KR102646424B1 - Snp marker set for identifying cucurbita moschata cultivars and method for identifying cucurbita moschata cultivars using the same - Google Patents

Abstract

The present invention relates to a single nucleotide polymorphism marker set and its use for purity testing and variety identification of Asian pumpkin traits, and can reduce the time and cost required for purity testing and variety identification of Asian pumpkin traits or increase the accuracy of judgment. .

Description

Single nucleotide polymorphism marker set and its use for purity testing and variety identification of Asian pumpkins {SNP MARKER SET FOR IDENTIFYING CUCURBITA MOSCHATA CULTIVARS AND METHOD FOR IDENTIFYING CUCURBITA MOSCHATA CULTIVARS USING THE SAME}

The present invention relates to a single nucleotide polymorphism marker set and its use for purity testing and variety identification of Asian pumpkins.

Distinguishing between plant varieties is a necessary study for various purposes. For example, after the establishment of the International Union for the Protection of New Varieties of Plant (UPOV), establishing variety protection rights became a major concern for seed companies and breeders, and Korea, a member of UPOV, also took steps to protect varieties. The need for quantification, which establishes or quantifies objective standards for classification, has been recognized.

General breed classification has been based on phenotype. However, because phenotypes are greatly influenced by environmental factors, many statistical experiments are required. In addition, when distinguishing between breeds, the objective criteria for phenotypes are unclear and do not reflect perceived differences. To overcome these difficulties, we began developing DNA molecular markers.

There are several types of DNA molecular markers, such as restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR), and single-nucleotide polymorphism (SNP). Among these, SNP markers are the most common type of DNA base mutation and are one of the methods with high success in developing polymorphic markers.

The cultivation area of pumpkin (Cucurbita spp.) continued to increase until 2007 due to increased demand from 2003, and then continued to increase and decrease. The cultivation area increased from 8,791 ha in 2003 to 9,206 ha in 2018, and production amounted to 310,000 tons in 2018. This is an increase of approximately 1.1 times compared to 270,000 tons in 2000. The domestic pumpkin seed market is also expected to grow by more than 1.5 times to KRW 11.7 billion in 2018 compared to 2014 (Seed Association, 2019). As the economic importance of pumpkin increases, the cultivation of new varieties is actively carried out, and the importance of strengthening the variety protection rights, which are intellectual property rights, and blocking the development of similar clones is emphasized.

1. KR 10-2264690B1

An object of the present invention is to test the purity of Asian pumpkin, including an agent for detecting all single nucleotide polymorphisms (SNPs) located at the 61st base of each base sequence in the base sequences of SEQ ID NOs. 1 to 41, and The purpose is to provide a composition for variety identification.

Another object of the present invention is to provide a kit for purity testing and variety identification of Asian pumpkin, comprising the above composition.

Another object of the present invention is a) isolating a polynucleotide from an Asian pumpkin sample and determining the genotype of all single nucleotide polymorphisms present at the 61st position of each base sequence represented by SEQ ID NOs. 1 to 41; and b) analyzing the genotype of the single nucleotide polymorphism to identify the purity and variety of the Asian pumpkin.

One aspect of the present invention is a purity test for Asian pumpkin, comprising an agent for detecting all single nucleotide polymorphisms (SNPs) located at the 61st base of each base sequence in the base sequences of SEQ ID NOs. 1 to 41, and A composition for variety identification is provided.

The base sequences of SEQ ID NOs: 1 to 41 are as follows:

