KR20210058610A - A set of SNP molecular markers for discriminating radish accessions using KASP assay and uses thereof - Google Patents

Abstract

The present invention relates to a set of SNP markers for KASP assay for determining radish resources, and uses thereof. Since SNP markers for classifying 16 species of the present invention can distinguish 34 radish core group varieties, it is possible to easily and quickly distinguish various radish genetic resources for each variety, and to be usefully used for development of high-quality improved radish varieties, identification of the export and import origin of agricultural and fishery products, maintenance of radish genome, etc.

Description

{A set of SNP molecular markers for discriminating radish accessions using KASP assay and uses thereof}

The present invention relates to a set of SNP markers for KASP analysis and use thereof to determine free resources.

Radish is a vegetable crop belonging to the Cabbage family like Chinese cabbage and is one of the three major crops in Korea along with rice and red pepper. Radish is one of the most important vegetable crops in Korea for the last 7 years in cumulative seed exports, ranking second only to peppers as a single crop. Radish production amounts to about 2% of the world's vegetable crop production, which is estimated to be 7 million tons per year. The commercially valuable part of radish is the root, which provides a wide range of nutrients such as sugar, minerals, vegetable extracts and edible fiber, and other parts are also provided as ingredients for edible vegetables and oils.

Radish for cultivation is classified into 4 groups after morphological analysis of edible parts such as root leaves. 1.Big radish group: East Asian long radish ( Raphanus. sativus convar. hortensis ), 2.Small radish group: European small radish ( R. sativus convar. s ativus ) and Back radish ( R. sativus var. niger ), 3 Oil radish group: R. sativus convar. oleifer , 4. Rat-tailed radish: R. sativus convar. caudatus . Wild radish is R. raphanistrum , native to the Mediterranean coastal region. subsp. landra , raphanistrum subsp. maritimus , R. raphanistrum subsp. raphanistrum and R. sativus var. Raphanistroides are included, and wild radishes are also classified by morphological analysis.

Radish is a crop used as a root vegetable in Asia. It has various root shapes in morphology, and physiologically, it is essential to secure or maintain genetic resources in order to produce high quality due to various differences in flowering time and nutritional composition. Therefore, it is essential to develop accurate and highly versatile molecular markers to foster non-new varieties through molecular breeding. In particular, radish is an economically important crop such as rice and Chinese cabbage, so studies of various types of molecular markers have been reported. In addition, four types of genomes of radish have been reported. However, morphological or physicochemical methods have been used to identify species and varieties of organisms developed up to now, but crops including Chinese radish have a complex chromosome composition, making it difficult to determine analytical indicators, so that varieties or species can be discriminated almost subjectively. This is the situation that is being made.

With the development of molecular genetics and sequencing technology, diversity of plant species can be distinguished at the genome level, and molecular markers developed using these information are very important for the classification and evaluation of genetic resources. In general, methods for identifying plant varieties are largely divided into a method for morphological characteristics through a cultivation test, an assay method through differences in isotopes between species or varieties, and an assay method using a molecular marker based on DNA. Among them, the assay method using molecular markers provides useful information for genetic diversity and lineage relationship analysis in crop genetic and breeding studies, and the conservation and management of plant genetic resources. Compared to traditional phenotype-based identification methods, the cultivation environment And it is not affected by the growth stage of the crop, so it is objective and has the advantage of high reproducibility.

Resource classification using markers is important for efficient management of crop genetic resources and breeding using them. Single nucleotide polymorphism (SNP) is a high throughput genotyping that can analyze a large amount of resources in a short time, and has low errors and high reproducibility. SNP refers to the insertion, deletion, or substitution of one nucleotide in DNA. The KASP assay, a technique used for genotyping analysis, is a Kompetitive Allele Specific PCR genotyping system and uses the principle of competitive allele-specific PCR. Compared to other genotyping assay methods, the KASP method takes less time to receive the results and the experiment method is simple. It has an economic advantage. It is also suitable for large-scale, large-scale analysis where bi-allelic scoring of SNPs and indels of specific regions must be identified.

On the other hand, radish, which can be said to be a major food crop, has not yet been reported in a systematic taxonomic system, and there is no molecular marker that can quickly distinguish the details of genetic resources such as the place of origin and cultivar. To date, the development of the SNP marker system for large-capacity analysis that can systematically classify non-resource or lineage in Korea is urgently needed in terms of introduction or arrangement of resources.

Korean Patent Publication No. 10-2018-0098508 (published on September 4, 2018)

An object of the present invention is a SNP marker set composition for discriminating radish varieties, a composition for discriminating radish varieties containing a probe specifically binding to the SNP marker or a primer for amplifying the polynucleotide, and discriminating radish varieties containing the composition It is to provide a kit for use and a method of identifying radish varieties using the same.

In order to achieve the above object, the present invention is a polynucleotide consisting of 10-99 consecutive DNA sequences including a single nucleotide polymorphism (SNP) marker of the 51st nucleotide of the nucleotide sequence represented by SEQ ID NO: 1 to SEQ ID NO: 16 Or it provides a SNP marker set composition for discriminating non-variety containing its complementary polynucleotide.

In addition, the present invention is a polynucleotide consisting of 10-99 consecutive DNA sequences including a single nucleotide polymorphism (SNP) marker of the 51st nucleotide of the nucleotide sequence represented by SEQ ID NO: 1 to SEQ ID NO: 16, or a complementary poly thereof. It provides a composition for discriminating radish varieties containing a probe that specifically binds to a nucleotide or a primer for amplifying the polynucleotide.

In addition, the present invention provides a kit for discriminating radish varieties containing the composition.

In addition, the present invention (1) the step of separating the DNA from radish; (2) identifying the genotype of the 51st nucleotide of the nucleotide sequence represented by SEQ ID NO: 1 to SEQ ID NO: 16 from the separated DNA; And (3) determining the radish variety based on the identified genotype.

The present invention relates to a set of SNP markers for KASP analysis for determining non-resource and use thereof, and the SNP markers for discriminating 16 species according to the present invention are capable of distinguishing 34 non-core group varieties. In particular, when the molecular marker set of the present invention is used, a variety of radish resources such as wild radish, rat-tailed radish, black radish, East Asian long radish, European small radish, oil seed, etc. Star classification is possible. Therefore, if the present invention is used, it is possible to easily and quickly classify various non-genetic sources for each variety, and thus, it may be usefully used for the development of high-quality radish improved varieties, determination of the origin of imports and exports of agricultural and marine products, and maintenance of the non-genome.

1 shows the results for 80 SNPs applied to 34 non-core group resources.
2 shows the analysis results of 34 radish sources of 16 selected SNP molecular markers. A: both alleles have the same sequence as the non-standard genome WK10039 (no SNP occurrence), B: both alleles have SNPs, H: one of the alleles has SNPs, -: have not been analyzed.
3 shows the results of phylogenetic tree analysis of a set of 16 SNP markers selected through the present invention.

Accordingly, in the present invention, a set of 16 KASP-based SNP markers was proposed to distinguish various non-core group resources. As for SNP markers, 16 of the existing 702 SNP markers, which is the minimum number, were selected. Using this marker set, 34 resources of the radish core group (9 wild radish, 1 rat-tailed radish, 1 black radish, 9 East Asian long radish, European small) When applied to 12 types of radish and 2 types of oil radish), it was confirmed that all of them were well separated by system, and the present invention was completed.

