KR102572418B1 - Single nucleotide polymorphism (SNP) marker composition for predicting or diagnosing onion gray mold resistance trait and method for prediction or diagnosis using the same - Google Patents

Abstract

A polynucleotide consisting of 5 to 400 consecutive nucleotides containing a 297th single nucleotide polymorphism (SNP) nucleotide marker of the nucleotide sequence represented by SEQ ID NO: 1 or a complementary polynucleotide containing onion gray mold resistance trait diagnosis or A predictive marker composition is provided.

Description

Single nucleotide polymorphism (SNP) marker composition for predicting or diagnosing onion gray mold resistance trait and method for prediction or diagnosis using same using the same}

The present invention relates to a single nucleotide polymorphism marker composition for predicting or diagnosing onion gray mold resistance traits and a prediction or diagnosis method using the same, and more particularly, to the 297th SNP (single nucleotide polymorphism) of the nucleotide sequence represented by SEQ ID NO: 1 ) A composition for predicting or diagnosing onion gray mold resistance traits using a primer set or a probe set that specifically binds to a polynucleotide consisting of 5 to 400 consecutive nucleotides including a nucleotide marker or a polynucleotide complementary thereto, and a composition for predicting or diagnosing the onion gray mold resistance trait It relates to the predictive or diagnostic method used.

Onion ( Allium cepa L) is one of the major vegetable crops consumed worldwide. However, the growth cycle of one generation of onion is not only 2 years, but because it is an omnivorous crop, the breeding period is long compared to other crops, and the size of the genome is as large as 16 Gb, and breeding is difficult due to its complexity. Therefore, for onion breeding, the seed market is monopolized by multinational companies in developed countries that have excellent breeding technology and biotechnology capable of accessing molecular genetics of breeding. In particular, the direction of onion breeding, especially in China, is changing from fixed species to fostering F1 varieties with high purity and good utilization, and multinational companies and advanced countries such as Japan are encroaching on the market. To prepare for market encroachment and monopoly by multinational companies and developed countries, securing genetic resources of onions with various breeding traits distributed in Korea and breeding using molecular genetic analysis of the genetic resources at the genome level Securing capacity is essential for stabilizing the domestic onion market and breeding new varieties. Therefore, it is necessary to analyze the genetic characteristics in a more effective and objective way to more easily distinguish the desired breeding traits in the onion breeding stage.

Methods for testing the varieties of breeding materials in the breeding process are largely divided into a method of testing morphological characteristics through cultivation tests in breeding fields and an assay method using molecular markers based on DNA obtained by extracting DNA from breeding materials. can However, in the case of onion, which is an omnivorous crop with a growth cycle of 2 years per generation, there are difficulties in investigating morphological characteristics during the breeding process. Therefore, the construction of a genome database for each onion variety through whole genome sequencing or RNA sequencing analysis and the shortening of the breeding period through the selection of molecular markers for the classification of various breeding traits using the database are expected to compete for seed sovereignty with multinational companies and developed countries and breed varieties with various traits. It is essential for breeding new breeds that can respond quickly to the market where demand for

Molecular markers developed using a database secured based on molecular genetic analysis can shorten the breeding period through early selection and systematic analysis of breeding materials with desired traits in the breeding stage, as well as reduce the time required in the breeding process. Labor and costs can also be reduced. Molecular markers typically include random amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), simple sequence repeat (SSR), and single nucleotide polymorphism (SNP). In the International Union for the Protection of New Varieties of Plants (UPOV), which presents guidelines for the establishment of databases for each variety by molecular markers, among several molecular markers, SSR or The use of SNP markers is recommended (UPOV, 2010).

Currently, there is a high resolution melting (HRM) curve analysis method using a real-time polymerase chain reaction for mutation analysis using a SNP marker based on one nucleotide sequence polymorphism between two comparison groups. presented (Wilhelm et al., 2003, Anal. Biochem. 317:218-225). This method is used by inserting a fluorescent dye such as 'LCGreen' or 'EvaGreen', which is constantly inserted into the SNP region during chain polymerization, instead of 'SYBR green', which is randomly inserted between the DNA double helix structures. Thereafter, in order to analyze the homology of the amplification product, after the completion of the chain polymerization reaction, the temperature is slowly raised at a constant rate, and the change in the melting temperature (Tm) of the amplified product is measured to a difference of 0.5 ° C or less. The shape of the dissociation curve of the amplification product in the genetic mutation region is influenced by differences in the GC ratio, length, and nucleotide sequence of the amplification product. Polymorphisms by breed can be analyzed by comparing the similarity values of dissociation curve shapes between individuals. In addition, this method is used for SNP mutation analysis because it can detect even changes in a single nucleotide sequence. However, since the real-time polymerase chain reaction, which is essential for high resolution melting (HRM) curve analysis, is expensive equipment, there are cost difficulties in SNP marker analysis.