sequence number Assay ID Chr. SNP Pos (bp) Sequence One CmoSPT01 Cmo_Chr01 1152621 TCTGATGGCA ACGAACATTC TTCAGCCGAC AGTTTACAAG CCGTCGGTGA AGATCGCTGC [T/C]AAGGTAATCGGGCGTAAGTGGGCACCGTTGCCGGCGGTGATGGCGACGTTTTTAGTATCG 2 CmoSPT02 Cmo_Chr01 3245142 ATCTCCTACCCCGATTAGGCAAAAAGGATCCGTTGCTTAGGAATGTCGATTTATTCATAT[A/T]ATTGCAACTTTTTCTTCAGGGGGCCTGTATTTTCAACCTCTTTCATGGAGCCTTCGCTTG 3 CmoSPT3 Cmo_Chr01 12189252 AAAGCTGGAGGCTCTGGAGTTCCCCCAGTGCTGCGTTGTAGTGTGTGTTATTATATGGGT[T/C]TCTTATAACCCTTTCTTTTTTGGATGAACTGAATTCCTCTCATGGAGGGAGTTATGATGAT 4 CmoSPT4 Cmo_Chr02 485611 TCCCCTCGATGTTCCACCGCTGCCTTCCGTTCATCTCCATCCCTCTCTTATCTCTTCCTC[A/T]CTCTCAGAAGGAATCTATGCGAATTTATAGGAATGCAGGCGGAGACGACGACGGAACCGG 5 CmoSPT5 Cmo_Chr02 8290850 CTATCCTAGATCCCCGCGCTCCACCAGCGGATCTGACGGAACCAAGCAGCAGAGCTACTG[T/G]AAAGAAGACGAACTCCAATTCAAAAATCGAGACAATACAACGGAGAAGCAATTCAAATAT 6 CmoSPT6 Cmo_Chr03 3235775 TATCTTTGGGGAAGCCAAGATTGAAGATTTGAGCTCCCAACTACAAACCCAAGCTGCAGA[A/G]CAGTTCAAGGCACCTAATTTGAGTAACTTGACATCAAAGCCTGAGCCATCAACCGTGGTT 7 CmoSPT7 Cmo_Chr03 8003317 ATGTCCATCCCTTTTACCTTATTGTACTCGAGTGACGAGAAAAAAACAGTAAAAAAAAGG[A/G]ACTTTTGCTTCATAGCTTTTCTCGTGGTGCCTTCGAGGAGTGAATTATGGGTACCCTTTA 8 CmoSPT8 Cmo_Chr04 4262843 TTATGGGCTTGGCTCTGTATCGTTGGTTGGGGCACCATAACAGATTGATATGAATGGTTA[T/G]TGGGAGCAGGATGCGGCTGCATAGACATGAAAACATTAGGATCTGAACCAATGGCCAACG 9 CmoSPT9 Cmo_Chr04 7816936 CTGGCCGTTCGTCACGATCGCGCTCAAAATACTCTCAATACGCTCCACCAGTTCCTCATC[A/G]ACGCCCTCCGCTGCCTCCAATACGCCATCAATCGACTGGAGAAACTCCACATTCGAATCG 10 CmoSPT10 Cmo_Chr04 17956800 GCGAATGAGCTAGTGCAGAAGTTTCATATAGCAATGGAGACTGTTTCTGAAAACATTAAT[A/T]GCAGTGAAGCTGGAGATAAATTTACCCAGCAGCATAAAGAACTTCTGTCTGTTCTCGATC 11 CmoSPT11 Cmo_Chr05 272092 TTTAAGATGCTTTGTTGGGTCACTGAAGTCAAACAAAACGCAGCATAGATTTAGCTTCTT[A/G]ACAAACAAATTCTGTTTTTCAGAGCTTGGAACTTCCCTAAACCCAGGCAAATGCCTCATATAT 12 CmoSPT12 Cmo_Chr05 5280339 TTATGAGTCGGTATGATTCTGCTAGCCAGAGTCACTTTTCACTTCTCTTTAGATCAGTGC[A/T]GCAGACAATAATGTTCTAGTCATTAGTAGGGTAGAAACTGATGTCTCCAATTTTTTTTTT 13 CmoSPT13 Cmo_Chr06 8873197 CAGAAGTGCTTCAGTAAATCTTGGCTAGCCAGGGTTGAAACGTGGTTCTTCACCCCGATC[A/G]TAATCGACCCCAAGAGAAACGTGGCCTCATCATCTGCTCGCAGCTTACAGCCATCCTTCAA 14 CmoSPT14 Cmo_Chr07 2866018 TTTGATGACTTTGCTGGCTTCTTGAGTTACTGGGTCGACAAGATAGTCAGGCACGTCACG[T/C]AGATGAGGAGCAGGGGGCTTCTTGCTGAGAATCTTATCATGTTTCAACAAATCTGCCCAA 15 CmoSPT15 Cmo_Chr07 4120945 CCCACCGAGTAAACAAAGAACAATGAGTGACAGACCAATCCACTGGTGGCCTTAGCAGCT[A/C]AGAAGCCCCCTCGGGCCCCAACGGAGGAAGTGGGTACAGAGAAGGAAAGGCCAACGCCAA 16 CmoSPT16 Cmo_Chr08 721844 TTCTTATACTCATGTAGCTGCACTGAGGAAGGAGATCTCAGACGAGACTGATAGAGAGAC[A/C]GGGCGCAGTAAACAAATTTCAAGTGTTCCAATCCATCTGAGTATCTACTCTCCCAATGGT 17 CmoSPT17 Cmo_Chr09 910035 TGACTTTGATGAGTCTAGCTGTAGGCTTAATGGTCGTTGCACTTGTTACAAAGGATCTAA[T/C]GAGGAACAAGAAATGCTGCAACTTGAGCCATCACAGAATTTCTTGTGCAGCTACGGTTAGG 18 CmoSPT18 Cmo_Chr09 2176802 TATAAAATCCCTTTACCCCTAAGTTTTGGGTACTGACCTCTTGCTGCATGATGATCATCCT[A/G]GTCGGTTGTTGCGAACAAAAGGCACGAGAATGGTGACGGTTGGACGGGGATGGAACGAAA 19 CmoSPT19 Cmo_Chr09 6066333 GATGAACAGGGTTGGCTTTTCTTAACCACTTGAATTCTGTATCTAAGTAATCCTTACTGT[T/C]GTTTTGTTTCTTGTTTACATCTATGATTTGTTTCTTTGTTTCAATTGGTGGAATCTTCTG 20 CmoSPT20 Cmo_Chr10 1050490 TTCAGGTCCAGAAACCTCCTGATTTCTGGGACTTGTTTTTGAATTTGAATTGTTGCTCTG[T/C]TTTTTAATCCACTGATCAAATGAGATCTTTTATGCCCACCAAGTGCTTGACCTGATGCGA 21 CmoSPT21 Cmo_Chr10 2077280 TTTTTCAAGATTGAAATCAATCTCAATCTCATGGTTGTCTTCTATAGGAAGCACGGATGC[A/C]ACTCCTCCAGTTGATTTGTTTTTGTAGTCGGATCTTGCTTTAAGCGTGGCTGCTCCTTTC 22 CmoSPT22 Cmo_Chr10 10779291 ATTGTTAACTGTATGTGAGATTCACTGTTCAGCACAGAATAGAATCTATGGAACGGAAAA[T/C]GATTGGTATGGGATTCTTCAAAATTGATCAATCAGCAGACGAAACAATTATAAAAAAAGCAA 23 CmoSPT23 Cmo_Chr11 11671486 CATAGACAGTGACAATGAGTTTAAGAAATCGGATTCTCATGGGCATATCGTCGACGCGTT[T/C]GGGGCGTCAATGGTAGAGCCTTGGCTTGCAGCCCATGGAGACCCAACAAGGTTTTCATTT 24 CmoSPT24 Cmo_Chr12 188855 GACTGGGCCTTCAACGGTATCGTTGCTACCACCGGGAGCCGCGGCCCCGTCGGTGGTGTT[T/C]CCAATCTTGCCATCGGCGCCGGGAGCCTCGGTTTTATTAGCACCGGCCTTGGGAGTTTCA 25 CmoSPT25 Cmo_Chr12 9389415 CTTCCCACACGGCGGAGCCATGGACTCAATGGATCATGGGGTTTACAGCGGGGGGTGGAAG[T/C]GGAGACGGGTACGGCGGGAACTTCCTGATTTCGATGGCGGCCGGGGCGGAAGCCGGGAGC 26 CmoSPT26 Cmo_Chr13 6150899 CACCAAAGGAGCAATTCTTCTCAAAATTTTAAAAGGTACTAAGTTGGAATCCCATTCTAA[G/C]TAAAGATTCTTGTGGTACAAGGACTACAAATTAATAGGAAGAAAAAACAATGTTGACTACT 27 CmoSPT27 Cmo_Chr14 1094562 CTCCTTAAGAACGGACACAAGCTCAATATTTGATTCTTGACTTCTCTTTAGCTGCAGAGC[T/G]AAATCAGCATTGGATTCTTTTTGGAACTTCAGTTCATCTTTCAATTCATCTAAGATTTTT 28 CmoSPT28 Cmo_Chr14 5494858 TAACTTTGAAAGGTCCGAAAAGTTGCAGCTCCGAAAGCAATTCGAATCAGATTTTTATTC[A/G]ACTTTTGGTTTTTCTTACGAAGTAGTCGGGTCGGGAGGTTTACAGTGTTTTTAACAACAG 29 CmoSPT29 Cmo_Chr15 3627784 ACATTTCTTACCCGAAATTGCAGCATTTAAGGCCGGTCTTCTTCTGCTCCCACAACAGTT[T/G]ACACCCGCCGTAGATATCGATGCGGCGGCAACGGTAGACCTGGTGATGCTGGTTCCGGTG 30 CmoSPT30 Cmo_Chr16 320626 TCGAGCTGCTTGTTCTCGCAAGCGATGGACTTTGGGACGTGGTACGCAACGAGGTATGCT[G/C]ACTACATCATTACCCCATGTATCTAAGATGCTCTAAATTGTGAACTCCACATACGAACTT 31 CmoSPT31 Cmo_Chr16 9148953 ATCATCTTCGGCCGAAAACTCCTCTGTTTCGTCGCAGACGATATCGGTGTCGTCAAATTC[A/C]CCGCTGCCAAGCTGACAATGGGGGAGTCATTAGTAATGTCGATAAGCGCCGATCGATCTT 32 CmoSPT32 Cmo_Chr17 2436117 TGACGTTTTCACTGCAGATTTCAATTCCAGTAAAACCAGTGCCAATTTTCCTCCGTCTC[G/C]GCATCTACAACTGCAAAAGCTGCTGGAAATATGCCATCTTCCCCATCCACAGCTGTGGGCA 33 CmoSPT33 Cmo_Chr17 7898700 ATTAAATTCACATTCGAGAGGAGTGGAAGCAGCCGTAGGATTGAAGCAAGGGTAGGATAG[T/G]AAATTAAGACAAAAGTGAAGGGCAGGGGTCAGTCGCACGTGTGAAAAAGATTGAAGTCAC 34 CmoSPT34 Cmo_Chr18 856797 AGATTCAGATTTACAGGTGATGAAGGAGAAATTAAACCCTGCAATCTTCCAGGGAAACGG[A/G]GAGGTTGAAGGTAAGAAAATCAGTGGGAGAAGTAACGGGGAAAGTTTCTCGGTGTCGAAG 35 CmoSPT35 Cmo_Chr18 5506608 ACAGATGCCCTTCTTCATTGACATTTGTCAGTCTGTAGAAAGATAGCCAAGCATGACAGC[T/C]TTGGATAAATTTGGTGACTAAACTGTATAAAATCTTTAAAAAGTCCTTTAGTCTTACATT 36 CmoSPT36 Cmo_Chr18 12459404 CTCTGACATTTTTCTTCTCAGCTGCGGCTCATCCTCCAGTCCCACCTTCTTTAACCGCAC[T/C]TTCGTCGGCGATTCAGTCAAACCCGCCTCCGACTTTCTCACTGCCGGCCGATCGGTGGCG 37 CmoSPT37 Cmo_Chr19 139944 TCGTAGAGCTACAAGAACGAAGATGATGATTCAATGTTTTTTGACATTTGGGCTTCTGAAC[T/G]TTTTAAATGCTTGCTCTGTCTTTGGTTTTTGTAGTTACAGATCTTTTATATTGTTCATCGAA 38 CmoSPT38 Cmo_Chr19 8523415 TATATACACTCATCCATCATATACACTCATCCATCGCCCTCTCGCTCTCTCTCGTGCGTT[T/G]TGTATGTACAGGAAGATTGTAACAGTGGAAGAGATCTTAACAATTGACATAAAACCAGGT 39 CmoSPT39 Cmo_Chr20 124230 GTTGGAGCAGCTCTTTGACAGCTCCCTCGTGGGTTGAGTATTACTCATCTATTTGTAGCT[T/C]TCCACTTATCAATGGATATTTTCTCGTTTCTTATCTAAAATATTTGCCGTCAACTTATCC 40 CmoSPT40 Cmo_Chr20 2167111 ACAGAATCAAACAGTCTCTATGAGACTTATAATCACCACTCATATGGTTTCTTCACCGGCC[G/C]ATTCGTTCTTCGATGGAGTTTCATCGCCAGATTTCTCCACTCCAAACATGGCTGATTCAA 41 CmoSPT41 Cmo_Chr20 4092624 TCATTTACTGGTGATCGATCATCATTGATATTTGAACAGCACGTACCTGGAGAGAGTGTC[A/G]ATGAGCTGAAAAAGAAAATTCAGTCACAGGTGATTTTAACATGCTTCAATTTGTGTTATC

