912 22,530 602_03 190,842 151,221 15,875 23,746 602_04 58,546 51,014 3,089 4,443 601_01 103,489 85,287 7,477 10,725 601_02 66,729 57,560 3,726 5,443 601_03 123,593 100,632 9,324 13,637 601_04 126,822 102,498 9,944 14,380 GF3_01 232,880 180,383 19,426 33,071 GF3_02 375,031 279,621 33,067 62,343 GF3_03 55,613 48,218 3,085 4,310 GF3_04 118,370 97,610 8,480 12,280 GF2_01 182,844 148,343 13,541 20,960 GF2_02 108,315 91,477 6,662 10,176 GF2_03 256,284 199,346 21,492 35,446 GF2_04 256,699 199,333 21,510 35,856 GF_01 171,339 135,493 13,981 21,865 GF_02 244,596 190,448 20,709 33,439 GF_03 74,607 63,419 4,568 6,620 GF_04 348,598 261,539 32,219 54,840 KWR_01 168,850 134,079 13,913 20,858 KWR_02 82,656 69,360 5,347 7,949 KWR_03 126,348 103,132 9,401 13,815 KWR_04 412,013 295,706 42,977 73,330 AE_01 170,847 138,308 12,784 19,755 AE_02 197,420 163,240 12,452 21,728 AE_03 220,598 176,080 16,966 27,552 AE_04 218,459 173,395 17,146 27,918 HS104_01 295,216 230,112 24,164 40,940 HS104_02 75,215 64,840 4,199 6,176 HS104_03 204,739 161,628 17,029 26,082 HS104_04 218,793 171,227 18,817 28,749 HC_01 257,382 204,395 19,245 33,742 HC_02 198,777 160,944 14,575 23,258 HC_03 118,006 100,546 6,915 10,545 HC_04 189,222 153,513 14,079 21,630 HS102_01 107,836 88,943 7,777 11,116 HS102_02 137,452 111,562 10,306 15,584 HS102_03 27,239 24,202 1,365 1,672 HS102_04 134,099 107,997 10,491 15,611 HS101_01 294,478 220,586 27,757 46,135 HS101_02 188,347 145,506 16,788 26,053 HS101_03 75,385 63,763 4,898 6,724 HS101_04 130,969 105,758 10,130 15,081 KM_01 127,088 103,321 9,721 14,046 KM_02 209,184 168,372 14,887 25,925 KM_03 150,273 122,600 10,804 16,869 KM_04 151,921 123,898 11,299 16,724 KG_01 97,177 81,470 6,452 9,255 KG_02 104,461 85,859 7,471 11,131 KG_03 267,736 207,308 22,536 37,892 KG_04 134,001 109,656 9,960 14,385 FLO_01 122,959 100,811 9,269 12,879 FLO_02 39,271 34,460 2,047 2,764 FLO_03 53,362 46,358 3,014 3,990 FLO_04 74,002 63,350 4,339 6,313 BK_01 123,054 100,383 9,246 13,425 BK_02 53,958 46,499 3,121 4,338 BK_03 41,617 36,438 2,185 2,994 BK_04 26,510 23,709 1,192 1,609 Rio_01 87,307 71,977 6,465 8,865 Rio_02 75,928 64,652 4,759 6,517 Rio_03 59,810 50,627 3,856 5,327 Rio_04 21,932 19,908 888 1,136

Example 4-3: SNP filtering

The SNP filter process was performed using the SNP matrix of 96 Italian ryegrass samples under the conditions in Table 14 below.

SNP loci of biallelic
Minor allele frequency > 5%
Missing data < 30%

Table 15 below shows the results of the SNP filter process using the SNP matrix of 96 Italian ryegrass samples.

filter stage filter item SNP matrix loci One Total SNP matrix 3,675,167 2 MAF (minor allele frequency) >5% ^*1 2,131,719 3 Missing data <30% ^*2 65,498 4 MAF >5% and Missing data <30% 13,196 5 Random 5,000 5,000 *1) MAF (Minor Allele Frequency) > 5%: SNPs with a minor allele frequency greater than 5% are selected from all samples of the locus.
*2) Missing data < 30%: SNPs with missing data of less than 30% are selected from the entire sample for the corresponding locus.

Random 5,000 disclosed in Table 15 means that random selection was performed on 5,000 SNP loci at a level available for analysis from the filtered SNP 13,196 loci.

Example 5: Construction of phylogenetic tree

Example 5-1: Phylogenetic tree

Inter-individual correlation analysis was performed using the MEGA6 program on 13,196 SNP loci selected through the filtering process previously described in Example 4.

In more detail, the linkage analysis was performed using the Neighbor-joining method using the 13,196 SNP loci disclosed previously. As a result, an optimal tree with a sum of branch lengths of 14.09591913 was completed. Evolutionary distances were calculated using the Maximum Composite Likelihood method and are expressed in units of the number of base substitutions per site.

Figure 4 shows a phylogenetic tree that is the result of analysis of inter-individual kinship relationships according to an embodiment of the present invention.

Referring to Figure 4, it can be seen that the classification of Italian ryegrass 96 individuals according to an embodiment of the present invention is structured. EMC (Early Maturing Crop) disclosed in the phylogenetic tree refers to an early maturing crop, LMC (Late Maturing Crop) refers to a long-maturing crop, MMC (Medium Maturing Crop) refers to a medium-maturing crop, and SEMC (Super Early Maturing Crop) refers to an extremely early maturing crop. It means an early maturing species, and Imported means an imported species.

However, it should be taken into account that the phylogenetic tree is a consensus tree obtained by performing the bootstrap method, and is the result without applying a cut-off value.