The main object of the present invention is to provide a SNP molecular marker composition that can easily diagnose the presence or absence of gray mold resistance traits in onion, and to use the SNP molecular marker for breeding of onion varieties resistant to gray mold disease.

Another object of the present invention is to identify SNPs (Single Nucleotide Polymorphism Sites) related to onion gray mold resistance traits, and based on this, to identify the SNPs through AS PCR (Allele-Specific PCR) primers to determine gray mold resistance traits of onions It is to provide a method for easily diagnosing the presence or absence.

In order to solve the above problems, the present invention is a polynucleotide consisting of 5 to 400 consecutive nucleotides containing a 297th single nucleotide polymorphism (SNP) nucleotide marker of the nucleotide sequence represented by SEQ ID NO: 1 or a polynucleotide complementary thereto It provides a marker composition for diagnosing or predicting onion gray mold resistance trait comprising.

In one embodiment of the present invention, the single nucleotide polymorphism (SNP) nucleotide is adenine (A) specifically present in gray mold resistant onion lines or cytosine (C) specifically present in gray mold susceptible onion lines ), and the present invention also provides a primer pair for diagnosing or predicting onion gray mold resistance traits for amplifying the marker composition for diagnosing or predicting onion gray mold resistance traits described above.

In one embodiment of the present invention, the primer pair consists of a 5' primer of SEQ ID NO: 3 and a 3' primer of SEQ ID NO: 4, and is for AS-PCR (Allele-specific PCR) analysis.

The present invention also has a nucleotide sequence of any one of (a) to (c) below and can be used as a primer or probe for detecting a single nucleotide polymorphism (SNP) for selecting gray mold resistant onion It provides a polynucleotide that does. (a) a base sequence consisting of 5 to 400 consecutive nucleotides including the 297th SNP nucleotide of the base sequence represented by SEQ ID NO: 1; (b) a nucleotide sequence complementary to the nucleotide sequence of (a); (c) In the nucleotide sequence of (a) or (b), a nucleotide sequence in which one to several bases other than the SNP are deleted, substituted, or added, and a polynucleotide containing the nucleotide sequence is ) or (b) a base sequence capable of hybridizing with a polynucleotide containing the base sequence under stringent conditions.

The present invention comprises the steps of (a) isolating nucleic acid molecules from onions; (b) confirming the base type of a single nucleotide polymorphism (SNP) corresponding to the 297th nucleotide of SEQ ID NO: 1 in the isolated nucleic acid molecule; and (c) checking whether the 297th nucleotide is adenine (A) or cytosine (C), whether or not there is gray mold resistance resistance.

In one embodiment of the present invention, the method for predicting or diagnosing gray mold resistant onion, when the 297th nucleotide is adenine (A), confirms that the onion line is resistant to ?F light mold disease, and the 297th nucleotide is cytosine In the case of (C), it is confirmed that the onion line is susceptible to ?F light mold disease.

The present invention provides a composition containing a SNP marker useful for diagnosing the presence or absence of gray mold resistance traits in onions, and primers or probes for detecting the corresponding SNPs, and using them to determine the presence or absence of gray mold resistance traits in onion breeding lines. Provides a method for diagnosing whether Using the SNP marker of the present invention, it is possible to easily check the presence or absence of gray mold resistance traits in the early stage of growth of onion plants, and based on this, it can be usefully used to improve onion varieties with excellent quality.

1 is the result of confirming the extracted total RNA quality.
Figure 2 shows the results of A / C SNP location confirmation at 297 bp of SEQ ID NO: 1 and SEQ ID NO: 2.
3 is an AS PCR analysis result.

Hereinafter, the preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

Before explaining the present invention in detail, the terms or words used in this specification should not be construed unconditionally in a conventional or dictionary sense, and in order for the inventor of the present invention to explain his/her invention in the best way Concepts of various terms can be appropriately defined and used.