The single nucleotide polymorphism (SNP) refers to the insertion, deletion, or substitution of one nucleotide in DNA. The term 'polymorphism' refers to the arrangement in the sequence of a gene that varies within a population. Polymorphisms consist of different “alleles.” “Allele” refers to a gene corresponding to an allele located in a corresponding region on a pair of homologous chromosomes, that is, a homologous locus. Two or more of one gene occupying the same chromosomal location (same chromolocsomalus). It means one of the above alternative forms. The placement of this polymorphism can be identified by its location in the gene and the different amino acids or bases found therein. This amino acid variation is the result of two possible variant bases, C and T, which are two different alleles. Because a genotype consists of two distinct alleles, any of several possible variants can be observed in any one individual (e.g., CC (denoted A), CT (denoted H), or TT (marked B)). Individual polymorphisms are also known to those skilled in the art.

In the present invention, the single nucleotide polymorphism may be located at the 61st base of each base sequence in SEQ ID NOs: 1 to 41.

The 61st base is the 61st base in any one of SEQ ID NOs: 1 to 41. The base sequences of SEQ ID NOs: 1 to 41 include single nucleotide polymorphisms and flanking sequences that exist on the chromosomes of Asian pumpkins, respectively. In SEQ ID NOs. 1 to 41, each 61st base is a single nucleotide polymorphism that exists on the chromosome of Asian pumpkins, and those related to the purity and variety identification of Asian pumpkins were selected. Analyzing the allele type of the single nucleotide polymorphism can be useful in determining the purity and variety of Asian pumpkins by comparing the nucleotide differences between parents and offspring.

The agent may contain a polynucleotide consisting of a base sequence identical to or complementary to contiguous bases including bases corresponding to the selected single nucleotide polymorphism. The contiguous bases may consist of 5 to 121 bases. For example, the agent is a polynucleotide consisting of all or part of the base of any one of the alleles located at the 61st position in any one of the base sequences of SEQ ID NOs. 1 to 41 and all or part of its flanking sequence, or a polynucleotide consisting of a sequence complementary thereto. It may be a nucleotide. In the nucleotide sequence of each sequence number, a single nucleotide polymorphism is located at the 61st base, and flanking sequences of 60 bp each are linked to the SNP in the upstream and downstream directions based on the 61st base. One single nucleotide polymorphism is located in the base sequence of each sequence number. The length of the consecutive bases may vary depending on the SNP genotype determination method.

According to one embodiment, the agent may be used to identify a single nucleotide polymorphism located at the 61st base of the sequence number corresponding to the polynucleotide to be detected or amplified. The agent may be a primer containing the base of any one of the single nucleotide polymorphism alleles corresponding to the 61st base of the sequence number and all or part of the flanking sequences contiguous thereto.

According to one embodiment, the agent may be any one of a probe, a primer, and a combination thereof.

The term “probe” refers to a polynucleotide that specifically binds to a particular target sequence. The probe may be complementary to or capable of specific binding to a polynucleotide containing a single nucleotide polymorphism site. The probe may have a difference in binding ability depending on the base difference in the single nucleotide polymorphism. The probe may be labeled so that the presence or absence of a specific nucleic acid can be confirmed.

The term “primer” refers to a single-stranded polynucleotide that can serve as a starting point in the polymerization reaction of nucleotides by a polymerase. The primers can be produced using methods known to those skilled in the art according to the selected SNP analysis method. The primer sequence may be identical to or complementary to some consecutive bases among the base sequences of SEQ ID NOs. 1 to 41. The length of the primer may vary depending on various factors, such as temperature and the purpose of the primer. Primers for identification of SEQ ID NOs: 1 to 41 are shown in Table 2 below:

sequence number Assay ID Chr. SNP Pos (bp) Primer Seq Allele X (FAM) Primer Seq Allele Y (HEX) Primer Seq common One CmoSPT01 Cmo_Chr01 1152621 CCCACTTACGCCCGATTACCTTA CCACTTACGCCCGATTACCTTG CAGTTTACAAGCCGTGGTGGAAGAT 2 CmoSPT02 Cmo_Chr01 3245142 GTTGCTTAGGAATGTCGATTTATTCATATA GTTGCTTAGGAATGTCGATTTATTCATATT GGCCCCCTGAAGAAAAAAGTTGCAAT 3 CmoSPT3 Cmo_Chr01 12189252 GTTCATCCAAAAAAGAAAGGGTTATAAGAA CATCCAAAAAAAGAAAGGGTTATAAGAG GTGCTGCGTTGTAGTGTGTGTTATTATAT 4 CmoSPT4 Cmo_Chr02 485611 AAATTCGCATAGATTCCTTCTGAGAGT AAATTCGCATAGATTCCTTCTGAGAGA CCGTTCATCTCCATCCCTCTCTTAT 5 CmoSPT5 Cmo_Chr02 8290850 GAACCAAGCAGCAGAGCTACTGT AACCAAGCAGCAGAGCTACTGG GTCTCGATTTTTGAATTGGAGTTCGTCTT 6 CmoSPT6 Cmo_Chr03 3235775 TACTCAAATTAGGTGCTTGAACTGT ACTCAAATTAGGTGCTTGAACTGC GATTTGAGCTCCCAACTACAAACCCAA 7 CmoSPT7 Cmo_Chr03 8003317 CACGAGAAAAGCTATGAAGCAAAAGTT ACGAGAAAAGCTATGAAGCAAAAGTC TTACCTTATTGTACTCGAGTGACGAGAAA 8 CmoSPT8 Cmo_Chr04 4262843 CAGCCGCATCCTGCTCCCAA AGCCGCATCCTGCTCCCAC GGCACCATAACAGATTGATATGAATGGTT 9 CmoSPT9 Cmo_Chr04 7816936 ACGCTCCACCAGTTCCTCATCA CGCTCCACCAGTTCCTCATCG GTTTCTCCAGTCGATTGATGGCGTA 10 CmoSPT10 Cmo_Chr04 17956800 GTAAATTTATCTCCAGCTTCACTGCT GTAAATTTATCTCCAGCTTCACTGCA GCAATGGAGACTGTTTCTGAAAACATTAAT 11 CmoSPT11 Cmo_Chr05 272092 CAAAACGCAGCATAGATTTAGCTTCTTA AAAACGCAGCATAGATTTAGCTTCTTG GGAAGTTCCAAGCTCTGAAAAACAGAATT 12 CmoSPT12 Cmo_Chr05 5280339 TTTCACTTCTCTTTAGATCAGTGCA CTTTTCACTTCTCTTTAGATCAGTGCT CAGTTTCTACCCTACTAATGACTAGAACAT 13 CmoSPT13 Cmo_Chr06 8873197 ACGTGGTTCTTCACCCCGATCA CGTGGTTCTTCACCCCGATCG GCAGATGATGAGGCACGTTTCTCTT 14 CmoSPT14 Cmo_Chr07 2866018 CAAGATAGTCAGGCACGTCACGT AAGATAGTCAGGCACGTCACGC CAAGAAGCCCCCTGCTCCTCAT 15 CmoSPT15 Cmo_Chr07 4120945 CACTGGTGGCCTTAGCAGCTA CACTGGTGGCCTTAGCAGCTC TTCCTGTACCCACTTCCTCCGTT 16 CmoSPT16 Cmo_Chr08 721844 CACTTGAAATTTGTTTACTGCGCCCT ACTTGAAATTTGTTACTGCGCCCG GAAGGAGATCTCAGACGAGACTGAT 17 CmoSPT17 Cmo_Chr09 910035 CAAGTTGCAGCATTTCTGTTCCTCA AAGTTGCAGCATTTCTGTTCCTCG GTCGTTGCACTTGTTACAAAGGATCTAA 18 CmoSPT18 Cmo_Chr09 2176802 CTTGCTGCATGATGATCATCCTA CTTGCTGCATGATGATCATCCTG CCATTCTCGTGCCTTTTGTTCGCAA 19 CmoSPT19 Cmo_Chr09 6066333 AACAAATCATAGATGTAAACAAGAAACAAAACA CAAATCATAGATGTAAACAAGAAACAAAACG CCACTTGAATTCTGTATCTAAGTAATCCTT 20 CmoSPT20 Cmo_Chr10 1050490 TGTTTTTGAATTTGAATTGTTGCTCTGT GTTTTTGAATTTGAATTGTTGCTCTGC GGCATAAAAGATCTCATTTGATCAGTGGAT 21 CmoSPT21 Cmo_Chr10 2077280 CTTCTATAGGAAGCCACGGATGCA CTTCTATAGGAAGCACGATGCC CCACGCTTAAAGCAAGATCCGACTA 22 CmoSPT22 Cmo_Chr10 10779291 GATCAATTTGAAGAATCCCATACCAATCA CAATTTGAAGAATCCCATACCAATCG AGCACAGAATAGAATCTATGGGAACGGAAA 23 CmoSPT23 Cmo_Chr11 11671486 GGGCATATCGTCGACGCGTTT GGCATATCGTCGACGCGTTC GCTGCAAGCCAAGGCTCTACCAT 24 CmoSPT24 Cmo_Chr12 188855 GGGCGCGATGGGCAAGATTGGA GCGCCGATGGCAAGATTGGG CGGCCCGTCGGTGGTGTT 25 CmoSPT25 Cmo_Chr12 9389415 CCGCCGTACCCGTCTCCA CCGCCGTACCCGTCTCCG CATGGACTCAAATGGATCATGGGGTT 26 CmoSPT26 Cmo_Chr13 6150899 AGTCCTTGTACCACAAGAATCTTTAC AGTCCTTGTACCACAAGAATCTTTAG AAAAGGTACTAAGTTGGAATCCCATTCTAA 27 CmoSPT27 Cmo_Chr14 1094562 TGACTTCTCTTTAGCTGCAGAGCT GACTTCTCTTTAGCTGCAGAGCG CTGAAGTTTCCAAAAAGAATCCAATGCTGAT 28 CmoSPT28 Cmo_Chr14 5494858 CGACTACTTCGTAAGAAAAAACCAAAAGTT GACTACTTCGTAAGAAAAAACCAAAAGTC AGCTCCGAAAGCAATTCGAATCAGATTT 29 CmoSPT29 Cmo_Chr15 3627784 CATCGATATCTACGGCGGGTGTA ATCGATATCTACGGCGGTGTC GTCTTCTTCTGCTCCCACAACAGTT 30 CmoSPT30 Cmo_Chr16 320626 GGTACGCAACGAGGTATGCTG GGTACGCAACGAGGTATGCTC GCATCTTAGATACATGGGGTAATGATGTA 31 CmoSPT31 Cmo_Chr16 9148953 GACGATATCGGTGTCGTCAAATTCA ACGATATCGGTGTCGTCAAATTCC CTAATGACTCCCCCATTGTCAGCTT 32 CmoSPT32 Cmo_Chr17 2436117 GTGCCAATTTTCCTCCGTCTCG GTGCCAATTTTCCTCCGTCTCC TATTTCCAGCAGCTTTTGCAGTTGTAGAT 33 CmoSPT33 Cmo_Chr17 7898700 AGGATTGAAGCAAGGGTAGGATAGT GAAGCAAGGGTAGGATAGG CCCCTGCCCTTCACTTTTGTCTTAA 34 CmoSPT34 Cmo_Chr18 856797 GCAATCTTCCAGGGAAACGGA GCAATCTTCCAGGGAAACGGG ACTTCTCCCACTGATTTTCTTACCTTCAA 35 CmoSPT35 Cmo_Chr18 5506608 TTTATACAGTTTAGTCACCAAATTTATCCAAA ATACAGTTTAGTCACCAAATTTATCCAAAG CATTGTCAGTCTGTAGAAAGATAGCCAA 36 CmoSPT36 Cmo_Chr18 12459404 GTCCCACCTTCTTTAACCGCACT CCCACCTTTCTTTAACCGCACC GGTTTGACTGAATCGCCGACGAA 37 CmoSPT37 Cmo_Chr19 139944 AAAAACCAAAGACAGAGCAAGCATTTAAAA AACCAAAGACAGAGCAAGCATTTAAAC GAGCTACAAGAACGAAGATGATGATTCAA 38 CmoSPT38 Cmo_Chr19 8523415 CACTGTTACAATCTTCCTGTACATACAA ACTGTTACAATCTTCCTGTACATACAC CTCTCGCTCTCTCTCTGTGCGTT 39 CmoSPT39 Cmo_Chr20 124230 AAACGAGAAAATATCCATTGATAAGTGGAA CGAGAAAATATCCATTGATAAGTGGAG CTCGTGGGTTGAGTATTACTCATCTATTT 40 CmoSPT40 Cmo_Chr20 2167111 CATATGGTTCTTCACCGGCCG CATATGGTTCTTCACCGGCCC GCGATGAAACTCCATCGAAGAACGAA 41 CmoSPT41 Cmo_Chr20 4092624 CACGTACCTGGAGAGAGTGTCA ACGTACCTGGAGAGAGTGTCG CCTGTGACTGAATTTTTCTTTTTCAGCTCAT