Example 5-2: Principal Component Analysis (PCA)

Principal component analysis (PCA) was performed using the R package SNPRelate on 13,196 SNP loci selected through the filter process previously described in Example 4. As a result, it was confirmed that the two main components (PC1 and PC2) could explain approximately 17.5% of genetic variation.

Figures 5a and 5b show the results of principal component analysis based on two factors according to an embodiment of the present invention.

Referring to Figures 5a and 5b, it can be seen that the Italian ryegrass 96 individual samples according to an embodiment of the present invention are differentiated to some extent depending on the variety. However, in the plot written in 2D, there were many overlapping parts, so it was somewhat difficult to clearly distinguish them.

Figure 6 shows the results of principal component analysis based on three factors according to an embodiment of the present invention.

Referring to FIG. 6, it can be seen that the Italian ryegrass 96 individual samples according to an embodiment of the present invention are more clearly distinguished according to variety than in the previous 2D plot.

Example 5-3: Population structure

Population structure analysis was performed by performing random selection three times to select 5,000 SNP loci at a level usable for analysis from the 13,196 SNP loci selected through the filter process previously described in Example 4. .

Table 16 below discloses the conditions used in the population structure analysis.

SNP loci of biallelic
Bayesian model-based approach (MCMC: Markov Chain Monte Carlo)
Burn-in period: 10,000
Number of MCMC Reps: 10,000
Ancestry Model: Admixture model
Allele Frequency Model: correlated allele frequency
Number of populations (K): 1 to 10
Number of iterations: 10

In more detail, population structure analysis performs analysis with multiple K values to find an appropriate K value (population) and performs this repeatedly using the delta-K method, an ad hoc quantity ( Δdescribed by Evanno et al. 2005.), the appropriate K value is calculated. At this time, as the number of repetitions increases, more time and resources are required, so 5,000 SNP loci at a level usable for analysis were selected three times and all three results were calculated. It was confirmed that repeatable results were obtained. The number of structure repetitions was set to 10 and analyzed.

Figure 7 is a graph showing the results of the delta-K method according to an embodiment of the present invention.

Referring to FIG. 7, as a result of calculating the structure by repeating it 10 times, a peak was confirmed when K = 2. Accordingly, below you can see a graph containing the results at the corresponding peak.

Table 17 below shows the results of population structure analysis for each variety when K = 2.

Sample Group Missing (%) Pop1 Pop2 GF3_02 SEMC 3 0.935 0.065 GF_04 SEMC 2 0.918 0.082 GF2_04 SEMC 6 0.746 0.254 GF2_03 SEMC 6 0.745 0.255 GF_02 SEMC 6 0.697 0.303 GF3_01 SEMC 8 0.596 0.404 GF2_01 SEMC 13 0.549 0.451 GF_01 SEMC 12 0.494 0.506 GF3_04 SEMC 24 0.163 0.837 GF2_02 SEMC 28 0.043 0.957 GF_03 SEMC 39 0.005 0.995 GF3_03 SEMC 48 0.002 0.998 GC2_01 EMC One 0.999 0.001 GC_02 EMC 2 0.949 0.051 GC2_02 EMC 3 0.926 0.074 KS_01 EMC 4 0.864 0.136 KW_01 EMC 4 0.857 0.143 GC_01 EMC 4 0.855 0.145 GC_03 EMC 4 0.829 0.171 KS_02 EMC 5 0.791 0.209 KG_03 EMC 4 0.788 0.212 GC2_03 EMC 9 0.694 0.306 KS_03 EMC 8 0.686 0.314 KW_02 EMC 4 0.683 0.317 GC_04 EMC 12 0.39 0.61 KW_04 EMC 13 0.356 0.644 KW_03 EMC 16 0.314 0.686 GC2_04 EMC 17 0.299 0.701 KG_04 EMC 20 0.244 0.756 KG_02 EMC 36 0.146 0.854 KS_04 EMC 36 0.009 0.991 KG_01 EMC 32 0.008 0.992 604_03 MMC One 0.999 0.001 605_01 MMC 2 0.882 0.118 603_01 MMC 4 0.881 0.119 604_02 MMC 4 0.828 0.172 KM_02 MMC 9 0.624 0.376 603_04 MMC 12 0.562 0.438 603_02 MMC 5 0.561 0.439 605_04 MMC 14 0.478 0.522 602_03 MMC 15 0.474 0.526 KM_03 MMC 17 0.447 0.553 602_02 MMC 13 0.43 0.57 604_04 MMC 10 0.403 0.597 603_03 MMC 13 0.349 0.651 KM_04 MMC 17 0.331 0.669 602_01 MMC 19 0.294 0.706 605_02 MMC 16 0.219 0.781 KM_01 MMC 21 0.163 0.837 601_04 MMC 20 0.141 0.859 605_03 MMC 29 0.141 0.859 601_03 MMC 24 0.1 0.9 601_01 MMC 29 0.008 0.992 604_01 MMC 35 0.006 0.994 601_02 MMC 46 0.001 0.999 602_04 MMC 52 0.001 0.999 901_01 L.M.C. 0 One 0 KWR_04 L.M.C. 2 0.934 0.066 901_02 L.M.C. 4 0.872 0.128 901_03 L.M.C. 2 0.813 0.187 HS101_01 L.M.C. 3 0.759 0.241 HS104_01 L.M.C. 5 0.723 0.277 HS104_04 L.M.C. 10 0.497 0.503 HS104_03 L.M.C. 13 0.41 0.59 HS101_02 L.M.C. 11 0.363 0.637 KWR_01 L.M.C. 15 0.299 0.701 HS102_02 L.M.C. 21 0.29 0.71 HS101_04 L.M.C. 20 0.203 0.797 HS102_04 L.M.C. 23 0.147 0.853 901_04 L.M.C. 23 0.122 0.878 HS102_01 L.M.C. 25 0.122 0.878 KWR_03 L.M.C. 22 0.099 0.901 KWR_02 L.M.C. 35 0.002 0.998 HS101_03 L.M.C. 40 0.001 0.999 HS102_03 L.M.C. 71 0.001 0.999 HS104_02 L.M.C. 42 0.001 0.999 HC_01 Imported 5 0.741 0.259 AE_02 Imported 10 0.589 0.411 AE_03 Imported 10 0.517 0.483 HC_02 Imported 11 0.462 0.538 HC_04 Imported 13 0.442 0.558 AE_01 Imported 14 0.431 0.569 AE_04 Imported 10 0.364 0.636 BK_01 Imported 21 0.267 0.733 FLO_01 Imported 24 0.213 0.787 HC_03 Imported 27 0.179 0.821 Rio_01 Imported 32 0.104 0.896 Rio_02 Imported 40 0.004 0.996 FLO_04 Imported 43 0.002 0.998 BK_02 Imported 49 0.001 0.999 BK_03 Imported 62 0.001 0.999 BK_04 Imported 73 0.001 0.999 FLO_02 Imported 66 0.001 0.999 FLO_03 Imported 51 0.001 0.999 Rio_03 Imported 55 0.001 0.999 Rio_04 Imported 79 0.001 0.999