Furthermore, it should be noted that these terms or words should be interpreted as meanings and concepts consistent with the technical idea of the present invention.

That is, the terms used in this specification are only used to describe preferred embodiments of the present invention, and are not intended to specifically limit the contents of the present invention.

It should be noted that these terms are terms defined in consideration of various possibilities of the present invention.

Also, in this specification, a singular expression may include a plurality of expressions unless the context clearly indicates otherwise.

In addition, it should be noted that similarly, even if expressed in a plurality, it may include a singular meaning.

Throughout this specification, when a component is described as "including" another component, it does not exclude any other component, but further includes any other component, unless otherwise stated. It can mean you can do it.

Furthermore, when a component is described as "existing inside or connected to and installed" of another component, this component may be directly connected to or installed in contact with the other component.

In addition, it may be installed at a certain distance, and in the case of being installed at a certain distance, a third component or means for fixing or connecting the corresponding component to another component may exist. .

Meanwhile, it should be noted that the description of the third component or means may be omitted.

On the other hand, when it is described that a certain element is "directly connected" to another element, or is "directly connected", it should be understood that no third element or means exists.

Similarly, other expressions describing the relationship between components, such as "between" and "directly between", or "adjacent to" and "directly adjacent to" have the same meaning. should be interpreted as

In addition, in the present specification, terms such as "one side", "the other side", "one side", "the other side", "first", and "second" refer to one component with respect to another component. It is used to make it clearly distinguishable from the elements.

However, it should be noted that the meaning of a corresponding component is not limitedly used by such a term.

In addition, in this specification, terms related to positions such as “upper”, “lower”, “left”, “right”, etc., if used, are to be understood as indicating relative positions of corresponding components in the drawing.

In addition, unless an absolute location is specified for these locations, these location-related terms should not be understood as referring to an absolute location.

Moreover, in the specification of the present invention, terms such as "... unit", "... unit", "module", and "device", if used, mean a unit capable of processing one or more functions or operations.

It should be noted that this may be implemented in hardware or software, or a combination of hardware and software.

In the drawings accompanying this specification, the size, position, coupling relationship, etc. of each component constituting the present invention is partially exaggerated, reduced, or omitted in order to sufficiently clearly convey the spirit of the present invention or for convenience of explanation. may be described, and therefore the proportions or scale may not be exact.

In addition, in the following description of the present invention, a detailed description of a configuration that is determined to unnecessarily obscure the subject matter of the present invention, for example, a known technology including the prior art, may be omitted.

In order to solve the above problems, the present invention is a high resolution analysis of SNP markers for predicting or diagnosing onion gray mold resistant traits using an expensive real-time polymerase chain reaction. Allele specific PCR (AS) that can easily determine the presence or absence of onion resistance to gray mold disease in a short time by using polymerase chain reaction (PCR) instead of melting (HRM) curve analysis method -PCR) was intended to develop primers, and as a result, an AS-PCR primer pair was developed using a SNP marker that exhibits a specific polymorphism between resistant and susceptible individuals, and the SNP was identified through the AS-PCR primer pair In this case, the present invention was completed by confirming that the presence or absence of gray mold resistance of onions can be easily diagnosed.

Hereinafter, the present invention will be described in more detail by examples. These examples are only for explaining the present invention, it is apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

The present inventors collected the leaves of gray mold resistant phenotypic onion ( Allium cepa L) objects and gray mold resistant phenotypic onion objects provided by Seeds and People, a farming corporation, from the breeding field where gray mold disease occurred.

Transcriptome analysis was performed by isolating the RNA of the collected seeds, and as a result of clustering the onion individuals with a gray mold resistant phenotype and the onion individuals with a gray mold susceptible phenotype, the SNP location was confirmed by comparative analysis of the contrasting transcriptome sequences did The process of extracting DNA from each individual and confirming the SNP was performed by AS PCR (Allele-Specific PCR) to develop a molecular marker related to gray mold resistance trait.

[Example]

Example 1: Confirmation of gray mold resistant phenotype and gray mold susceptible phenotype

Four onion individuals, S&P 7521, S&P 7522, S&P 7129, and S&P 7168, in which gray mold resistance phenotypes were confirmed in the onion breeding field of Seed and People, a farming association, were selected. Dog onion individuals S&P 7483, S&P 7130, and S&P 7175 were selected and used as controls for gray mold resistant phenotype individuals, and the phenotypes of the selected gray mold resistant onions and gray mold susceptible onions were visually confirmed.