The primer may be a primer for amplifying the KASP marker for determining the purity and variety of Asian pumpkin.

The “KASP (kompetitive allele specific PCR)” is one of the PCR-based analysis methods and is a homogenous and fluorescence-based genotyping technology. KASP is a technology based on fluorescence resonance energy transfer for allele-specific oligo extension and signal generation.

Another aspect of the present invention provides a kit for purity testing and variety identification of Asian pumpkin, comprising the above composition.

In one embodiment of the present invention, the kit may be a PCR kit, microarray, or KASP kit.

The PCR kit may include a primer set capable of detecting the single nucleotide polymorphism. The PCR kit may further include DNA polymerase, dNTP mixture, and PCR buffer solution. The DNA polymerase may be, for example, a klenow fragment of E. coli DNA polymerase I, a thermostable DNA polymerase, or bacteriophage T7 polymerase. The PCR buffer solution may contain KCl, Tris-HCl, and MgCl2. Additionally, the PCR kit may include components necessary for confirmation of the amplification product. The PCR kit may include a guide. The guide may be a printed document explaining how to use the kit, for example, how to prepare a PCR buffer solution, proposed reaction conditions, etc.

The microarray may be one in which polynucleotides, for example, probes capable of detecting the single nucleotide polymorphism, are immobilized at a high density in distinct areas of the substrate surface.

The KASP analysis kit includes a primer set for KASP; and a reagent for performing an amplification reaction, and the reagent for performing the amplification reaction may include, but is not limited to, DNA polymerase, dNTPs, and a buffer. The dNTPs include dATP, dCTP, dGTP, and dTTP. DNA polymerase is a heat-stable DNA polymerase, and commercially available polymerases such as Taq DNA polymerase and Tth DNA polymerase can be used.

Another aspect of the present invention includes the steps of: a) isolating a polynucleotide from an Asian pumpkin sample and determining the genotype of all single nucleotide polymorphisms present at the 61st position of each base sequence represented by SEQ ID NOs. 1 to 41; and b) analyzing the genotype of the single nucleotide polymorphism to identify the purity and variety of the Asian pumpkin.

Step a) may further include amplifying the polynucleotide isolated from the sample. The amplification step can be performed using any method known to those skilled in the art. For example, polymerase chain reaction (PCR), ligase chain reaction, nucleic acid sequence-based amplification, transcription-based amplification system, strand displacement amplification. (strand displacement amplification) or replicase may be used, but are not limited thereto.

In step b), the purity and variety of Asian pumpkin can be identified by analyzing the genotype of the analyzed single nucleotide polymorphism. Specifically, the genotype of the analyzed single nucleotide polymorphism of the Asian pumpkin F ₁ may be compared with the genotype of its parent pumpkin to test purity and determine the variety.

The composition for testing the purity and identifying varieties of Asian pumpkins according to one embodiment of the present invention can determine the F ₁ variety of Asian pumpkins, and can accurately and quickly determine whether or not they are crossed by both parents by testing the purity of the F ₁ seeds. there is.

Therefore, the present invention can be used in various fields, such as testing the authenticity of varieties in the event of a dispute over Asian pumpkin seeds, selecting control varieties when applying for variety protection, and managing seed distribution.

Figure 1 shows the results of genotyping (Company A and Company B) for 33 out of 38 Asian pumpkin core lines (red: FAM fluorescence, green: HEX fluorescence, blue: heterozygous type, white: missing value).
Figure 2 shows the results of genotyping (C company) for Asian pumpkin F ₁ varieties (4) and parental lines.
Figure 3 shows the results of genotyping of 45 lines of various breeding materials used in poultry pumpkin breeding (Company A, Company B, and Company D).
Figure 4 shows the results of genotyping using Asian pumpkin SNP molecular markers (example: results for 4 primers).
Figure 5 shows a physical map showing the locations of the 41 marker sets of Asian pumpkin SNPs whose utility was tested (green: locations of 41 SNPs whose utility was tested).
Figure 6 shows the results of genetic phylogenetic tree analysis and classification for Asian pumpkin core lines and growing materials (78 in total).
Figure 7 shows the results of clustering analysis for Asian pumpkin core lines and growing materials (78 in total).
Figure 8 shows the results of genetic phylogenetic tree analysis and classification for Asian pumpkin F ₁ and parental lines (total 12).

Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are intended to illustrate one or more embodiments and the scope of the present invention is not limited to these examples.

Example 1: Materials and Methods

1-1. test material

In order to select SNPs that can be used in various breeding combinations of Asian pumpkins, 226 pumpkin resources were collected and 38 Asian pumpkin core lines with non-overlapping characteristics in various traits were selected (sold by companies A and B).

As materials for testing the marker set to be developed, four cultivars of zucchini and rootstock (oriental) F ₁ , their eight parent lines, and the remaining 45 breeding lines were used (distributed by companies A, B, C, and D). .

1-2. GBS analysis of Asian pumpkin core lines, SNP search and selection between lines

High purity gDNA was isolated using the CTAB method, and using Nanodrop, it was confirmed that the A260/280 value was over 1.8 and the concentration met the standards of over 100ng/μL.

A GBS library was constructed using the restriction enzyme ApeKI, and the library was subjected to base sequence analysis for each core lineage of Asian pumpkin using Illumina Nextseq 500 equipment.

SNP calling of core lines was performed using the SAM Tools program, and SNP filtering was performed to select SNPs that satisfied the selection conditions.

1-3. Developed in the form of a KASP marker set for seedling company breeding lines and F _One Applies to breeds

The selected SNPs were developed as a set of primers (or probes) customized for the KASP ^TM genotyping system, and using this, it is possible to test multiple molecular markers on multiple samples in a short period of time.