Figure 8 is a graph showing the results of population structure analysis at a peak with a K value of 2 according to an embodiment of the present invention.

Referring to Figure 8, it can be seen that the proportion of SNPs of each individual contributed from the ancestral population is shown in segments. It was confirmed that the Pop1 and Pop2 ratios within each species of extremely early, early, intermediate, late, and imported species appeared in a similar pattern with a gradual decrease. In addition, the maximum Pop1 ratio was confirmed to be 0.935 to 1 for domestic species, including extremely early-maturing species, early-maturing species, mid-maturing species, and late-maturing species, while it was 0.741 for imported species, which was slightly different.

Example 6: Pairwise F _S.T. analyze

Using the 13,196 SNP loci selected through the filter process previously described in Example 4, pairwise F _ST Analysis was performed. Additionally, pairwise F _ST analysis was performed between samples within each group to predict the distance between objects within the group. The F _ST value is calculated between 0 and 1, and the closer it is to 1, the greater the distance between groups can be predicted.

Table 18 below shows the results of pairwise F _ST analysis within the group.

group name Extremely early maturing species early maturing species midlife species Late species imported species Extremely early maturing species - 0.025 0.029 0.040 0.033 early maturing species 0.025 - 0.007 0.028 0.020 midlife species 0.029 0.007 - 0.015 0.011 Late species 0.040 0.028 0.015 - 0.017 imported species 0.033 0.020 0.011 0.017 -

Referring to Table 18 above, it can be seen that the extremely early maturing species, early maturing species, mid-growing species, late maturing species, and imported species are of the same genus, Lolium multiflorum , so the F _ST values are quite close. In other words, it can be seen that the distance between individuals of extremely early-maturing species, early-maturing species, intermediate-growing species, late-maturing species, and imported species is quite close. Accordingly, it was confirmed that it is very difficult to distinguish between extremely early-maturing species, early-maturing species, mid-maturing species, late-maturing species, and imported species.

Table 19 below shows the results of pairwise F _ST analysis within extremely early maturing species.

Extremely early maturing species GF GF2 GF3 GF - 0.014 0.009 GF2 0.014 - 0.016 GF3 0.009 0.016 -

Table 20 below shows the results of pairwise F _ST analysis within early maturing species.

early maturing species GC GC2 K.G. K.S. K.W. GC - 0.004 0.034 0.027 0.033 GC2 0.004 - 0.043 0.033 0.035 K.G. 0.034 0.043 - 0.031 0.046 K.S. 0.027 0.033 0.031 - 0.042 K.W. 0.033 0.035 0.046 0.042 -

Table 21 below shows the results of pairwise F _ST analysis within mesozoic species.

midlife species 601 602 603 604 605 K.M. 601 - 0.084 0.060 0.062 0.082 0.091 602 0.084 - 0.036 0.038 0.033 0.037 603 0.060 0.036 - -0.003 0.021 0.036 604 0.062 0.038 -0.003 - 0.022 0.036 605 0.082 0.033 0.021 0.022 - 0.042 K.M. 0.091 0.037 0.036 0.036 0.042 -

Table 22 below shows the results of pairwise F _ST analysis within late-ripening species.

Late species 901 HS101 HS102 HS104 KWR 901 - 0.039 0.068 0.033 0.029 HS101 0.039 - 0.026 0.020 0.004 HS102 0.068 0.026 - 0.031 0.019 HS104 0.033 0.020 0.031 - 0.020 KWR 0.029 0.004 0.019 0.020 -

Table 23 below shows the results of pairwise F _ST analysis within imported species.

imported species A.E. B.K. FLO HC Rio A.E. - 0.043 0.046 -0.011 0.052 B.K. 0.043 - 0.015 0.049 0.000 FLO 0.046 0.015 - 0.059 -0.007 HC -0.011 0.049 0.059 - 0.070 Rio 0.052 0.000 -0.007 0.070 -

Referring to Tables 19 to 23 above, the cultivars in each group of extremely early maturing species, early maturing species, mid-maturing species, late maturing species, and imported species are of the same genus Lolium multiflorum and have a common feature of having similar heading times, F _S.T. You can see that the values are quite close. In other words, it can be seen that the distances between individuals within each group of extremely early-maturing species, early-maturing species, mid-maturing species, late-maturing species, and imported species are quite close. Accordingly, it was confirmed that it was very difficult to distinguish cultivars within each group of extremely early-maturing species, early-maturing species, mid-maturing species, late-maturing species, and imported species.