Example 2: RNA sequencing of selected onion populations

RNeasy®Kit (QIAGEN, Germany) was used to perform RNA sequencing of the selected gray mold resistant phenotype S&P 7521, S&P 7522, S&P 7129, S&P 7168 and the gray mold susceptible phenotype S&P 7483, S&P 7130, S&P 7175. RNA was extracted using

Before analyzing RNA sequencing, the extracted total RNA quality was confirmed (Table 1, Figure 1).

Sample ID Conc.(ng/ul) A260/280 A260/230 Ratio (28S:18S) RIN S&P 7521 114.79 2.09 2.08 2.1 5.9 S&P 7522 173.19 2.11 2.27 1.9 5.7 S&P 7129 79.72 2.08 1.08 2.0 5.5 S&P 7168 438.08 2.2 2.38 1.8 6.2 S&P 7483 166.45 2.10 1.79 1.9 5.9 S&P 7130 101.18 2.08 2.05 1.9 5.7 S&P 7175 378.40 2.20 2.45 1.9 6.2

A cDNA library was constructed using the isolated RNA, and RNA sequence raw data of each onion lineage was obtained by performing RNA sequencing using the constructed library information and HiSeqX (Illumina, USA). The RNA sequence raw data obtained thereafter were preprocessed using Trimmomatic and Trinity software, and the data obtained through the preprocessing process were mapped to onion reference transcript data. For the data after mapping, protein sequence clustering was performed using the CD-HIT-EST package of CD-HIT software to reduce sequence redundancy and improve analysis performance by clustering biological sequences. Then, using TransDecoder, sequences with more than 100 amino acids in length were extracted from the sequences generated by Trinity, and the sequences of each onion lineage were aligned with HISAT2 using the onion reference transcript data as a reference. Finally, using StringTie software 87,427 transcripts were obtained.

Example 3: Identification of SNP location through comparative analysis of RNA sequence results of gray mold resistant phenotypic onion subjects and gray mold susceptible phenotypic onion subjects

The transcripts data obtained through RNA sequencing of the gray mold resistant phenotypic objects S&P 7521, S&P 7522, S&P 7129, S&P 7168 and the gray mold susceptible phenotypic objects S&P 7483, S&P 7130, and S&P 7175 were analyzed using haplotypeCaller of the Genome Analysis Tool Kit. After producing mutation information using this method, the final raw vcf (variants call file) file was created by integrating the mutation files of 7 individuals. Afterwards, filtering was performed to remove low quality genotypes, and finally, SNPs (Single Nucleotide Polymorphism site, single nucleotide polymorphism) significantly present between gray mold resistant phenotype individuals and gray mold susceptible phenotype individuals were identified. did

As a result of comparing the sequence of SEQ ID NO: 1 in individuals with a gray mold resistant phenotype and the sequences of SEQ ID NO: 2 in individuals with a gray mold susceptible phenotype, the A / C SNP position could be identified at 297 bp of SEQ ID NOs: 1 and SEQ ID NO: 2 (Fig. 2). The identified SNP site was named AS_1833. That is, the single nucleotide polymorphism (SNP) nucleotide is adenine (A) specifically present in gray mold resistant onion lines or cytosine (C) specifically present in gray mold susceptible onion lines, and the corresponding SNP It is predicted to be translated into phenylalanine, one of the amino acids, in gray mold resistant onion lines, but it is predicted to be translated into cysteine, one of the amino acids, in gray mold susceptible onion lines, which is judged to affect gray mold resistance traits. do.

Therefore, the marker composition for diagnosing or predicting onion gray mold resistance traits according to the present invention is a polynucleotide consisting of 5 to 400 consecutive nucleotides including the 297th SNP nucleotide of the nucleotide sequence represented by SEQ ID NO: 1 or its complement A polynucleotide is included, and all primer pairs for amplifying it also fall within the scope of the present invention.

Example 4: Development and verification of markers for selection of gray mold resistant strains of onion through AS PCR analysis

AS (Allele-Specific) PCR analysis was performed to verify SNPs for selection of gray mold resistant strains of onions identified in Example 3 and to develop molecular markers for SNP confirmation.