The present invention provides a polynucleotide consisting of 10-99 consecutive DNA sequences including a single nucleotide polymorphism (SNP) marker of the 51st nucleotide of the nucleotide sequence represented by SEQ ID NO: 1 to SEQ ID NO: 16, or a complementary polynucleotide thereof. It provides a SNP marker set composition for discriminating non-species containing.

Preferably, the radish varieties are wild radish, rat-tailed radish, black radish, East Asian long radish, European small radish or oil. It may be radish (oil radish), but is not limited thereto.

Preferably, the SNP marker set composition may be for use in a competitive allele specific PCR (Kompetitive Allele Specific PCR; KASP) analysis.

In the polynucleotide including the 51st nucleotide of SEQ ID NO: 1, the polymorphic SNP mutation can be described by denoting RsHH025-1_13109, and the allele is C/T. The 51st nucleotide of SEQ ID NO: 1 was prepared according to the multi-base material method, but since it may be C or T, it is described as "y".

In the polynucleotide comprising the 51st nucleotide of SEQ ID NO: 2, a polymorphic SNP mutation can be described by denoting RS2CL3703s_516, and the allele is G/C. The 51st nucleotide of SEQ ID NO: 2 was prepared according to the multi-base material method, but since it may be G or C, it is described as "s".

In the polynucleotide including the 51st nucleotide of SEQ ID NO: 3, the polymorphic SNP mutation can be described by denoting COS0566_179, and the allele is T/C. The 51st nucleotide of SEQ ID NO: 3 was prepared according to the multi-base material method, and since it may be T or C, it is described as "y".

In the polynucleotide comprising the 51st nucleotide of SEQ ID NO: 4, the polymorphic SNP mutation can be described by denoting COS0151_283, and the allele is A/T. The 51st nucleotide of SEQ ID NO: 4 was prepared according to the multi-base material method, and it may be A or T, so it is described as "w".

In the polynucleotide including the 51st nucleotide of SEQ ID NO: 5, the polymorphic SNP mutation can be described by denoting RsSNP07531_14513, and the allele is G/C. The 51st nucleotide of SEQ ID NO: 5 was prepared according to the multi-base material method, and since it may be G or C, it is described as "s".

In the polynucleotide comprising the 51st nucleotide of SEQ ID NO: 6, the polymorphic SNP mutation can be described by denoting RS2CL1544s_2285, and the allele is T/G. The 51st nucleotide of SEQ ID NO: 6 was prepared according to the multi-base material method, and since it may be T or G, it is described as "k".

The polymorphic SNP mutation in the polynucleotide including the 51st nucleotide of SEQ ID NO: 7 can be described by denoting RsSNP06056_1252, and the allele is T/C. The 51st nucleotide of SEQ ID NO: 7 was prepared according to the multi-base material method, and since it may be T or C, it is described as "y".

In the polynucleotide including the 51st nucleotide of SEQ ID NO: 8, the polymorphic SNP mutation can be described by denoting COS0530_10656, and the allele is G/A. The 51st nucleotide of SEQ ID NO: 8 was prepared according to the multi-base material method, and since it may be G or A, it is described as "r".

The polymorphic SNP mutation in the polynucleotide including the 51st nucleotide of SEQ ID NO: 9 can be described by denoting RSS2884_12650, and the allele is G/A. The 51st nucleotide of SEQ ID NO: 9 was prepared according to the multi-base material method, and since it may be G or A, it is described as "r".

The polymorphic SNP mutation in the polynucleotide including the 51st nucleotide of SEQ ID NO: 10 can be described by denoting RS2CL683s_6854, and the allele is T/G. The 51st nucleotide of SEQ ID NO: 10 was prepared according to the multi-base material method, but since it may be T or G, it is described as "k".

The polymorphic SNP mutation in the polynucleotide including the 51st nucleotide of SEQ ID NO: 11 can be described by denoting RS2CL2646_8211, and the allele is G/T. The 51st nucleotide of SEQ ID NO: 11 was prepared according to the multi-base material method, and since it may be G or T, it is described as "k".

The polymorphic SNP mutation in the polynucleotide including the 51st nucleotide of SEQ ID NO: 12 can be described by denoting RSS1854_5572, and the allele is G/A. The 51st nucleotide of SEQ ID NO: 12 was prepared according to the multi-base material method, and since it may be G or A, it is described as "r".

The polymorphic SNP mutation in the polynucleotide including the 51st nucleotide of SEQ ID NO: 13 can be described by denoting RsSNP08808_10107, and the allele is G/A. The 51st nucleotide of SEQ ID NO: 13 was prepared according to the multi-base material method, and since it may be G or A, it is described as "r".

The polymorphic SNP mutation in the polynucleotide including the 51st nucleotide of SEQ ID NO: 14 can be described by denoting RSS3548_8083, and the allele is A/C. The 51st nucleotide of SEQ ID NO: 14 was prepared according to the multi-base material method, and since it may be A or C, it is described as "m".

In the polynucleotide including the 51st nucleotide of SEQ ID NO: 15, the polymorphic SNP mutation can be described by denoting RsSNP14867_2430, and the allele is A/G. The 51st nucleotide of SEQ ID NO: 15 was prepared according to the multi-base material method, and since it may be A or G, it is described as "r".

In the polynucleotide comprising the 51st nucleotide of SEQ ID NO: 16, the polymorphic SNP mutation can be described as COS0279_2650, and the allele is G/T. The 51st nucleotide of SEQ ID NO: 16 was prepared according to the multi-base material method, and since it may be G or T, it is described as "k".

In the present invention, “marker” refers to a nucleotide sequence used as a reference point when identifying a genetically unspecified related locus. The term also applies to nucleic acid sequences that are complementary to a marker sequence, such as nucleic acids used as a set of primers capable of amplifying the marker sequence. The location of a molecular marker on the genetic map is called a genetic locus.

In the present invention, the "molecular marker" can be usefully used because it is not affected by the cultivation environment or growth timing of the crop and can quickly identify the variety. Molecular markers are a method of indicating polymorphism between individuals by targeting differences in the nucleotide sequence of DNA, which is the essence of genetic phenomena. Evaluation of genetic resources by external morphological characteristics is affected by crop growth status and various environmental factors, but evaluation based on DNA markers is not affected by the environment, so it is highly useful for replacing and supplementing phenotypic characteristics. . The DNA marker is a useful technique for discriminating the same cultivar with the same cultivar name (different cultivar name) or several different cultivar names (different cultivar names) and similar cultivars despite different cultivars. Therefore, breed identification by DNA markers can prevent cultivar incorporation in the seedling distribution process because it is possible to easily and accurately classify cultivars using extracted DNA without examining morphological traits.

In the present invention, "polymorphism" refers to a case where two or more alleles are present in a single locus, and "polymorphic site" refers to a locus in which the allele is present. Among the polymorphic sites, the thing that only a single base differs from person to person is called "single nucleotide polymorphism," or SNP (single nucleotide polymorphism).

In addition, the present invention is a polynucleotide consisting of 10-99 consecutive DNA sequences including a single nucleotide polymorphism (SNP) marker of the 51st nucleotide of the nucleotide sequence represented by SEQ ID NO: 1 to SEQ ID NO: 16, or a complementary poly thereof. It provides a composition for discriminating radish varieties containing a probe that specifically binds to a nucleotide or a primer for amplifying the polynucleotide.