Example 7: Genotyping

In order to develop a marker specific to the HS104 variety sample, common loci between repeat samples of the HS104 variety were selected among the 13,196 SNP loci selected through the filter process previously described in Example 4, and the results are shown in Table 24 below.

filter stage filter item No. of SNPs One Total number of SNPs secured 13,196 2 Common Homo-type SNP locus between HS104 repeat samples*1 1,060

As shown in Table 24 above, 1,060 common homo-type SNP loci were selected between repeat samples of the HS104 variety.

Using the previously selected common loci, identifiable barcode SNPs were selected between the HS104 variety and the remaining 92 individual samples.

Table 25 below shows a barcode SNP set consisting of 6 barcodes that can identify the HS104 variety among 96 Italian ryegrass individuals.

HS104 classification marker set Marker Set No. One 2 3 4 5 6 Number of candidate markers 2 One One One One One HS104 b b b b b b KW_01 a a b a a a KW_02 a b a a a - KW_03 h a h a a a KW_04 h - a - a a KS_01 b a b h h - KS_02 b a b b a a KS_03 h a - h a a KS_04 h - a - a b GC2_01 h h b a a h GC2_02 h h - h a h GC2_03 h a b a h h GC2_04 h a a a b - GC_01 h a h h a a GC_02 b a h a a h GC_03 b a b a a b GC_04 h - a a a h 901_01 b h b h h a 901_02 b a - h h h 901_03 b a b a a h 901_04 b h a a h a 605_01 a h - a h a 605_02 h - a a h a 605_03 a - a b b - 605_04 a h a a - a 604_01 h h a a a - 604_02 b a - h a - 604_03 h h b h h a 604_04 b a a h h b 603_01 b a - a h b 603_02 b a a a a h 603_03 b a a b h - 603_04 b a - a h - 602_01 b h a a a - 602_02 b a - a a - 602_03 b a b b h - 602_04 b a a - - - 601_01 h - a - a a 601_02 b - a - - - 601_03 b - a b a a 601_04 h a a - a - GF3_01 b h a a a a GF3_02 b h b a a a GF3_03 b - a a - - GF3_04 b a a - a a GF2_01 b - a h a - GF2_02 b - a - - a GF2_03 b a h a a a GF2_04 b a a b a a GF_01 b - a a a a GF_02 b - b a a h GF_03 b a a - - - GF_04 b a b b a h KWR_01 b a a - a - KWR_02 b - a - h a KWR_03 h a a - - h KWR_04 h b a a h b AE_01 a a - - a - AE_02 a a - - a b AE_03 a a - a a b AE_04 a a a b a b HC_01 a h h a h b HC_02 a a b - a b HC_03 a a - - a - HC_04 a a b b a b HS102_01 b - - a a - HS102_02 b a - a a a HS102_03 b - a - - a HS102_04 b - a b h b HS101_01 b a b b h h HS101_02 b a a a h h HS101_03 b a a a a b HS101_04 b a a b - h KM_01 a a a - a a KM_02 h a - - a a KM_03 a a b b - a KM_04 a a - - a a KG_01 h a a a a b KG_02 - - b h a b KG_03 h h b a h a KG_04 h - b b a h FLO_01 h - b a - b FLO_02 h a a - - - FLO_03 a a a - b h FLO_04 h - a a a a BK_01 b h - a - b BK_02 b a a - - - BK_03 - - - a - - BK_04 b - a - - - Rio_01 a - - b b h Rio_02 a - - - a - Rio_03 h - a - - a Rio_04 h a a - - -

Referring to Table 25 above, the barcode SNP set that can identify the HS104 variety consists of 6 barcodes, with 2 candidate loci in marker set 1 and 1 locus in marker sets 2 to 7. You can check it. As shown in Table 25 above, since there is no cultivar whose marker type is 'bbbbbb' like the HS104 variety, a barcode SNP set for identifying the HS104 variety according to an embodiment of the present invention was added to the DNA of Italian ryegrass. If used, you can check whether the Italian ryegrass is the HS104 variety.

The 'a' refers to a locus in the selected SNP that is the same allele as the reference genome and the depth ratio is homozygote, and the 'b' refers to the selected SNP It refers to a locus that is an allele that is different from the reference genome and the depth ratio is homozygote, and the 'h' refers to the locus in the selected SNP compared to the reference genome. It refers to a locus with a different allele and a heterozygote depth ratio.

Accordingly, the base sequence information of the barcode SNP set that can identify the HS104 variety of the present invention is shown in Table 26 below.