For As PCR analysis, individuals of S&P 7521, S&P 7522, S&P 7129, and S&P 7168 resistant to gray mold disease and individuals of S&P 7483, S&P 7130 and S&P 7175 susceptible to gray mold disease were selected and analyzed.

The primers used in the AS PCR analysis place the A/C-specific polymorphism at the AS_1833 SNP position at the 3' end of the primer, so that a PCR product with a size of 123 bp is amplified only in gray mold-resistant onion individuals during PCR analysis, resulting in gray color. In order to confirm that the onion was resistant to fungal disease, forward primers (5' primers) and reverse primers (3' primers) specific to onions with a gray mold resistance phenotype were prepared. The prepared primers were named AS_1833_F (SEQ ID NO: 3) and AS_1833_R (SEQ ID NO: 4) and are shown in Table 2.

primer base sequence AS_1833_F (SEQ ID NO: 3) 5'- CAA ACT GTC CTT CTC AAA CCA GAG A - 3' AS_1833_R (SEQ ID NO: 4) 5'-TGC AGC AGA AGG TGT GTA AGA GCA G - 3'

For a total of 7 individuals selected in this way, AS PCR analysis was performed with the primers of SEQ ID NO: 3 and SEQ ID NO: 4 to verify the possibility of using the AS_1833 SNP site as a molecular marker for selecting onion strains with a gray mold resistance phenotype. AS PCR The analysis was performed using Biofact 2X H-Star Taq PCR Pre-Mix, with a composition of 1 ul primer each and 1 ul DNA for each individual, and after pre-denaturation at 95 ° C for 15 minutes through a PCR machine, at 95 ° C for 20 seconds A total of 35 cycles were performed, including denaturation, annealing at 59°C for 30 seconds, and extension at 72°C for 30 seconds. After that, final-extension was performed at 72° C. for 5 minutes.

As a result of the AS PCR analysis, distinct bands could be identified in individuals with gray mold resistance phenotypes S&P 7521, S&P 7522, S&P 7129, and S&P 7168, and bands could be observed in individuals with gray mold susceptibility phenotypes S&P 7483, S&P 7130, and S&P 7175. no (Fig. 3).

Through these results, it was confirmed once again that the AS_1833 SNP site is a molecular marker significantly associated with the onion gray mold resistance trait, and in order to select onion gray mold resistant phenotypic lines through a simple AS PCR method, the above It was confirmed that the primers of SEQ ID NO: 3 to SEQ ID NO: 4 can be usefully utilized.

As described above, the present invention provides a polynucleotide that can be used as a primer or probe for detecting the above-described single nucleotide polymorphism (SNP), and this polynucleotide is the 297th SNP of the nucleotide sequence represented by SEQ ID NO: 1 In the nucleotide sequence consisting of 5 to 400 consecutive nucleotides including nucleotides, the nucleotide sequence complementary to the nucleotide sequence of (a), or the nucleotide sequence of (a) or (b) above, one to several other than SNPs A nucleotide sequence in which a base is deleted, substituted or added, and a polynucleotide comprising the nucleotide sequence can hybridize to a polynucleotide comprising the nucleotide sequence of (a) or (b) under stringent conditions can have

The present invention is not limited by the foregoing embodiments and accompanying drawings. It will be clear to those skilled in the art that the components according to the present invention can be substituted, modified, and changed without departing from the technical spirit of the present invention.