The designed SNP marker set was applied to 33 out of 38 core lines, and genotype analysis was performed on 4 F ₁ cultivars, 8 parent lines, and 41 lines of various breeding materials used for breeding Asian pumpkins.

Through the genotyping results of the high-throughput marker set, SNPs showing polymorphism between lines used when breeding Asian pumpkins were additionally selected, which allows for purity testing and variety identification.

Genetic tree analysis was performed to analyze the genetic diversity of each lineage.

Example 2: Study Results

2-1. Selection and collection of Asian pumpkin core lines

Among 226 resources containing various characteristics such as pumpkin traits such as fruit size, fruit shape, skin color, leaf shape, presence of leaf patterns, and first age, 38 were selected as core Asian strains and young leaves (shoots) were collected (Company A) and sale by Company B).

2-2. GBS analysis and SNP search of Asian pumpkin core lineage

High purity gDNA was extracted and fragmented into reads using ApeKI restriction enzyme to construct a GBS library. Different adapters were attached to both ends of the fragment and ligated with T4 DNA ligase.

The base sequence of the constructed GBS library was sequenced for each pumpkin resource using Illumina Nextseq 500 equipment, and then the adapter sequence was removed and trimming was performed using the trimmomatic program. As a result, the average number of reads for 192 resources (Company A) and 34 resources (Company B) collected were 1,765,076 and 2,523,739, respectively.

Read mapping was performed on the standard genome using the Bowtie2 program, and the average overall read mapping rate was 84.0% for Company A and 81.8% for Company B.

Using the VCFtools program, SNP calling was performed to search for genome variation information between the standard genome and Asian resources (167) among the collected pumpkin resources, and as a result, 10,549 final SNPs were discovered.

The number of SNPs for each chromosome was calculated using in-house scrip, and the home/hetero genotype ratio for each sample was also calculated.

2-3. High-throughput customized SNP selection and marker set development

A total of 10,549 SNPs searched by companies A and B were selected for genotype results corresponding to a depth of 5x or more, and those with different genotypes between strains were classified as SNPs to select a total of 2,693 SNPs, and SNPs with a homozygosity of 0.96 or more were selected. 2,214 were selected.

Among the selected SNPs, in order to select SNPs suitable for tasks such as purity testing and breed identification, the PIC (Polymorphism Information Content) value is approximately 0.35 to 0.5, and 48 SNPs that are evenly distributed across 20 chromosomes were ultimately secured. did.

A KASP ^TM primer (or probe) set for PCR amplification was synthesized based on the flanking or amplicon sequence information, which is a 60bp nucleotide sequence adjacent to the SNP position in both directions.

2-4. Efficacy testing by applying KASP marker set to Asian pumpkin strains and varieties

The effectiveness of the designed 48 KASP ^TM primers was tested by applying them to various breeding lines and F ₁ varieties used in the development of Asian pumpkin varieties.

Genotyping was performed on 33 of 38 core lines, 4 F ₁ cultivars, 8 parent lines, and 45 lines of various breeding materials used for breeding Asian pumpkins (Figures 1 to 3).

As a result of the analysis, it was confirmed that among the 48 developed KASP ^TM marker sets, 41, excluding 7 that showed heterozygote genotypes in most lines or were difficult to detect with a fluorescent probe, functioned as molecular markers (Figure 4). A physical map was created for 41 SNP marker sets whose utility was tested (Figure 5).

Information on the final 41 KASP marker sets whose utility was tested through genotyping analysis is shown (Table 1).

Through the results of polymorphic genotyping of the F ₁ variety and breeding lines, it was confirmed that the developed 41 Asian pumpkin SNP marker set can be used for purity testing and variety identification (Figures 1 to 3).

Phylogenetic tree (dendrogram) analysis was performed to analyze the genetic diversity of each lineage used in the analysis. Phylogenetic analysis was performed using Darwin6 software (ver. 6.0.10) (http://darwin) using the unweighted pair group method with arithmetic mean among the hierarchical clustering methods. Carried out in .cirad.fr/). Bootstraps were determined at 1,000 replicates.

As a result of genetic diversity analysis of the core strains and breeding materials of Asian pumpkins distributed by Company A, Company B, and Company D, they were broadly classified into three groups. The first and second groups included strains of Asian pumpkins that were mainly edible (zucchini, etc.), and the third group included many growing materials used as rootstock (Figure 6).

In addition, we analyzed the distribution of core lines and foster lines through factorial analysis in Darwin6 software, and it was confirmed that they were largely classified into three groups, as in the flexible relationship analysis (Figure 7).

Genetic diversity was analyzed using a total of 41 marker sets for a total of 12 Asian pumpkin F ₁ and parental lines distributed from Company C. Both F ₁ and parental lines were genetically similar but classified (Figure 8).

The above results can be summarized as follows:

Thirty-eight Asian pumpkin core lines with various characteristics (fruit shape, fruit color, leaf ash and spine, etc.) were selected, DNA was extracted from young leaves, and a GBS library was constructed using ApeKI restriction enzyme. The corresponding base sequence was analyzed using Illumina Nextseq 500.

380 SNPs were selected based on criteria such as sequence reliability (depth), polymorphism information content (PIC), homozygosity, separation ratio, and SNP proximity, and 48 SNP primers (or probes) customized to the KASP ^TM genotyping analysis system. A set was developed.

The developed marker set was applied to 90 of 95 resources. It was applied to 33 of the 38 core lines, and as a result of testing on a total of 45 breeding lines such as zucchini and rootstock (oriental) used in breeding at seed companies, as well as 12 Asian F ₁ and parent lines, out of a total of 48 It was confirmed that 41 primers (or probes) function as molecular markers.

The 41 marker sets whose effectiveness was ultimately tested can be used to test the purity of seeds and identify varieties because they can test the genotype for each breeding line.

So far, the present invention has been examined focusing on its preferred embodiments. A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.