marker number Related information Flanking sequence 1(b) ID: LM_39437611_10876_151303 GTTAGACTTTCTGCATTACTCTTAACTAATAGTTGGAAAGAAAAAGTGGATCTGTCGTTGGTGGAAAGTTGGAGTAAGCCCATTGAATTTTCCATTTTAACATATTCAGATTGTCATGGTGTCGCCAAGTCATCCCTGAGGGCAGACGACAGTTTTTTTTGCATCCACTGTGTGTGCGTGCGGGGGCTTGAGGATGTACTTGTACTACCCTAAAGAGGGGATCAATTAATGGGGTTCATTTTTCTTAAGGCAAACCTTGAAT CACAGGGTTTGTACAGCAGATGCTGGATATCTTACACGTAG[G/A]CTTGTTGAAGTAGTTCAACATATTATTGTGCGTAGAAGAGATTGTGGTACTATCCGAGGTATTTCTGTGAATCCTCAAAATGGGATGACGGAAAAACTTTTTGTCCAAACACTAATTGGTCGTGTATTACCAGACGATATAGGGGTAGGGAGGTTATGTACTCGTTTGGTACCAGTTTTATCTTTGACACCCTGGACAGGAAGATATGAATCGTAATAACCTTGTTTCCAAGCTGTTCTACTTTTTATTCAGCTTAT ACTCCTTCCATTTCTGAAAAGAATGTCTATAAGTTTTTTCCTG Location: 442 Reference allele:
A Allele:
[G/A] 1(b) ID: LM_39437611_10876_151303 AGTTGGAAGAAAAAGTGGATCTGTCGTTGGTGGAAAGTTGGAGTAAGCCCATTGAATTTTCCATTTTAACATATTCAGATTGTCATGGTGTCGCCAAGTCATCCCTGAGGGCAGACGACAGTTTTTTTTGCATCCACTGTGTGTGCGTGCGGGCTTGAGGATGTACTTGTACTACCCTAAAGAGGGAATCAATTAATGGGGTTCATTTTTCTTAAGGCAAACCTTGAATCACAGGGTTTGTACAGCAGATGCTGGATA TCTTACACGTAGACTTGTTGAAGTAGTTCAACATATTATTGT[C/G]CGTAGAAGAGATTGTGGTACTATCCGAGGTATTTCTGTGAATCCTCAAAATGGGATGACGGAAAAACTTTTTGTCCAAACACTAATTGGTCGTGTATTACCAGACGATATAGGGGTAGGGAGGTTATGTACTCGTTTGGTACCAGTTTTATCTTTGACACCCTGGACAGGAAGATATGAATCGTAATAACCTTGTTTCCAAGCTGTTCTACTTTTTATTCAGCTTATACTCCTTCCATTTCTGAAAAGAATGTCT ATAAGTTTTTCCTGAGTCAGACAATGTAAAGTCTTGACCAAGTT Location: 472 Reference allele:G Allele:[C/G] 2(b) ID: LM_39420842_4812_72573 GGTGTTGGTTTTGCAAGGGGATCACCGGAGCTACGCCGACGATGAGCACTGCGGGCGGTGCCACTCCGGCGAGAATCGAATCAGAGGCTACAGGAGGTTCTAGGATGCGAGATCTAGCTCTACACATGCGTATGCTCACCCTGAACATGATGGCGTGGTCGGCTCCGGCGATCGTCGATGGAGACGACATCGATTGGCGATCTCCTGGTGAACTACTGCGGAAGGGAACGCCGAAATTGAGCCGATTCCTT GCTCCCCTTGATGCTCTGCTCCTCCAGGGTGCTCCTTGAGTCCAGGCGCT[T/C]CTCCTGGACCGCTCAATCGAGCTTGGGGTAGCCTCCTCCGTCGGCTTCGTCTTCGAGTCCAGCGACAGAGAGTGGAAGAAAACGAGAGATAGAAAGCATCTGAGAAGAAATGGAGTGGCGGAGGCGAACAAGAAGTGCACGCAGTGCTGGCTGGACATATTTATAGAACACGGCGAGGTCCATCTCGTCATTCCACCGGGCGGCAATGGCCGAGATGCAGCGGCGACTATGTAGTGAATATGGGGCGTGAATCT TGATGCTCCTGGATAAGCTCGGACGCGAGAGAGATAGTGGAGGGTTCTA Location: 2301 Reference allele: C Allele:[T/C] 3(b) ID: LM_39162661_3016_41723 GATCCCGGCCTATGGCCCCGATCAACATCAGGTTCTTGAAAAAGTACTTTGCGTGTTTTTTTTATTCGCTTTGCCATTTTGCCAAAAAGGGGTTTTCCGATTGCCTATAGAAGTCTTCAGTGCACCTACCATTGCATATGTAGGCTTACTGATGATCGCTTTTCTTCCCCCTTTTCATGAACTACAAAGTCCTACAAAAAAAATCTAGCTTTTTCTAATCACGCTTAGCTTGTGACTGATGATTTGACTGCCTTATGACTTGTC TAGCGGACACACGGTGTATCTCGTGGTAAGTGGAGC [A/G] GCACCTACACCCACAACACACGACACACACACACAATACATGCTCCGAGAGATGGTGGAGAAGACTACTTGGGTACTTCCGAGTAATGAGGTCCCACACCCCCGTTCTTTCGGATGTGTCTCGAGTTCACGGAGACACCATGCCTC Location: 2870 Reference allele:G Allele:[A/G] 4(b) ID: LM_39400933_8530_121559 TGTTGGGATTTTCACTCGATTTGAGGATGATCTTCATTGTTTGCACTGGTATGGTCGATCATGTGCTCGAAAATAATGAGATGGAAAACCATATGCATCATCATCCTGGTGTTCCCCCCAAGCTGAAGAAGATCTCCAATTGTGAGTATACCAACTTAGCCTTGCATCAACTTTGTATGGAGATCTTTTAGTCTGGTTCTTGGTCTATTGGTCATGTTGCCCGAAGCCTGAACTATTTCACCAGGAATCATGTCAGTTGTGTGAA CCAGTGGAGCAAAGTTAGTATGATTGTTACATCTC[T/C]GCCACATTGTTATGTTTGCACACAAAATTCTTCAATCAATGGTAGTCCAATAGACATAAGTGAGCAAGCTTATCTTATTTTCCATTAGTGCACAAGGTCATCGTTGATTGATGGCACTCATTCTGAAGATGTCGTTCCCTTGCTGTTTCGGGCTGTGTCACCGGAGGTGGACGATAGCACCTTCGTTGTTGATCCTCAGATTTAACAGCGAGGACCCTTTAAGTTTGAGCTCGGTGAAGGCTATTTAATATCTACA GCTCAATCACAAACCTCAAGTGCATATGCCAGATAGACCGTTGGG Location: 1941 Reference allele: C Allele:[T/C] 5(b) ID: LM_39416471_10931_180747 TGGACGGAGGATGACCCCGGAGACGTTCACTGCTGTGTCGAGGCTCTCGTCAAGGCAGGGAAGGAGGACGAGGCTGTTCGGTTGTTTAGAGGCTTGGAGAGGCAGAAATTGCTACCTCAGCGAAGTGTTGGTGCTGGAGGGGAAGGCATATGGTCAAGCAGCCTTGCCATGGTCCAGGTACTGTGCATGAAGGGCTACGCTCGTGAGGGCTCAGGGTGTGGTGTGGCACCACAAGAGCGAGCTCTCGA CGGAGCCCATGGGTAGCATCGTTCAGCGTAGCCTCCTCCATGGATGGTGTGT[A/G]CATGGAAATGCTAAAGAAGCTCGGAGAGTCCTTGATGACATCAAGTCCTCAGGCTTCCCACTAGGGTTGCCGTCGTTCAATGATTTTCTCAACTGTGTTTGCCATAGAAACCTCAAGTTTAACCCTTCTGCGCTTGTTCCCGAAGCTATGGATATTTTAACAGAGATGAGGTTCTGTGGTGTCACCCCTGCTGCATCCAGCTTCAACATCTTGCTTTCTTGTATGGGACGAGCCAGAAGAGTGAAAGAAGCATAC AGAATCCTTTATTTGATGAGGGAAGGAAAAGAGGGCGTGCTCACCT Location: 6901 Reference allele:G Allele:[A/G] 6(b) ID: LM_39185741_5015_67238 TCCCAGAAAAGAATTGAGCACTTGATGTACCACAGAATATGCTTGACATTTAGTTCGAGGAATCCTACAACTGTGTAGATCATGGACCATATGGGTCTCATGGTGACCAGTACTCAAAGCACTATCTTCGGAGGAGGGGACGGTGGCCGAGGTTGTGCGGTAGCATGATTCACGTTGGCGAGGGGTCGCTGGCCACCAGGACTCGTCGACGCAAACGTGAACCATGGAGAGTTGTTTGTCCGCCACGAGCTGTAAGTAG GAGCTTCCTGGCGGCTGCCTCCTCCTCGAGCACATTGGACC[T/A]AGGTGAGCCCTGGTCCTTCTCTCGTGTTACTCTAACTAGCGCTGGTCTTCGTTTAAATCTGTGATAGTGGTTTGTTCTTCCTGCAGGTTCAATCGTGGTTATGTTTTGTATTAACATGGACCCAACAAATGGGCTACTATGGCAACTTGAATCATAGCCTGAATTTCTAAGTTCGATACCATCGGAGATAGGCAAGCTGACACAACTTACTCACTTGGATGTCTTTCATTGTTTTACATTAGTTGGTTATTAGTAGTCACT TCTGAGAGGCTTCACTTTTGTATCAGTTTCTCACGGCCCCAC Location: 1154 Reference allele:A Allele:[T/A] * A: Adenien; G: Guanine; C: Cytosine; T: Thymine; N: Any nucleotide;
a: In the selected SNP, the locus is the same allele as the reference genome and the depth ratio is homozygote.
b: In the selected SNP, an allele that is different from the reference genome and a locus with a depth ratio of homozygote