Preferably, the primer is a primer set represented by SEQ ID NO: 17, SEQ ID NO: 33, and SEQ ID NO: 49, a primer set represented by SEQ ID NO: 18, SEQ ID NO: 34, and SEQ ID NO: 50, SEQ ID NO: 19, SEQ ID NO: 35, and sequence A primer set represented by SEQ ID NO: 51, SEQ ID NO: 20, SEQ ID NO: 36, and SEQ ID NO: a primer set represented by SEQ ID NO: 52, a primer set represented by SEQ ID NO: 21, SEQ ID NO: 37 and SEQ ID NO: 53, SEQ ID NO: 22, SEQ ID NO: 38 and A primer set represented by SEQ ID NO: 54, SEQ ID NO: 23, SEQ ID NO: 39, and a primer set represented by SEQ ID NO: 55, SEQ ID NO: 24, a primer set represented by SEQ ID NO: 40 and SEQ ID NO: 56, SEQ ID NO: 25, SEQ ID NO: 41 And a primer set represented by SEQ ID NO: 57, a primer set represented by SEQ ID NO: 26, SEQ ID NO: 42, and SEQ ID NO: 58, a primer set represented by SEQ ID NO: 27, SEQ ID NO: 43 and SEQ ID NO: 59, SEQ ID NO: 28, SEQ ID NO: A primer set represented by 44 and SEQ ID NO: 60, SEQ ID NO: 29, a primer set represented by SEQ ID NO: 45 and SEQ ID NO: 61, a primer set represented by SEQ ID NO: 30, SEQ ID NO: 46 and SEQ ID NO: 62, SEQ ID NO: 31, sequence A primer set represented by SEQ ID NO: 47 and SEQ ID NO: 63; It may be a primer set represented by SEQ ID NO: 32, SEQ ID NO: 48, and SEQ ID NO: 64, but is not limited thereto.

Preferably, the radish varieties are wild radish, rat-tailed radish, black radish, East Asian long radish, European small radish or oil. It may be radish (oil radish), but is not limited thereto.

Preferably, the composition may be for use in Kompetitive Allele Specific PCR (KASP) analysis.

In the present invention, "probe" refers to a nucleic acid fragment such as RNA or DNA corresponding to a short number of bases to hundreds of bases capable of specifically binding to an mRNA, and is labeled so that the presence or absence of a specific mRNA, The amount of expression can be confirmed. The probe may be manufactured in the form of an oligonucleotide probe, a single strand DNA (DNA) probe, a double strand DNA (DNA) probe, an RNA probe, or the like. Selection of an appropriate probe and conditions for hybridization can be appropriately selected according to techniques known in the art.

In the present invention, "primer" refers to a single-stranded oligonucleotide sequence that is complementary to a nucleic acid strand to be amplified, and may serve as a starting point for the synthesis of a primer extension product. The length and sequence of the primers should allow the synthesis of the extension product to begin. The specific length and sequence of the primers will depend on the conditions of use of the primer, such as temperature and ionic strength, as well as the complexity of the DNA or RNA target required. In the present invention, the oligonucleotide used as a primer may also include a nucleotide analogue, such as phosphorothioate, alkylphosphorothioate or peptide nucleic acid, or It may contain an intercalating agent.

In addition, the present invention provides a kit for discriminating radish varieties comprising the composition.

The kit is preferably a PCR kit or a DNA chip kit, but is not limited thereto.

In addition to the primer set of the present invention, the kit of the present invention may include conventional components included in the PCR kit. Typical components included in the kit may be a reaction buffer solution, a polymerase, dNTP (dATP, dCTP, dGTP and dTTP), and a cofactor such as Mg2+. Various DNA polymerases can be used in the amplification step of the present invention, and include Klenow fragment of E. coli DNA polymerase I, thermostable DNA polymerase, and bacteriophage T7 DNA polymerase. Preferably, the polymerase is a thermostable DNA polymerase obtainable from a variety of bacterial species. Most of these polymerases can be isolated from the bacteria themselves or can be purchased commercially.

In addition, the present invention (1) the step of separating the DNA from radish; (2) identifying the genotype of the 51st nucleotide of the nucleotide sequence represented by SEQ ID NO: 1 to SEQ ID NO: 16 from the separated DNA; And (3) determining the radish variety based on the identified genotype.

Preferably, the step of identifying the genotype of the 51st nucleotide of the nucleotide sequence represented by SEQ ID NO: 1 to SEQ ID NO: 16 in step (2) includes a primer set represented by SEQ ID NO: 17, SEQ ID NO: 33, and SEQ ID NO: 49, A primer set represented by SEQ ID NO: 18, SEQ ID NO: 34 and SEQ ID NO: 50, a primer set represented by SEQ ID NO: 19, SEQ ID NO: 35, and SEQ ID NO: 51, a primer set represented by SEQ ID NO: 20, SEQ ID NO: 36, and SEQ ID NO: 52 , A primer set represented by SEQ ID NO: 21, SEQ ID NO: 37, and SEQ ID NO: 53, a primer set represented by SEQ ID NO: 22, SEQ ID NO: 38, and SEQ ID NO: 54, a primer set represented by SEQ ID NO: 23, SEQ ID NO: 39, and SEQ ID NO: 55 Set, a primer set represented by SEQ ID NO: 24, SEQ ID NO: 40 and SEQ ID NO: 56, a primer set represented by SEQ ID NO: 25, SEQ ID NO: 41 and SEQ ID NO: 57, SEQ ID NO: 26, SEQ ID NO: 42 and SEQ ID NO: 58 Primer set, primer set represented by SEQ ID NO: 27, SEQ ID NO: 43 and SEQ ID NO: 59, primer set represented by SEQ ID NO: 28, SEQ ID NO: 44 and SEQ ID NO: 60, represented by SEQ ID NO: 29, SEQ ID NO: 45 and SEQ ID NO: 61 A primer set represented by SEQ ID NO: 30, SEQ ID NO: 46 and SEQ ID NO: 62, a primer set represented by SEQ ID NO: 31, SEQ ID NO: 47, and SEQ ID NO: 63; It may be a step of performing PCR amplification using a primer set represented by SEQ ID NO: 32, SEQ ID NO: 48, and SEQ ID NO: 64, and analyzing the PCR amplification product, but is not limited thereto.

Preferably, the radish varieties are wild radish, rat-tailed radish, black radish, East Asian long radish, European small radish or oil. It may be radish (oil radish), but is not limited thereto.

Preferably, the composition may be for use in Kompetitive Allele Specific PCR (KASP) analysis.

As a method of separating DNA from radish, a method known in the art may be used. In addition, PCR is a method of amplifying a target nucleic acid from a primer set that specifically binds to a target nucleic acid using a polymerase. Such PCR methods are well known in the art, and commercially available kits may be used.

Hereinafter, the present invention will be described in detail according to examples that do not limit the present invention. It goes without saying that the following examples of the present invention are intended to embodi the present invention and do not limit or limit the scope of the present invention. Therefore, what can be easily inferred by experts in the technical field to which the present invention pertains from the detailed description and examples of the present invention is interpreted as belonging to the scope of the present invention.

< Experimental example >

The following experimental examples are intended to provide experimental examples commonly applied to each of the examples according to the present invention.