<110> Kyung Hee University Industry Academic Cooperation Foundation <120> Single nucleotide polymorphism (SNP) marker composition for predicting or diagnosing onion gray mold resistance trait and method for prediction or diagnosis using the same <130> K-1001 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 1833 <212> DNA <213> allium cepa <400> 1 ccacaaaaca aaaacaaaaa cgttctattt ctgtttcaat ttgcatttca tttttacaac 60 catcattgta tttacaacct gcactgttac attgcatatt tcactgattc acatttcaag 120 ttgcaatttc tgttcactgt ctgttcacag tgccttcata ccttctctgt tcacaaccac 180 cacatcaaac cattcctctg tcccctgcat aacattcaac taccatcagc ctgcacacat 240 cccttcagac aatccaccct caacttcaag ttcaaactgt ccttctcaaa ccagagaaca 300 tacaactcca ccacccagca agaaacacat cactgaccat catttcaaca tccattgcaa 360 aatcatttct ctgctcttac acaccttctg ctgcaacatc atatccagtg ccttactgtc 420 accatttacc cacttcataa cctctacact tcatatccac caactcaccc ataaacacaa 480 cttcccactg cctattccc ttaccactga gccaaatcat ttacaacttc ctacagcaac 540 atcaaccatt cttcccctat atcttcacag ctccaaacca tcaccgctta acattgcaac 600 acaatacatc acaacccacc atcatcatca acagtcatct ccatctccac tgaaaaactt 660 ccactactgc ccaaaatcca acaaacatca tcagccataa ccctgtaccc aaaccactat 720 ccaatctcca ccccttcata caaagtttta caacacagca gcatcatgtc tctgttcatc 780 caacaaccca ataaacttct ccatttccac agcactactt caacattcac actctcttct 840 gcttctacag ctaccagcct tctgccatcc tcctgaaaca cgactgacca tacaacatca 900 acttccgcaa cagtgcccaa acaactacaa gcaactgcag catcacaact tcactcccct 960 gcctcacttc acaagtacaa agcagctcca ccacatacac aaacgactta catacaacac 1020 ttcatcaaca aacttcaaca tacaattcat ctctactacc ctaccgacca tcaacaacaa 1080 gcagagagcc tcatcatcac tgcaacagca gtaccatcct tcaaacaaat aactactcgc 1140 actgcactgc atacatacca tgtcactgac gtttcatctc cttctacaat caataccagc 1200 aaccttcttc tgacatcccc attattgcct gcattcattc tccatccaac acaacccata 1260 cttctgcctg aacaaaacac tacttctgct cttcaacaac ccaatcactc atacaccctc 1320 accactgtct ctgccaatac atcaacctct gccatctcct tcgacctcac cacgtagcag 1380 tccaagcaaa ccaccaacct tcagccaatc accgaagcaa cccccatcca aagaaatcaa 1440 caatcaatcg aaatcgcgaa gaccatctac caatcctcaa atcgaaccac agcgatcctc 1500 accacaacca gaaatcacag ttcgcatcac ctcattgaac cgcgagcacg caacgcaatc 1560 gcctccatct tctaacccga accactgtca tcaccaaaac actccaattc gaactcactt 1620 gaccaagacg ccgtgcctga agcgaacaaa ctcatccaca cgcgaccact tcgtcccacc 1680 gcagtgaatc gaacagtcgc gaacaagttt ttcaatcttt gaaccaactc gatttgtgaa 1740 caaaaaaaaa taaaaataaa aataaagagt attcgcttgg caattctcaa ctcgattgga 1800 gattttgggaa atttcataat tggggattat ttg 1833 <210> 2 <211> 1833 <212> DNA <213> allium cepa <400> 2 ccacaaaaca aaaacaaaaa cgttctattt ctgtttcaat ttgcatttca tttttacaac 60 catcattgta tttacaacct gcactgttac attgcatatt tcactgattc acatttcaag 120 ttgcaatttc tgttcactgt ctgttcacag tgccttcata ccttctctgt tcacaaccac 180 cacatcaaac cattcctctg tcccctgcat aacattcaac taccatcagc ctgcacacat 240 cccttcagac aatccaccct caacttcaag ttcaaactgt ccttctcaaa ccagagcaca 300 tacaactcca ccacccagca agaaacacat cactgaccat catttcaaca tccattgcaa 360 aatcatttct ctgctcttac acaccttctg ctgcaacatc atatccagtg ccttactgtc 420 accatttacc cacttcataa cctctacact tcatatccac caactcaccc ataaacacaa 480 cttcccactg cctattccc ttaccactga gccaaatcat ttacaacttc ctacagcaac 540 atcaaccatt cttcccctat atcttcacag ctccaaacca tcaccgctta acattgcaac 600 acaatacatc acaacccacc atcatcatca acagtcatct ccatctccac tgaaaaactt 660 ccactactgc ccaaaatcca acaaacatca tcagccataa ccctgtaccc aaaccactat 720 ccaatctcca ccccttcata caaagtttta caacacagca gcatcatgtc tctgttcatc 780 caacaaccca ataaacttct ccatttccac agcactactt caacattcac actctcttct 840 gcttctacag ctaccagcct tctgccatcc tcctgaaaca cgactgacca tacaacatca 900 acttccgcaa cagtgcccaa acaactacaa gcaactgcag catcacaact tcactcccct 960 gcctcacttc acaagtacaa agcagctcca ccacatacac aaacgactta catacaacac 1020 ttcatcaaca aacttcaaca tacaattcat ctctactacc ctaccgacca tcaacaacaa 1080 gcagagagcc tcatcatcac tgcaacagca gtaccatcct tcaaacaaat aactactcgc 1140 actgcactgc atacatacca tgtcactgac gtttcatctc cttctacaat caataccagc 1200 aaccttcttc tgacatcccc attattgcct gcattcattc tccatccaac acaacccata 1260 cttctgcctg aacaaaacac tacttctgct cttcaacaac ccaatcactc atacaccctc 1320 accactgtct ctgccaatac atcaacctct gccatctcct tcgacctcac cacgtagcag 1380 tccaagcaaa ccaccaacct tcagccaatc accgaagcaa cccccatcca aagaaatcaa 1440 caatcaatcg aaatcgcgaa gaccatctac caatcctcaa atcgaaccac agcgatcctc 1500 accacaacca gaaatcacag ttcgcatcac ctcattgaac cgcgagcacg caacgcaatc 1560 gcctccatct tctaacccga accactgtca tcaccaaaac actccaattc gaactcactt 1620 gaccaagacg ccgtgcctga agcgaacaaa ctcatccaca cgcgaccact tcgtcccacc 1680 gcagtgaatc gaacagtcgc gaacaagttt ttcaatcttt gaaccaactc gatttgtgaa 1740 caaaaaaaaa taaaaataaa aataaagagt attcgcttgg caattctcaa ctcgattgga 1800 gattttgggaa atttcataat tggggattat ttg 1833 <210> 3 <211> 25 <212> DNA <213> artificial sequence <220> <223> 5'Primer <400> 3 caaactgtcc ttctcaaacc agaga 25 <210> 4 <211> 25 <212> DNA <213> artificial sequence <220> <223> 3''Primer <400> 4 tgcagcagaa ggtgtgtaag agcag 25