<110> Republic of Korea (Management: Rural Development Administration) <120> SNP MARKER SET FOR IDENTIFYING CUCURBITA MOSCHATA CULTIVARS AND METHOD FOR IDENTIFYING CUCURBITA MOSCHATA CULTIVARS USING THE SAME <130>RDA-BPN210028 <160> 41 <170> KoPatentIn 3.0 <210> 1 <211> 121 <212> DNA <213> Artificial Sequence <220> <223> CmoSPT01 <400> 1 tctgatggca acgaacattc ttcagccgac agtttacaag ccgtcggtga agatcgctgc 60 yaaggtaatc gggcgtaagt gggcaccgtt gccggcggtg atggcgacgt ttttagtatc 120 g 121 <210> 2 <211> 121 <212> DNA <213> Artificial Sequence <220> <223> CmoSPT02 <400> 2 atctcctacc ccgattaggc aaaaaggatc cgttgcttag gaatgtcgat ttattcatat 60 wattgcaact ttttcttcag ggggcctgta ttttcaacct ctttcatgga gccttcgctt 120 g 121 <210> 3 <211> 121 <212> DNA <213> Artificial Sequence <220> <223> CmoSPT3 <400> 3 aaagctggag gctctggagt tcccccagtg ctgcgttgta gtgtgtgtta ttatatgggt 60 ytcttataac cctttctttt ttggatgaac tgaattcctc tcatggagga gttatgatga 120 t 121 <210> 4 <211> 121 <212> DNA <213> Artificial Sequence <220> <223> CmoSPT4 <400> 4 tcccctcgat gttccaccgc tgccttccgt tcatctccat ccctctctta tctcttcctc 60 wctctcagaa ggaatctatg cgaatttata ggaatgcagg cggagacgac gacggaaccg 120 g 121 <210> 5 <211> 121 <212> DNA <213> Artificial Sequence <220> <223> CmoSPT5 <400> 5 ctatcctaga tccccgcgct ccaccagcgg atctgacgga accaagcagc agagctactg 60 kaaagaagac gaactccaat tcaaaaatcg agacaataca acggagaagc aattcaaata 120 t 121 <210> 6 <211> 121 <212> DNA <213> Artificial Sequence <220> <223> CmoSPT6 <400> 6 tatctttggg gaagccaaga ttgaagattt gagctcccaa ctacaaaccc aagctgcaga 60 rcagttcaag gcacctaatt tgagtaactt gacatcaaag cctgagccat caaccgtggt 120 t 121 <210> 7 <211> 121 <212> DNA <213> Artificial Sequence <220> <223> CmoSPT7 <400> 7 atgtccatcc cttttacctt attgtactcg agtgacgaga aaaaaacagt aaaaaaaaagg 60 racttttgct tcatagcttt tctcgtggtg ccttcgagga gtgaattatg ggtacccttt 120 a 121 <210> 8 <211> 121 <212> DNA <213> Artificial Sequence <220> <223> CmoSPT8 <400> 8 ttatgggctt ggctctgtat cgttggttgg ggcaccataa cagattgata tgaatggtta 60 ktgggagcag gatgcggctg catagacatg aaaacattag gatctgaacc aatggccaac 120 g 121 <210> 9 <211> 121 <212> DNA <213> Artificial Sequence <220> <223>CmoSPT9 <400> 9 ctggccgttc gtcacgatcg cgctcaaaat actctcaata cgctccacca gttcctcatc 60 racgccctcc gctgcctcca atacgccatc aatcgactgg agaaactcca cattcgaatc 120 g 121 <210> 10 <211> 121 <212> DNA <213> Artificial Sequence <220> <223>CmoSPT10 <400> 10 gcgaatgagc tagtgcagaa gtttcatata gcaatggaga ctgtttctga aaacattaat 60 wgcagtgaag ctggagataa atttacccag cagcataaag aacttctgtc tgttctcgat 120 c 121 <210> 11 <211> 121 <212> DNA <213> Artificial Sequence <220> <223> CmoSPT11 <400> 11 tttaagatgc tttgttgggt cactgaagtc aaacaaaacg cagcatagat ttagcttctt 60 racaaacaaa ttctgttttt cagagcttgg aacttcccta aacccaggca atgcctcata 120 t 121 <210> 12 <211> 121 <212> DNA <213> Artificial Sequence <220> <223> CmoSPT12 <400> 12 ttatgagtcg gtatgattct gctagccaga gtcacttttc acttctcttt agatcagtgc 60 wgcagacaat aatgttctag tcattagtag ggtagaaact gatgtctcca attttttttt 120 t 121 <210> 13 <211> 121 <212> DNA <213> Artificial Sequence <220> <223> CmoSPT13 <400> 13 cagaagtgct tcagtaaaatc ttggctagcc agggttgaaa cgtggttctt caccccgatc 60 rtaatcgacc ccaagagaaa cgtgcctcat catctgctcg cagcttacag ccatccttca 120 a 121 <210> 14 <211> 121 <212> DNA <213> Artificial Sequence <220> <223> CmoSPT14 <400> 14 tttgatgact ttgctggctt cttgagttac tgggtcgaca agatagtcag gcacgtcacg 60 yagatgagga gcagggggct tcttgctgag aatcttatca tgtttcaaca aatctgccca 120 a 121 <210> 15 <211> 121 <212> DNA <213> Artificial Sequence <220> <223> CmoSPT15 <400> 15 cccaccgagt aaacaaaagaa caatgagtga cagaccaatc cactggtggc cttagcagct 60 magaagcccc ctcgggcccc aacggaggaa gtgggtacag agaaggaaag gccaacgcca 120 a 121 <210> 16 <211> 121 <212> DNA <213> Artificial Sequence <220> <223> CmoSPT16 <400> 16 ttcttatact catgtagctg cactgaggaa ggagatctca gacgagactg atagagagac 60 mgggcgcagt aaacaaattt caagtgttcc aatccatctg agtatctact ctcccaatgg 120 t 121 <210> 17 <211> 121 <212> DNA <213> Artificial Sequence <220> <223> CmoSPT17 <400> 17 tgactttgat gagtctagct gtaggcttaa tggtcgttgc acttgttaca aaggatctaa 60 ygaggaacag aaatgctgca acttgagcca tcacagaatt tcttgtgcag ctacggttag 120 g 121 <210> 18 <211> 121 <212> DNA <213> Artificial Sequence <220> <223> CmoSPT18 <400> 18 tataaatccc tttaccccta agttttgggt actgacctct tgctgcatga tgatcatcct 60 rgtcggttgt tgcgaacaaa aggcacgaga atggtgacgg ttggacgggg atggaacgaa 120 a 121 <210> 19 <211> 121 <212> DNA <213> Artificial Sequence <220> <223>CmoSPT19 <400> 19 gatgaacagg gttggctttt cttaaccact tgaattctgt atctaagtaa tccttactgt 60 ygttttgttt cttgtttaca tctatgattt gtttctttgt ttcaattggt ggaatcttct 120 g 121 <210> 20 <211> 121 <212> DNA <213> Artificial Sequence <220> <223> CmoSPT20 <400> 20 ttcaggtcca gaaacctcct gatttctggg acttgttttt gaatttgaat tgttgctctg 60 ytttttaatc cactgatcaa atgagatctt ttatgcccac caagtgcttg acctgatgcg 120 a 121 <210> 21 <211> 121 <212> DNA <213> Artificial Sequence <220> <223> CmoSPT21 <400> 21 tttttcaaga ttgaaatcaa tctcaatctc atggttgtct tctataggaa gcacggatgc 60 mactcctcca gttgatttgt ttttgtagtc ggatcttgct ttaagcgtgg ctgctccttt 120 c 121 <210> 22 <211> 121 <212> DNA <213> Artificial Sequence <220> <223>CmoSPT22 <400> 22 attgttaact gtatgtgaga ttcactgttc agcacagaat agaatctatg gaacggaaaa 60 ygattggtat gggattcttc aaattgatca atcagcagac gaaacaatta taaaaaagca 120 a 121 <210> 23 <211> 121 <212> DNA <213> Artificial Sequence <220> <223>CmoSPT23 <400> 23 catagacagt gacaatgagt ttaagaaatc ggattctcat gggcatatcg tcgacgcgtt 60 yggggcgtca atggtagagc cttggcttgc agcccatgga gacccaacaa ggttttcatt 120 t 121 <210> 24 <211> 121 <212> DNA <213> Artificial Sequence <220> <223>CmoSPT24 <400> 24 gactgggcct tcaacggtat cgttgctacc accgggagcc gcggccccgt cggtggtgtt 60 yccaatcttg ccatcggcgc cgggagcctc ggttttatta gcaccggcct tgggagtttc 120 a 121 <210> 25 <211> 121 <212> DNA <213> Artificial Sequence <220> <223> CmoSPT25 <400> 25 cttcccacac ggcggagcca tggactcaat ggatcatggg gtttacagcg ggggtggaag 60 yggagacggg tacggcggga acttcctgat ttcgatggcg gccggggcgg aagccgggag 120 c 121 <210> 26 <211> 121 <212> DNA <213> Artificial Sequence <220> <223>CmoSPT26 <400> 26 caccaaagga gcaattcttc tcaaaatttt aaaaggtact aagttggaat cccattctaa 60 staaagattc ttgtggtaca aggactacaa attaatagga agaaaaaacaa tgttgactac 120 t 121 <210> 27 <211> 121 <212> DNA <213> Artificial Sequence <220> <223>CmoSPT27 <400> 27 ctccttaaga acggacacaa gctcaatatt tgattcttga cttctcttta gctgcagagc 60 kaaatcagca ttggattctt tttggaactt cagttcatct ttcaattcat ctaagatttt 120 t 121 <210> 28 <211> 121 <212> DNA <213> Artificial Sequence <220> <223>CmoSPT28 <400> 28 taactttgaa aggtccgaaa agttgcagct ccgaaagcaa ttcgaatcag atttttattc 60 racttttggt ttttcttacg aagtagtcgg gtcgggaggt ttacagtgtt tttaacaaca 120 g 121 <210> 29 <211> 121 <212> DNA <213> Artificial Sequence <220> <223>CmoSPT29 <400> 29 acatttctta cccgaaattg cagcatttaa ggccggtctt cttctgctcc cacaacagtt 60 kacaccgcc gtagatatcg atgcggcggc aacggtagac ctggtgatgc tggttccggt 120 g 121 <210> 30 <211> 121 <212> DNA <213> Artificial Sequence <220> <223>CmoSPT30 <400>30 tcgagctgct tgttctcgca agcgatggac tttgggacgt ggtacgcaac gaggtatgct 60 sactacatca ttacccccatg tatctaagat gctctaaatt gtgaactcca catacgaact 120 t 121 <210> 31 <211> 121 <212> DNA <213> Artificial Sequence <220> <223>CmoSPT31 <400> 31 atcatcttcg gccgaaaact cctctgtttc gtcgcagacg atatcggtgt cgtcaaattc 60 mccgctgcca agctgacaat gggggagtca ttagtaatgt cgataagcgc cgatcgatct 120 t 121 <210> 32 <211> 121 <212> DNA <213> Artificial Sequence <220> <223>CmoSPT32 <400> 32 tgacgttttc actgcagatt tcaattccag taaaaaccag tgccaatttt cctccgtctc 60 sgcatctaca actgcaaaag ctgctggaaa tatgccatct tccccatcca cagctgtggc 120 a 121 <210> 33 <211> 121 <212> DNA <213> Artificial Sequence <220> <223>CmoSPT33 <400> 33 attaaattca cattcgagag gagtggaagc agccgtagga ttgaagcaag ggtaggatag 60 kaaattaaga caaaagtgaa gggcaggggt cagtcgcacg tgtgaaaaag attgaagtca 120 c 121 <210> 34 <211> 121 <212> DNA <213> Artificial Sequence <220> <223>CmoSPT34 <400> 34 agattcagat ttacaggtga tgaaggagaa attaaaccct gcaatcttcc agggaaacgg 60 rgaggttgaa ggtaagaaaa tcagtgggag aagtaacggg gaaagtttct cggtgtcgaa 120 g 121 <210> 35 <211> 121 <212> DNA <213> Artificial Sequence <220> <223>CmoSPT35 <400> 35 acagatgccc ttcttcattg acatttgtca gtctgtagaa agatagccaa gcatgacagc 60 yttggataaa tttggtgact aaactgtata aaatctttaa aaagtccttt agtcttacat 120 t 121 <210> 36 <211> 121 <212> DNA <213> Artificial Sequence <220> <223>CmoSPT36 <400> 36 ctctgacatt tttcttctca gctgcggctc atcctccagt cccaccttct ttaaccgcac 60 yttcgtcggc gattcagtca aacccgcctc cgactttctc actgccggcc gatcggtggc 120 g 121 <210> 37 <211> 121 <212> DNA <213> Artificial Sequence <220> <223>CmoSPT37 <400> 37 tcgtagagct acaagaacga agatgatgat tcaatgtttt ttgacatttg gcttctgaac 60 ktttaaatgc ttgctctgtc tttggttttt gtagttacag atctttatat tgttcatcga 120 a 121 <210> 38 <211> 121 <212> DNA <213> Artificial Sequence <220> <223>CmoSPT38 <400> 38 tatatacact catccatcat atacactcat ccatcgccct ctcgctctct ctcgtgcgtt 60 ktgtatgtac aggaagattg taacagtgga agagatctta acaattgaca taaaaccagg 120 t 121 <210> 39 <211> 121 <212> DNA <213> Artificial Sequence <220> <223>CmoSPT39 <400> 39 gttggagcag ctctttgaca gctccctcgt gggttgagta ttactcatct atttgtagct 60 ytccacttat caatggatat tttctcgttt cttatctaaa atatttgccg tcaacttatc 120 c 121 <210> 40 <211> 121 <212> DNA <213> Artificial Sequence <220> <223>CmoSPT40 <400> 40 acagaatcaa acagtctcta tgagacttat aatcaccact catatggttc ttcaccggcc 60 sattcgttct tcgatggagt ttcatcgcca gatttctcca ctccaaacat ggctgattca 120 a 121 <210> 41 <211> 121 <212> DNA <213> Artificial Sequence <220> <223>CmoSPT41 <400> 41 tcatttactg gtgatcgatc atcattgata tttgaacagc acgtacctgg agagagtgtc 60 ratgagctga aaaagaaaat tcagtcacag gtgattttaa catgcttcaa tttgtgttat 120 c 121

Claims (6)

A composition for purity testing and variety identification of Asian pumpkin, comprising an agent for detecting all single nucleotide polymorphisms (SNPs) located at the 61st base of each base sequence in the base sequences of SEQ ID NOs: 1 to 41.
According to claim 1,
The agent contains a polynucleotide consisting of 5 to 121 consecutive bases including a single nucleotide polymorphism site located at the 61st base of each base sequence in SEQ ID NOs. 1 to 41, or a base sequence complementary thereto. Composition for purity testing and variety identification of pumpkin.
According to claim 1,
A composition for purity testing and variety identification of Asian pumpkin, wherein the agent is any one of a probe, a primer, and a combination thereof.
A kit for purity testing and variety identification of Asian pumpkin, comprising the composition of claim 1.
The kit for purity testing and variety identification of Asian pumpkin according to claim 4, wherein the kit is a PCR kit, microarray, or KASP kit.
a) isolating a polynucleotide from an Asian pumpkin sample and determining the genotype of all single nucleotide polymorphisms present at the 61st position of each base sequence represented by SEQ ID NOs: 1 to 41; and
b) A purity test and variety identification method for Asian pumpkins, comprising the step of identifying the purity and variety of Asian pumpkins by analyzing the genotype of the single nucleotide polymorphism.