1. Plant material genome and DNA extraction

Among the 125 resources of various radish core groups, _{34 resources that have progressed F 3} or higher and the line of radish resources are clearly stated (9 species of wild radish, 1 species of rat-tailed radish, and black radish) (1 black radish), 9 East Asian long radish, 12 European small radish, 2 oil radish) Radish resources were used (Table 1). Genomic DNA (gDNA) was extracted from non-resourced young leaf tissue. Each young leaf was crushed using a metal ball, and then gDNA was extracted using the Plant gDNA extraction kit (Biomedic) according to the manufacturer's manual.

Type Sample ID Root shape Root color Origin Accession Scientific name Black radish CUR096 Circular Black Unknown Kohlschwarzer plattrunder R. sativus var. niger East Asian long radish CUR066 Rectangular Red Afghanistan Unknown R. sativus convar . sativus CUR068 Narrow obtriangular Green and white Portugal Giant Japanese radish R. sativus convar . sativus CUR069 Elliptic Black Germany Dresdner grauer R. sativus convar . sativus CUR071 Elliptic Red NorthKorea Bang-uimu R. sativus convar . sativus CUR072 Narrow obtriangular Red and white Libyay Unknown R. sativus convar . sativus CUR074 Rectangular Green and white Czechoslovakia Blanc demi long de strasbourg R. sativus convar . sativus CUR075 Elliptic Purple Greece Unknown R. sativus convar . sativus CUR076 Rectangular White Greece Unknown R. sativus convar . sativus CUR078 Obovate Black North Korea Unknown R. sativus convar . sativus European small radish CUR079 Elliptic Red Switzerland Triumph R. sativus convar . sativus CUR081 Circular Red Czechoslovakia Maslova obri R. sativus convar . sativus CUR082 Rectangular Red Bulgaria Sofijskaja prevazhodna R. sativus convar . sativus CUR083 Elliptic Red Armenia Unknown R. sativus convar . sativus CUR084 Elliptic Red Hungary Szentesi hajtato R. sativus convar . sativus CUR085 Elliptic Red Mongolia Unknown R. sativus convar . sativus CUR086 Rectangular Red Kazakhstan Unknown R. sativus convar . sativus CUR087 Narrow rectangular Red Tajikistan Unknown R. sativus convar . sativus CUR088 Circular Red Tajikistan Calcham-ca (tadz.) R. sativus convar . sativus CUR089 Circular Red Russia Dunganskyi R. sativus convar . sativus CUR091 Circular Red Spain Unknown R. sativus convar . sativus CUR094 Obovate Red Turkey Radieschen R. sativus convar . sativus Oil radish CUR006 Narrow rectangular Red and white Germany Mator R. sativus convar . Oleifer CUR007 Narrow rectangular Red and white UK Slobolt R. sativus convar . Oleifer Rat-tailed radish CUR003 Rectangular White India Magri R. sativus convar . Caudatus Wild radish CUR099 Not expanded White Greece Unknown R. raphanistrum subsp. landra CUR103 Not expanded White Poland Unknown R. raphanistrum subsp. raphanistrum CUR105 Not expanded White Spain Unknown R. raphanistrum subsp. raphanistrum CUR109 Not expanded White Spain Unknown R. raphanistrum subsp. raphanistrum CUR111 Not expanded White Finland Unknown R. raphanistrum subsp. raphanistrum CUR117 Not expanded White Spain Unknown R. raphanistrum subsp. raphanistrum CUR121 Narrow obtriangular White South Korea Bukjeju-KHC-1999-80 R. sativus var. raphanistroides CUR122 Narrow obtriangular White South Korea Busanhaeundae-2003-26 R. sativus var. raphanistroides CUR124 Narrow obtriangular White South Korea Jejunamjeju-2003-208 R. sativus var. raphanistroides

2. SNP molecular label analysis

SNP molecular label analysis was performed using the KASP assay method using gDNA from 34 resources of the no core group. Primers required for KASP were synthesized from LGC, and genotyping was performed using the KASP master mix. PCR conditions were performed as shown in Table 2 below, and then the genotype of each sample was determined based on the FAM and HEX fluorescence values.

94℃ for 15 minutes Hot-start activation 94℃ for 20 seconds 10 cycles 61-55℃ for 60 seconds (dropping 0.6℃ per cycle) 94℃ for 20 seconds 26 cycles 55℃ for 60 seconds

Various non-core groups based on polymorphism, ease of analysis, and location within chromosomes in 31 SNP molecular markers discovered in the non-F1 variety previously possessed in this laboratory and 702 SNP molecular markers for Fluidigm developed for analysis of genetic resources 54 SNPs were selected to differentiate resources. KASP genotyping primers were prepared for these 85 SNPs, and applied to 34 resources of the <Non Core Group> to confirm whether resource classification was possible.

3. SNP minimum molecular marker selection and genetic analysis

In order to select the smallest molecular label that can distinguish various non-resource, genotyping data, which is the result of KASP assay, was selected by analyzing the minimum molecular label that can distinguish non-core groups using the PowerCore 1.0 program. The UPGMA phylogenetic tree was created using 2.1, and the genetic distance was analyzed.

<Example>

1. SNP molecular label selection and analysis

From the 702 SNP markers previously held in this laboratory, 85 SNP molecular markers were selected in consideration of polymorphism, ease of analysis, and chromosomal location, and genetic polymorphism analysis was performed by applying to various non-core group resources. As a result, there were 5 molecular markers that did not have polymorphism or were difficult to analyze, and 80 of them excluding them clearly showed analysis results (FIG. 1). Based on the results of genetic polymorphism analysis for 34 resources, 16 marker sets that can be classified as resource-free were selected. The sequence of the selected 16 SNPs was confirmed and summarized in Table 3 (the 51st base is mutated in each of the sequences of SEQ ID NOs: 1 to 16). The information of the primer set for this is shown in Tables 4 to 6 (ASI primer: forward primer for the reference sequence before mutation, AS2 primer: forward primer for SNP after mutation, and common primer: reverse primer for each SNP). I got it.

For the selected 16 SNP molecular markers, if there is a base mutation (SNP) in both chromosome pairs, it is referred to as B, if there is a base mutation (SNP) in only one chromosome pair, it is referred to as H, and both chromosomes. In the absence of a base mutation (SNP), the 34 non-core group resources could be classified by denoting A (FIG. 2).