Claims (7)

Onion gray mold resistance trait diagnosis or A marker composition for prediction. According to claim 1,
The single nucleotide polymorphism (SNP) nucleotide is a marker composition for diagnosing or predicting onion gray mold resistance trait, characterized in that adenine (A) present specifically in gray mold resistant onion lines. A pair of primers for diagnosing or predicting onion gray mold resistance traits, for amplifying the marker composition for diagnosing or predicting onion gray mold resistance traits according to claim 1 or 2. According to claim 3,
The primer pair consists of a 5′ primer of SEQ ID NO: 3 and a 3′ primer of SEQ ID NO: 4, and is characterized in that it is for AS-PCR (Allele-specific PCR) analysis. Primers for diagnosing or predicting onion gray mold resistance traits pair. A polynucleotide having the nucleotide sequence of any one of the following (a) to (c) and characterized in that it can be used as a primer or probe for detecting a single nucleotide polymorphism (SNP) for selecting gray mold resistant onion:
(a) a base sequence consisting of 5 to 400 consecutive nucleotides including the 297th SNP nucleotide of the base sequence represented by SEQ ID NO: 1;
(b) a nucleotide sequence complementary to the nucleotide sequence of (a);
(c) In the nucleotide sequence of (a) or (b), a nucleotide sequence in which one to several bases other than the SNP are deleted, substituted, or added, and a polynucleotide containing the nucleotide sequence is ) or (b) a base sequence capable of hybridizing with a polynucleotide containing the base sequence under stringent conditions. A method for predicting or diagnosing gray mold resistant onions comprising the following steps:
(a) isolating nucleic acid molecules from onions;
(b) confirming the base type of a single nucleotide polymorphism (SNP) corresponding to the 297th nucleotide of SEQ ID NO: 1 in the isolated nucleic acid molecule; and
(c) A method for predicting or diagnosing onion gray mold resistance traits comprising the step of determining whether the 297th nucleotide is adenine (A) or cytosine (C), whether or not gray mold resistance is present. The method of claim 6, wherein the method for predicting or diagnosing gray mold resistant onion is, when the 297th nucleotide is adenine (A), it is confirmed that the onion line is F light mold resistant, and the 297th nucleotide is cytosine ( C) A method for predicting or diagnosing onion gray mold resistance traits, characterized by confirming that the onion strain is susceptible to ?F light mold disease.