Through the above-described content, those skilled in the art will be able to see that various changes and modifications can be made without departing from the technical spirit of the present invention. Therefore, the technical scope of the present invention is not limited to what is described in the detailed description of the specification and should be defined by the scope of the patent claims.

<110> REPUBLIC OF KOREA(RURAL DEVELOPMENT ADMINISTRATION) <120> SNP MARKERS FOR DISCRIMINATING ITALIAN RYEGRASS HS104 VARIETIES AND METHOD FOR DISCRIMINATING ITALIAN RYEGRASS HS104 VARIETIES USING THE SAME <130> FP-2201004-KR <160> 7 <170> KoPatentIn 3.0 <210> 1 <211> 601 <212> DNA <213> Artificial Sequence <220> <223> Scaffold Sequence of Lolium multiflorum <400> 1 gttagacttt ctgcattact cttaactaat agttggaaga aaaagtggat ctgtcgttgg 60 tggaaagttg gagtaagccc attgaatttt ccattttaac atattcagat tgtcatggtg 120 tcgccaagtc atccctgagg gcagacgaca gttttttttg catccactgt gtgtgcgtgc 180 ggggcttgag gatgtacttg tactacccta aagagggatc aattaatggg gttcattttt 240 cttaaggcaa accttgaatc acagggtttg tacagcagat gctggatatc ttacacgtag 300 rcttgttgaa gtagttcaac atattattgt gcgtagaaga gattgtggta ctatccgagg 360 tatttctgtg aatcctcaaa atgggatgac ggaaaaactt tttgtccaaa cactaattgg 420 tcgtgtatta ccagacgata taggggtagg gaggttatgt actcgtttgg taccagtttt 480 atctttgaca ccctggacag gaagatatga atcgtaataa ccttgtttcc aagctgttct 540 actttttat cagcttatac tccttccatt tctgaaaaga atgtctataa gttttttcct 600 g 601 <210> 2 <211> 601 <212> DNA <213> Artificial Sequence <220> <223> Scaffold Sequence of Lolium multiflorum <400> 2 agttggaaga aaaagtggat ctgtcgttgg tggaaagttg gagtaagccc attgaatttt 60 ccattttaac atattcagat tgtcatggtg tcgccaagtc atccctgagg gcagacgaca 120 gttttttttg catccactgt gtgtgcgtgc ggggcttgag gatgtacttg tactacccta 180 aagagggatc aattaatggg gttcattttt cttaaggcaa accttgaatc acagggtttg 240 tacagcagat gctggatatc ttacacgtag acttgttgaa gtagttcaac atattattgt 300 scgtagaaga gattgtggta ctatccgagg tatttctgtg aatcctcaaa atgggatgac 360 ggaaaaactt tttgtccaaa cactaattgg tcgtgtatta ccagacgata taggggtagg 420 gaggttatgt actcgtttgg taccagtttt atctttgaca ccctggacag gaagatatga 480 atcgtaataa ccttgtttcc aagctgttct actttttat cagcttatac tccttccatt 540 tctgaaaaga atgtctataa gttttttcct gagtcagaca atgtaaagtc ttgaccaagt 600 t601 <210> 3 <211> 601 <212> DNA <213> Artificial Sequence <220> <223> Scaffold Sequence of Lolium multiflorum <400> 3 ggtgttggtt ttgcaagggg atcaccggag ctacgccgac gatgagcact gcggcggtgc 60 cactccggcg agaatcgaat cagaggctac aggaggttct aggatgcgag atctagctct 120 acacatgcgt atgctcaccc tgaacatgat ggcgtggtcg gctccggcga tcgtcgatgg 180 agacgacatc gattggcgat ctcctggtga actactgcgg aagggaacgc cgaaattgag 240 ccgattcctt gctccccttg atgctctgct cctccagggt gctccttgag tccaggcgct 300 yctcctggac cgctcaatcg agcttggggt agcctcctcc gtcggcttcg tcttcgagtc 360 cagcgacaga gagtggaaga aaacgagaga tagaaagcat ctgagaagaa atggagtggc 420 ggaggcgaac aagaagtgca cgcagtgctg ctggacatat ttatagaaca cggcgaggtc 480 catctcgtca ttccaccggc ggcaatggcc gagatgcagc ggcgactatg tagtgaatat 540 gggcgtgaat cttgatgctc ctggataagc tcggacgcga gagagatagt ggagggttct 600 a 601 <210> 4 <211> 447 <212> DNA <213> Artificial Sequence <220> <223> Scaffold Sequence of Lolium multiflorum <400> 4 gatcccggcc tatggccccg atcaacatca ggttcttgaa aaagtacttt gcgtgttttt 60 tttatcgct ttgccatttt gccaaaaagg ggttttccga ttgcctatag aagtcttcag 120 tgcacctacc attgcatatg taggcttact gatgatcgct tttcttcccc cttttcatga 180 actacaaagt cctacaaaaa aaatctagct ttttctaatc acgcttagct tgtgactgat 240 gatttgactg ccttatgact tgtctagcgg acacacggtg tatctcgtgg taagtggagc 300 rgcacctaca cccacaacac acgacacaca cacacaatac atgctccgag agatggtgga 360 gaagactact tgggtacttc cgagtaatga ggtcccacac ccccgttctt tcggatgtgt 420 ctcgagttca cggagacacc atgcctc 447 <210> 5 <211> 601 <212> DNA <213> Artificial Sequence <220> <223> Scaffold Sequence of Lolium multiflorum <400> 5 tgttgggatt ttcactcgat ttgaggatga tcttcattgt ttgcactggt atggtcgatc 60 atgtgctcga aaataatgag atggaaaacc atatgcatca tcatcctggt gttccccccca 120 agctgaagaa gatctccaat tgtgagtata ccaacttagc cttgcatcaa ctttgtatgg 180 agatctttta gtctggttct tggtctattg gtcatgttgc ccgaagcctg aactatttca 240 ccagggaatca tgtcagttgt gtgaaccagt ggagcaaagt tagtatgatt gttacatctc 300 ygccacattg ttatgtttgc acacaaaatt cttcaatcaa tggtagtcca atagacataa 360 gtgagcaagc ttatcttatt ttccattagt gcacaaggtc atcgttgatt gatggcactc 420 attctgaaga tgtcgttccc ttgctgttcg ggctgtgtca ccggaggtgg acgatagcac 480 cttcgttgtt gatcctcaga tttaacagcg aggacccttt aagtttgagc tcggtgaagg 540 ctatttaata tctacagctc aatcacaaac ctcaagtgca tatgccagat agaccgttgg 600 g 601 <210> 6 <211> 601 <212> DNA <213> Artificial Sequence <220> <223> Scaffold Sequence of Lolium multiflorum <400> 6 tggacggagg atgaccccgg agacgttcac tgctgtggtc gaggctctcg tcaaggcagg 60 gaaggaggac gaggctgttc ggttgtttag aggcttggag aggcagaaat tgctacctca 120 gcgaagtgtt ggtgctggag gggaaggcat atggtcaagc agccttgcca tggtccaggt 180 actgtgcatg aagggctacg ctcgtgaggc tcagggtgtg gtgtggcacc acaagagcga 240 gctctcgacg gagcccatgg gtagcatcgt tcagcgtagc ctcctccatg gatggtgtgt 300 rcatggaaat gctaaagaag ctcggagagt ccttgatgac atcaagtcct caggcttccc 360 actagggttg ccgtcgttca atgattttct caactgtgtt tgccatagaa acctcaagtt 420 taacccttct gcgcttgttc ccgaagctat ggatatttta acagagatga ggttctgtgg 480 tgtcacccct gctgcatcca gcttcaacat cttgctttct tgtatgggac gagccagaag 540 agtgaaagaa gcatacagaa tcctttatt gatgagggaa ggaaaagagg cgtgctcacc 600 t601 <210> 7 <211> 601 <212> DNA <213> Artificial Sequence <220> <223> Scaffold Sequence of Lolium multiflorum <400> 7 tcccagaaaa gaattgagca cttgatgtac cacagaatat gcttgacatt tagttcgagg 60 aatcctacaa ctgtgtagat catggaccat atgggtctca tggtgaccag tactcaaagc 120 actatcttcg gaggagggga cggtggccga ggttgtgcgg tagcatgatt cacgttggcg 180 aggggtcgct ggccaccagg actcgtcgac gcaaacgtga accatggaga gttgtttgtc 240 cgccacgagc tgtaagtagg agcttcctgg cggctgcctc ctcctcgagc acattggacc 300 waggtgagcc ctggtccttc tctcgtgtta ctctaactag cgctggtctt cgtttaaatc 360 tgtgatagtg gtttgttctt cctgcaggtt caatcgtggt tatgttttgt attaacatgg 420 acccaacaaa tgggctacta tggcaacttg aatcatagcc tgaatttcta agttcgatac 480 catcggagat aggcaagctg acaacttact cacttggatg tctttcattg ttttacatta 540 gttggttatt agtagtcact tctgagaggc ttcacttttg tatcagtttc tcacggccca 600 c 601