Sequence number chromosome location SNP name SNP sequence One R1 319352 RsHH025-1_13109 CTCTACTCGGGCAACTTACTTGCTCCTTACAACCAGTACCGAGCCCTCTA [C/T] CTTGCTGCCGACAAATACGACATTCCTTATCTCCAAGACGTTTGCCGCGA
(SEQ ID NO: 1) 2 R2 677185 RS2CL3703s_516 CAGAAATTTAGGCTTGGAAAGCAGCCGCACAAGGAGTACGGAGATCACTC [G/C] ACAAAGGAAGGTTCAAGAGGTGACCGCTCTTCTTTTCTAGATTTATTAAG
(SEQ ID NO: 2) 3 R2 10393209 COS0566_179 CTCCACTAGATGAGGTCACATTCCGTAGGTTACAGTCTCTCCAGAAAAAG [T/C] TAGTGGATGCTGTTCCTCATGTTGCCGGTCTGAACCCACGCTCTTTCCGT
(SEQ ID NO: 3) 4 R2 20441435 COS0151_283 AACTCTTCTCTAAGCCGTCTTCCCAAGTCAGGCATGTGGCCTCCTCCGTA [A/T] AGAATCGCAACTCTCTTGTGTCCTTGCTCTATTGCTCTTTTCAAAGCTTC
(SEQ ID NO: 4) 5 R3 2888842 RsSNP07531_14513 CGTAGAGGTCATGGCCTCTTCAACTAGTACAGTTCTTGATCAATAGTCTT [G/C] TGTATTTACATTTACCTTAGATATGTATTGGCGTCGTCTCATATTCTAGA
(SEQ ID NO: 5) 6 R3 19500287 RS2CL1544s_2285 TAAGTAAGAACATTGAGATATGTTGAAGTCGTACCGAAGATTTTGTAGTC [T/G] CCGTCGACAACAGCAGGGACGGTACCGAAAGGCTGCATGGGAAGAAACAA
(SEQ ID NO: 6) 7 R4 42354514 RsSNP06056_1252 AGCTTATTAGTTATCATCATTTGGTTATTAATTATTGAGCATACAATAGA [T/C] GGTTAACTTTATATATGGAAAATGCATGGAACATGACATAGCTATGGAGC
(SEQ ID NO: 7) 8 R6 14598282 COS0530_10656 TGAAGGTTACGCCAAGAGCTAAACGACATAACTGTGGGAAGAAGATCGAG [G/A] GCAAGTCAAAAGGGTTTAGACTAAACAGACCAAGGAAGCTGGTTCTTAAG
(SEQ ID NO: 8) 9 R6 41317536 RSS2884_12650 ACGAGTTTCAGACAGAGATTTGCTGATTAGGATCCATTGTGAAAAAAACA [G/A] AGGTTGTTTAGTTAAGATCTTGAGTTCACTGGAGAAGTTTCGTCTTGAAG
(SEQ ID NO: 9) 10 R7 690390 RS2CL683s_6854 AGAGGGATGACAGGGTACTTCGTGACCTTTCCATCTTCAGCAACAACCAC [T/G] GGGTCTGCCCCCTGTGTAATCACGGTGGTCCTCTTGTATGTTCCTGTGGC
(SEQ ID NO: 10) 11 R7 16677445 RS2CL2646_8211 GTGGACATTGCCTTTATAGGATTCTACAGTTGGTTCCGTACGTACGAGGT [G/T] ATTGCAAATTTCAGCATTGTGGCAGAGTATCCTAAGTTAATTGCTTGGGC
(SEQ ID NO: 11) 12 R7 18383868 RSS1854_5572 CACACAGGTGATCGGATTGCCACTAAGATCTTCGAGTCTTTGGAGGAATG [G/A] GGTTTAGAGAAAAAGATTTTCTCAATTATTCTAGACAATGCCACAAAAAA
(SEQ ID NO: 12) 13 R8 575156 RsSNP08808_10107 ATTTGAAACTAGTGGAAATATAAACCAAACTATAGAACAAAAATATTCAC [G/A] AAAATTGTAAAGATCAAATTATGAGTGGAATGAGAGTCAGTCCATCGAAT
(SEQ ID NO: 13) 14 R8 4214931 RSS3548_8083 AAAACTGTATGATGTTCACTACTTGATGCAAAATATCTACACCCGAATCT [A/C] AAATGTACTTACCGCAATGTTCATTAAATTTTGGGTGTCATTTCATATCG
(SEQ ID NO: 14) 15 R8 16571251 RsSNP14867_2430 TGTGCAGGACAGAGTATCAGCCCCCGAGATCTTGCTATACTCTGCCAAAA [A/G] GTTATAGTAAGGTCATTTCCTGAATTTGTGCAATGTTAATTTGTTTTTTC
(SEQ ID NO: 15) 16 R9 28041727 COS0279_2650 TGGGGAGATGATTGGGGCGATGGAGATGAAGAACAGCCGAACACTCCGGT [G/T] TTACCACTCACACCGAGTGTTTCTTCTAGAGGACTTGCTCCTAGACGACT
(SEQ ID NO: 16)

SNP marker Forward (FAM-labelled) primer (5' to 3') RsHH025-1_13109 GTCGTATTTGTCGGCAGCAAGG (SEQ ID NO: 17) RS2CL3703s_516 CAAGGAGTACGGAGATCACTCG (SEQ ID NO: 18) COS0566_179 CAACATGAGGAACAGCATCCACTAA (SEQ ID NO: 19) COS0151_283 AGGACACAAGAGAGTTGCGATTCTT (SEQ ID NO: 20) RsSNP07531_14513 AGTACAGTTCTTGATCAATAGTCTTG (SEQ ID NO: 21) RS2CL1544s_2285 AAGTCGTACCGAAGATTTTGTAGTCT (SEQ ID NO: 22) RsSNP06056_1252 ATTTGGTTATTAATTATTGAGCATACAATAGAT (SEQ ID NO: 23) COS0530_10656 ATAACTGTGGGAAGAAGATCGAGG (SEQ ID NO: 24) RSS2884_12650 GAACTCAAGATCTTAACTAAACAACCTC (SEQ ID NO: 25) RS2CL683s_6854 TTCCATCTTCAGCAACAACCACT (SEQ ID NO: 26) RS2CL2646_8211 TGGTTCCGTACGTACGAGGTG (SEQ ID NO: 27) RSS1854_5572 GAATAATTGAGAAAATCTTTTTCTCTAAACCC (SEQ ID NO: 28) RsSNP08808_10107 ATAAACCAAACTATAGAACAAAAATATTCACG (SEQ ID NO: 29) RSS3548_8083 AAAATTTAATGAACATTGCGGTAAGTACATTTT (SEQ ID NO: 30) RsSNP14867_2430 ACAAATTCAGGAAATGACCTTACTATAACT (SEQ ID NO: 31) COS0279_2650 GAACAGCCGAACACTCCGGTG (SEQ ID NO: 32)

SNP marker Forward (HEX-labelled) primer (5' to 3') RsHH025-1_13109 AATGTCGTATTTGTCGGCAGCAAGA (SEQ ID NO: 33) RS2CL3703s_516 CAAGGAGTACGGAGATCACTCC (SEQ ID NO: 34) COS0566_179 AACATGAGGAACAGCATCCACTAG (SEQ ID NO: 35) COS0151_283 AGGACACAAGAGAGTTGCGATTCTA (SEQ ID NO: 36) RsSNP07531_14513 CTAGTACAGTTCTTGATCAATAGTCTTC (SEQ ID NO: 37) RS2CL1544s_2285 AGTCGTACCGAAGATTTTGTAGTCG (SEQ ID NO: 38) RsSNP06056_1252 TGGTTATTAATTATTGAGCATACAATAGAC (SEQ ID NO: 39) COS0530_10656 CATAACTGTGGGAAGAAGATCGAGA (SEQ ID NO: 40) RSS2884_12650 GAACTCAAGATCTTAACTAAACAACCTT (SEQ ID NO: 41) RS2CL683s_6854 TCCATCTTCAGCAACAACCACG (SEQ ID NO: 42) RS2CL2646_8211 GTTGGTTCCGTACGTACGAGGTT (SEQ ID NO: 43) RSS1854_5572 AGAATAATTGAGAAAATCTTTTTCTCTAAACCT (SEQ ID NO: 44) RsSNP08808_10107 ATAAACCAAACTATAGAACAAAAATATTCACA (SEQ ID NO: 45) RSS3548_8083 TAATGAACATTGCGGTAAGTACATTTG (SEQ ID NO: 46) RsSNP14867_2430 CAAATTCAGGAAATGACCTTACTATAACC (SEQ ID NO: 47) COS0279_2650 AGAACAGCCGAACACTCCGGTT (SEQ ID NO: 48)

SNP marker Reverse primer (5' to 3') RsHH025-1_13109 TTACAACCAGTACCGAGCCCTCTA (SEQ ID NO: 49) RS2CL3703s_516 CGGTCACCTCTTGAACCTTCCTTT (SEQ ID NO: 50) COS0566_179 CCGTAGGTTACAGTCTCTCCAGAAA (SEQ ID NO: 51) COS0151_283 AGGCATGTGGCCTCCTCCGTA (SEQ ID NO: 52) RsSNP07531_14513 GACGACGCCAATACATATCTAAGGTAAAT (SEQ ID NO: 53) RS2CL1544s_2285 CTTTCGGTACCGTCCCTGCTGTT (SEQ ID NO: 54) RsSNP06056_1252 GCTATGTCATGTTCCATGCATTTTCCATA (SEQ ID NO: 55) COS0530_10656 GGTCTGTTTAGTCTAAACCCTTTTGACTT (SEQ ID NO: 56) RSS2884_12650 GAGATTTGCTGATTAGGATCCATTGTGAA (SEQ ID NO: 57) RS2CL683s_6854 GAACATACAAGAGGACCACCGTGAT (SEQ ID NO: 58) RS2CL2646_8211 TACTCTGCCACAATGCTGAAATTTGCAAT (SEQ ID NO: 59) RSS1854_5572 ACTAAGATCTTCGAGTCTTTGGAGGAAT (SEQ ID NO: 60) RsSNP08808_10107 TGGACTGACTCTCATTCCACTCATAATTT (SEQ ID NO: 61) RSS3548_8083 CTACTTGATGCAAAATATCTACACCCGAA (SEQ ID NO: 62) RsSNP14867_2430 CCGAGATCTTGCTATACTCTGCCAA (SEQ ID NO: 63) COS0279_2650 GAAGAAACACTCGGTGTGAGTGGTA (SEQ ID NO: 64)

2. No resources To distinguish KASP Development of SNP molecular label set based on

The present invention relates to a set of KASP-based SNP molecular markers for distinguishing various non-resource, based on the R. raphanistrum sequence (Origin: USA, Accession: Moghe et al. (2014)) reported in 2013, non-core Among the collective resources, black radish 1 (CUR096), East Asian long radish 9 (CUR066, CUR068, CUR069, CUR071, CUR072, CUR074, CUR075, CUR076, CUR078), 12 European small radish (CUR079, CUR081, CUR082) , CUR083, CUR084, CUR085, CUR086, CUR087, CUR088, CUR089, CUR091, CUR094), rat-tailed radish 1 type (CUR003), Oli radish 2 types (CUR006, CUR007), wild radish 9 types ( CUR099, CUR103, CUR105, CUR109, CUR111, CUR117, CUR121, CUR122, CUR124) were analyzed for polymorphism with KASP-based SNP markers. Among them, 16 KASP-based SNP molecular markers that can distinguish the 34 non-core group resources were selected.

When the SNP base mutation of the present invention is found in double-stranded gDNA (genomic DNA), it is interpreted to include a polynucleotide sequence complementary to the nucleotide sequence described above. Therefore, the base of the SNP position in the complementary polynucleotide sequence is also a complementary base.

The SNP marker set of the present invention is applied to radish, and by applying the SNP marker to unknown radish samples of unknown species or strain, Black radish, Rat-tailed radish, Oli radish, East Asian long radish, European small radish, Since it is possible to check whether it is a wild radish, it may be characterized in that it is possible to distinguish the species or lineage of the radish from the seed stage.

Of the 9 chromosomes of radish, 16 SNP polymorphism markers present on 8 chromosomes excluding chromosome 5 were selected.

As a result of the phylogenetic analysis of the 16 SNP marker sets selected through this study, coefficients of 0.5 to 6 groups can be classified. Group 1 has 1 rattail radish (CUR003) and Group 2 has 4 East Asian long radish ( CUR066, CUR072, CUR074, CUR075) and 8 European small radish types (CUR079, CUR081, CUR085, CUR084, CUR094, CUR082, CUR086, CUR091), and Group 3 includes 4 European small radish types (CUR083, CUR087, CUR088, CUR089), group 4 includes 5 East Asian long radish (CUR076, CUR068, CUR071, CUR069, CUR078), 2 oil radish (CUR006, CUR007), and 1 black radish (CUR096). And, in groups 5 and 6, 9 species of wild radish are included (FIG. 3).

As a result of analyzing 34 resources of various radish core groups with this marker set, various resources and lines of radish could be clearly identified.