Claims (10)

In a polynucleotide consisting of the base sequences of SEQ ID NO. 1 or SEQ ID NO. 2, and SEQ ID NO. 3 to 7, a single nucleotide polymorphism (SNP) marker at the 301st base of SEQ ID NO. among each base sequence can be detected. A composition for identification of Italian ryegrass HS104 variety, comprising an agent capable of identifying the variety. According to claim 1,
The single nucleotide polymorphism marker is,
In the polynucleotide represented by the base sequence of SEQ ID NO: 1, the 301st base is G or A,
In the polynucleotide represented by the base sequence of SEQ ID NO: 2, the 301st base is C or G,
In the polynucleotide represented by the base sequence of SEQ ID NO: 3, the 301st base is T or C,
In the polynucleotide represented by the base sequence of SEQ ID NO: 4, the 301st base is A or G,
In the polynucleotide represented by the base sequence of SEQ ID NO: 5, the 301st base is T or C,
In the polynucleotide represented by the base sequence of SEQ ID NO: 6, the 301st base is A or G,
A composition for identifying Italian ryegrass HS104, a single nucleotide polymorphism marker in which the 301st base is T or A in the polynucleotide represented by the base sequence of SEQ ID NO: 7. According to claim 1,
The agent capable of detecting the single nucleotide polymorphism marker includes a polynucleotide consisting of 10 to 400 consecutive DNA sequences containing a single nucleotide polymorphism marker site; A composition for identifying the Italian ryegrass HS104 variety, which is a polynucleotide that specifically hybridizes with a complementary polynucleotide thereof. According to clause 3,
The polynucleotide is one or more selected from the group consisting of primers, probes, and combinations thereof capable of detecting a single nucleotide polymorphism marker. A composition for identifying the Italian ryegrass HS104 variety. An Italian ryegrass HS104 variety identification kit comprising the composition according to any one of claims 1 to 4. Extracting DNA from Italian ryegrass;
Using the extracted DNA as a template and performing polymerase chain reaction (PCR) using the composition according to any one of claims 1 to 4; and
Identifying the variety of Italian ryegrass by analyzing the product produced by the polymerase chain reaction. According to clause 6,
The step of identifying the variety is,
Among the DNA of the Italian ryegrass, the base of the DNA corresponding to the position of the 301st base in the polynucleotide shown in SEQ ID NO: 1 is G, or the base corresponding to the position of the 301st base in the polynucleotide shown in SEQ ID NO: 2 The base of DNA is C,
The base of the DNA corresponding to the 301st base in the polynucleotide represented by SEQ ID NO: 3 is T,
The base of the DNA corresponding to the 301st base in the polynucleotide represented by SEQ ID NO: 4 is A,
The base of the DNA corresponding to the 301st base in the polynucleotide represented by SEQ ID NO: 5 is T,
The base of the DNA corresponding to the 301st base in the polynucleotide represented by SEQ ID NO: 6 is A,
When the base of the DNA corresponding to the position of the 301st base in the polynucleotide represented by SEQ ID NO: 7 is T, the Italian ryegrass is identified as the HS104 variety. A method for identifying the Italian ryegrass HS104 variety. According to clause 6,
The Italian ryegrass varieties include Kowinner, Kospeed, Green Call, Green Call No. 2, IR601, IR602, IR603, IR605, IR901, and Green Farm. ), Green Farm No. 2, Green Farm No. 3, Kowinearly, AE, Hwasan No. 101, Hwasan No. 102, Hwasan No. 104, Hercules (HC), Kowin Master How to identify Italian ryegrass HS104 cultivars: (Kowinmaster), Kogreen, Florida, Billiken, and Rio. In the polynucleotide consisting of the base sequences of SEQ ID NO. 1 and SEQ ID NO. 3 to 7, it includes an agent capable of detecting a single nucleotide polymorphism (SNP) marker at the 301st base of SEQ ID NO. among each base sequence. Composition for identification of Italian ryegrass HS104 variety. Italian ryegrass containing an agent capable of detecting a single nucleotide polymorphism (SNP) marker at the 301st base of SEQ ID NO:1 in a polynucleotide consisting of the base sequences of SEQ ID NOs: 2 to 7. HS104 Composition for variety identification.