<110> THE CATHOLIC UNIVERSITY OF KOREA INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> A set of SNP molecular markers for discriminating radish accessions using KASP assay and uses thereof <130> ADP-2019-0578 <150> KR 10-2019-0144662 <151> 2019-11-13 <160> 64 <170> KopatentIn 2.0 <210> 1 <211> 99 <212> DNA <213> Radish <400> 1 ctctactcgg gcaacttact tgctccttac aaccagtacc gagccctcta ytgctgccga 60 caaatacgac attccttatc tccaagacgt ttgccgcga 99 <210> 2 <211> 99 <212> DNA <213> Radish <400> 2 cagaaattta ggcttggaaa gcagccgcac aaggagtacg gagatcactc saaaggaagg 60 ttcaagaggt gaccgctctt cttttctaga tttattaag 99 <210> 3 <211> 99 <212> DNA <213> Radish <400> 3 ctccactaga tgaggtcaca ttccgtaggt tacagtctct ccagaaaaag ygtggatgct 60 gttcctcatg ttgccggtct gaacccacgc tctttccgt 99 <210> 4 <211> 99 <212> DNA <213> Radish <400> 4 aactcttctc taagccgtct tcccaagtca ggcatgtggc ctcctccgta waatcgcaac 60 tctcttgtgt ccttgctcta ttgctctttt caaagcttc 99 <210> 5 <211> 99 <212> DNA <213> Radish <400> 5 cgtagaggtc atggcctctt caactagtac agttcttgat caatagtctt statttacat 60 ttaccttaga tatgtattgg cgtcgtctca tattctaga 99 <210> 6 <211> 99 <212> DNA <213> Radish <400> 6 taagtaagaa cattgagata tgttgaagtc gtaccgaaga ttttgtagtc kgtcgacaac 60 agcagggacg gtaccgaaag gctgcatggg aagaaacaa 99 <210> 7 <211> 99 <212> DNA <213> Radish <400> 7 agcttattag ttatcatcat ttggttatta attattgagc atacaataga yttaacttta 60 tatatggaaa atgcatggaa catgacatag ctatggagc 99 <210> 8 <211> 99 <212> DNA <213> Radish <400> 8 tgaaggttac gccaagagct aaacgacata actgtgggaa gaagatcgag raagtcaaaa 60 gggtttagac taaacagacc aaggaagctg gttcttaag 99 <210> 9 <211> 99 <212> DNA <213> Radish <400> 9 acgagtttca gacagagatt tgctgattag gatccattgt gaaaaaaaca rgttgtttag 60 ttaagatctt gagttcactg gagaagtttc gtcttgaag 99 <210> 10 <211> 99 <212> DNA <213> Radish <400> 10 agagggatga cagggtactt cgtgaccttt ccatcttcag caacaaccac kgtctgcccc 60 ctgtgtaatc acggtggtcc tcttgtatgt tcctgtggc 99 <210> 11 <211> 99 <212> DNA <213> Radish <400> 11 gtggacattg cctttatagg attctacagt tggttccgta cgtacgaggt ktgcaaattt 60 cagcattgtg gcagagtatc ctaagttaat tgcttgggc 99 <210> 12 <211> 99 <212> DNA <213> Radish <400> 12 cacacaggtg atcggattgc cactaagatc ttcgagtctt tggaggaatg rtttagagaa 60 aaagattttc tcaattattc tagacaatgc cacaaaaaa 99 <210> 13 <211> 99 <212> DNA <213> Radish <400> 13 atttgaaact agtggaaata taaaccaaac tatagaacaa aaatattcac raattgtaaa 60 gatcaaatta tgagtggaat gagagtcagt ccatcgaat 99 <210> 14 <211> 99 <212> DNA <213> Radish <400> 14 aaaactgtat gatgttcact acttgatgca aaatatctac acccgaatct matgtactta 60 ccgcaatgtt cattaaattt tgggtgtcat ttcatatcg 99 <210> 15 <211> 99 <212> DNA <213> Radish <400> 15 tgtgcaggac agagtatcag cccccgagat cttgctatac tctgccaaaa rtatagtaag 60 gtcatttcct gaatttgtgc aatgttaatt tgttttttc 99 <210> 16 <211> 99 <212> DNA <213> Radish <400> 16 tggggagatg attggggcga tggagatgaa gaacagccga acactccggt kaccactcac 60 accgagtgtt tcttctagag gacttgctcc tagacgact 99 <210> 17 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> RsHH025-1_13109 Forward (FAM-labelled) primer <400> 17 gtcgtatttg tcggcagcaa gg 22 <210> 18 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> RS2CL3703s_516 Forward (FAM-labelled) primer <400> 18 caaggagtac ggagatcact cg 22 <210> 19 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> COS0566_179 Forward (FAM-labelled) primer <400> 19 caacatgagg aacagcatcc actaa 25 <210> 20 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> COS0151_283 Forward (FAM-labelled) primer <400> 20 aggacacaag agagttgcga ttctt 25 <210> 21 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> RsSNP07531_14513 Forward (FAM-labelled) primer <400> 21 agtacagttc ttgatcaata gtcttg 26 <210> 22 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> RS2CL1544s_2285 Forward (FAM-labelled) primer <400> 22 aagtcgtacc gaagattttg tagtct 26 <210> 23 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> RsSNP06056_1252 Forward (FAM-labelled) primer <400> 23 atttggttat taattattga gcatacaata gat 33 <210> 24 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> COS0530_10656 Forward (FAM-labelled) primer <400> 24 ataactgtgg gaagaagatc gagg 24 <210> 25 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> RSS2884_12650 Forward (FAM-labelled) primer <400> 25 gaactcaaga tcttaactaa acaacctc 28 <210> 26 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> RS2CL683s_6854 Forward (FAM-labelled) primer <400> 26 ttccatcttc agcaacaacc act 23 <210> 27 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> RS2CL2646_8211 Forward (FAM-labelled) primer <400> 27 tggttccgta cgtacgaggt g 21 <210> 28 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> RSS1854_5572 Forward (FAM-labelled) primer <400> 28 gaataattga gaaaatcttt ttctctaaac cc 32 <210> 29 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> RsSNP08808_10107 Forward (FAM-labelled) primer <400> 29 ataaaccaaa ctatagaaca aaaatattca cg 32 <210> 30 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> RSS3548_8083 Forward (FAM-labelled) primer <400> 30 aaaatttaat gaacattgcg gtaagtacat ttt 33 <210> 31 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> RsSNP14867_2430 Forward (FAM-labelled) primer <400> 31 acaaattcag gaaatgacct tactataact 30 <210> 32 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> COS0279_2650 Forward (FAM-labelled) primer <400> 32 gaacagccga acactccggt g 21 <210> 33 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> RsHH025-1_13109 Forward (HEX-labelled) primer <400> 33 aatgtcgtat ttgtcggcag caaga 25 <210> 34 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> RS2CL3703s_516 Forward (HEX-labelled) primer <400> 34 caaggagtac ggagatcact cc 22 <210> 35 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> COS0566_179 Forward (HEX-labelled) primer <400> 35 aacatgagga acagcatcca ctag 24 <210> 36 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> COS0151_283 Forward (HEX-labelled) primer <400> 36 aggacacaag agagttgcga ttcta 25 <210> 37 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> RsSNP07531_14513 Forward (HEX-labelled) primer <400> 37 ctagtacagt tcttgatcaa tagtcttc 28 <210> 38 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> RS2CL1544s_2285 Forward (HEX-labelled) primer <400> 38 agtcgtaccg aagattttgt agtcg 25 <210> 39 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> RsSNP06056_1252 Forward (HEX-labelled) primer <400> 39 tggttattaa ttattgagca tacaatagac 30 <210> 40 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> COS0530_10656 Forward (HEX-labelled) primer <400> 40 cataactgtg ggaagaagat cgaga 25 <210> 41 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> RSS2884_12650 Forward (HEX-labelled) primer <400> 41 gaactcaaga tcttaactaa acaacctt 28 <210> 42 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> RS2CL683s_6854 Forward (HEX-labelled) primer <400> 42 tccatcttca gcaacaacca cg 22 <210> 43 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> RS2CL2646_8211 Forward (HEX-labelled) primer <400> 43 gttggttccg tacgtacgag gtt 23 <210> 44 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> RSS1854_5572 Forward (HEX-labelled) primer <400> 44 agaataattg agaaaatctt tttctctaaa cct 33 <210> 45 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> RsSNP08808_10107 Forward (HEX-labelled) primer <400> 45 ataaaccaaa ctatagaaca aaaatattca ca 32 <210> 46 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> RSS3548_8083 Forward (HEX-labelled) primer <400> 46 taatgaacat tgcggtaagt acatttg 27 <210> 47 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> RsSNP14867_2430 Forward (HEX-labelled) primer <400> 47 caaattcagg aaatgacctt actataacc 29 <210> 48 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> COS0279_2650 Forward (HEX-labelled) primer <400> 48 agaacagccg aacactccgg tt 22 <210> 49 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> RsHH025-1_13109 Reverse primer <400> 49 ttacaaccag taccgagccc tcta 24 <210> 50 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> RS2CL3703s_516 Reverse primer <400> 50 cggtcacctc ttgaaccttc cttt 24 <210> 51 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> COS0566_179 Reverse primer <400> 51 ccgtaggtta cagtctctcc agaaa 25 <210> 52 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> COS0151_283 Reverse primer <400> 52 aggcatgtgg cctcctccgt a 21 <210> 53 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> RsSNP07531_14513 Reverse primer <400> 53 gacgacgcca atacatatct aaggtaaat 29 <210> 54 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> RS2CL1544s_2285 Reverse primer <400> 54 ctttcggtac cgtccctgct gtt 23 <210> 55 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> RsSNP06056_1252 Reverse primer <400> 55 gctatgtcat gttccatgca ttttccata 29 <210> 56 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> COS0530_10656 Reverse primer <400> 56 ggtctgttta gtctaaaccc ttttgactt 29 <210> 57 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> RSS2884_12650 Reverse primer <400> 57 gagatttgct gattaggatc cattgtgaa 29 <210> 58 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> RS2CL683s_6854 Reverse primer <400> 58 gaacatacaa gaggaccacc gtgat 25 <210> 59 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> RS2CL2646_8211 Reverse primer <400> 59 tactctgcca caatgctgaa atttgcaat 29 <210> 60 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> RSS1854_5572 Reverse primer <400> 60 actaagatct tcgagtcttt ggaggaat 28 <210> 61 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> RsSNP08808_10107 Reverse primer <400> 61 tggactgact ctcattccac tcataattt 29 <210> 62 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> RSS3548_8083 Reverse primer <400> 62 ctacttgatg caaaatatct acacccgaa 29 <210> 63 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> RsSNP14867_2430 Reverse primer <400> 63 ccgagatctt gctatactct gccaa 25 <210> 64 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> COS0279_2650 Reverse primer <400> 64 gaagaaacac tcggtgtgag tggta 25

Claims (12)

A polynucleotide consisting of 10-99 consecutive DNA sequences including a single nucleotide polymorphism (SNP) marker of the 51st nucleotide of the nucleotide sequence represented by SEQ ID NO: 1 to SEQ ID NO: 16, or a non-complementary polynucleotide thereof SNP marker set composition for cultivar identification. The method of claim 1, wherein the radish varieties are wild radish, rat-tailed radish, black radish, East Asian long radish, and European small radish. Or oil radish (oil radish), characterized in that the SNP marker set composition for identifying radish varieties. The composition of claim 1, wherein the SNP marker set composition is for use in Kompetitive Allele Specific PCR (KASP) analysis. Specific to a polynucleotide consisting of 10-99 consecutive DNA sequences containing a single nucleotide polymorphism (SNP) marker of the 51st nucleotide of the nucleotide sequence represented by SEQ ID NO: 1 to SEQ ID NO: 16 or a complementary polynucleotide thereof A composition for discriminating radish varieties containing a probe to bind or a primer for amplifying the polynucleotide. The method of claim 4, wherein the primer is a primer set represented by SEQ ID NO: 17, SEQ ID NO: 33, and SEQ ID NO: 49, a primer set represented by SEQ ID NO: 18, SEQ ID NO: 34, and SEQ ID NO: 50, SEQ ID NO: 19, SEQ ID NO: 35 And a primer set represented by SEQ ID NO: 51, a primer set represented by SEQ ID NO: 20, SEQ ID NO: 36, and SEQ ID NO: 52, a primer set represented by SEQ ID NO: 21, SEQ ID NO: 37, and SEQ ID NO: 53, SEQ ID NO: 22, SEQ ID NO: A primer set represented by 38 and SEQ ID NO: 54, SEQ ID NO: 23, a primer set represented by SEQ ID NO: 39 and SEQ ID NO: 55, a primer set represented by SEQ ID NO: 24, SEQ ID NO: 40 and SEQ ID NO: 56, SEQ ID NO: 25, sequence A primer set represented by SEQ ID NO: 41 and SEQ ID NO: 57, SEQ ID NO: 26, a primer set represented by SEQ ID NO: 42 and SEQ ID NO: 58, a primer set represented by SEQ ID NO: 27, SEQ ID NO: 43 and SEQ ID NO: 59, SEQ ID NO: 28, A primer set represented by SEQ ID NO: 44 and SEQ ID NO: 60, SEQ ID NO: 29, a primer set represented by SEQ ID NO: 45 and SEQ ID NO: 61, a primer set represented by SEQ ID NO: 30, SEQ ID NO: 46 and SEQ ID NO: 62, SEQ ID NO: 31 , A primer set represented by SEQ ID NO: 47 and SEQ ID NO: 63, and; A composition for discriminating radish varieties, characterized in that it is a primer set represented by SEQ ID NO: 32, SEQ ID NO: 48, and SEQ ID NO: 64. The method of claim 4, wherein the radish varieties are wild radish, rat-tailed radish, black radish, East Asian long radish, and European small radish. Or oil radish (oil radish), characterized in that the composition for determining radish varieties. The composition of claim 4, wherein the composition is for use in kompetitive allele specific PCR (KASP) analysis. A kit for discriminating radish varieties containing the composition of any one of claims 4 to 7. (1) separating DNA from radish;
(2) identifying the genotype of the 51st nucleotide of the nucleotide sequence represented by SEQ ID NO: 1 to SEQ ID NO: 16 from the separated DNA; And
(3) Radish variety discrimination method comprising the step of determining the radish variety based on the identified genotype. The method of claim 9, wherein the step of confirming the genotype of the 51st nucleotide of the nucleotide sequence represented by SEQ ID NO: 1 to SEQ ID NO: 16 in step (2) comprises a primer represented by SEQ ID NO: 17, SEQ ID NO: 33, and SEQ ID NO: 49. Set, a primer set represented by SEQ ID NO: 18, SEQ ID NO: 34 and SEQ ID NO: 50, a primer set represented by SEQ ID NO: 19, SEQ ID NO: 35 and SEQ ID NO: 51, SEQ ID NO: 20, SEQ ID NO: 36 and SEQ ID NO: 52 Primer set, primer set represented by SEQ ID NO: 21, SEQ ID NO: 37 and SEQ ID NO: 53, primer set represented by SEQ ID NO: 22, SEQ ID NO: 38 and SEQ ID NO: 54, represented by SEQ ID NO: 23, SEQ ID NO: 39, and SEQ ID NO: 55 A primer set represented by SEQ ID NO: 24, SEQ ID NO: 40, and SEQ ID NO: 56, a primer set represented by SEQ ID NO: 25, SEQ ID NO: 41 and SEQ ID NO: 57, SEQ ID NO: 26, SEQ ID NO: 42, and SEQ ID NO: 58 Primer set represented, SEQ ID NO: 27, SEQ ID NO: 43, and SEQ ID NO: 59, the primer set represented by SEQ ID NO: 28, SEQ ID NO: 44 and SEQ ID NO: 60, the primer set represented by SEQ ID NO: 29, SEQ ID NO: 45 and SEQ ID NO: 61 A primer set represented by SEQ ID NO: 30, SEQ ID NO: 46 and SEQ ID NO: 62, a primer set represented by SEQ ID NO: 31, SEQ ID NO: 47, and SEQ ID NO: 63; A method for discriminating radish varieties, characterized in that the step of performing PCR amplification using a primer set represented by SEQ ID NO: 32, SEQ ID NO: 48, and SEQ ID NO: 64, and analyzing the PCR amplification product. The method of claim 10, wherein the PCR amplification product analysis is a kompetitive allele specific PCR (KASP) analysis. The method according to any one of claims 9 to 11, wherein the radish variety is wild radish, rat-tailed radish, black radish, East Asian long radish, European small radish (European small radish) or oil radish (oil radish), characterized in that the radish variety identification method.

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