Method for determining the degree of quercetin content in onion seed plants, method for producing onion seed plants with high quercetin content, onion seed plants with high quercetin content, and molecular markers for quercetin content in onion seed plants.

Abstract

To provide a technique for determining the degree of quercetin content in individual onions.SOLUTION: The method for determining the degree of quercetin content in an onion seed plant comprises a step of determining at least one of a base itself (SNP) corresponding to any of the bases (a) to (d) below, or a continuous polynucleotide containing the base as a molecular marker for the quercetin content in onion seed plants: (a) the 41st base of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 1; (b) the 55th base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 2; (c) the 106th base of the polynucleotide consisting of the base sequence shown in SEQ ID NO:3; and (d) the 73rd base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 4.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for determining the degree of quercetin content in onion plants, a production method for producing onion plants with high quercetin content, onion plants with high quercetin content, and molecular markers related to quercetin content in onion plants.

Background Art

[0002] In recent years, quercetin has attracted attention as a functional component contained in onions. It has been clarified that quercetin has the effect of maintaining cognitive function and preventing dementia. As a conventional breeding method for varieties rich in quercetin, the following methods are known. For example, according to the technique disclosed in Non-Patent Document 1, onions are actually cultivated, and a part of the harvested bulbs is sampled to measure the quercetin content. Thereby, individuals with high quercetin content are selected, and they are systematized and bred as varieties. Regarding such measurement of quercetin content, Non-Patent Document 1 describes that the sample of the collected bulbs is freeze-dried, powdered, dissolved in a solvent, and the extract is subjected to quantitative analysis by high performance liquid chromatography (HPLC) or a simple evaluation method for absorbance measurement.

Prior Art Documents

Non-Patent Documents

[0003]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] However, conventional techniques, such as those described in Non-Patent Document 1, cannot identify individuals with high quercetin content until the onions are actually cultivated in the field and harvested. Since onions are a biennial crop, the labor and time costs involved in cultivation and measuring quercetin content are considerable. Furthermore, because onions exhibit strong self-pollination depression, the parent lines of varieties are maintained through mass seed collection and are not genetically fixed, resulting in large variations in quercetin content even among individuals of the same variety. Moreover, quercetin content is easily affected by cultivation environment factors such as field inconsistencies. Therefore, it is difficult to stably and efficiently breed and select onion varieties with high quercetin content using methods that involve sampling cultivated individuals.

[0005] One aspect of the present invention aims to provide a technique for determining the degree of quercetin content in individual onions, in view of the above-mentioned problems. [Means for solving the problem]

[0006] A method for determining the degree of quercetin content in an onion seed plant according to one aspect of the present invention is to determine in the onion seed plant whether the base itself (SNP) corresponds to any of the following bases (a) to (d), or a sequence of polynucleotides containing said base: (a) The 41st base of a polynucleotide consisting of the base sequence shown in Sequence ID No. 1; (b) The 55th base of a polynucleotide consisting of the base sequence shown in Sequence ID No. 2; (c) The 106th base of a polynucleotide consisting of the base sequence shown in Sequence ID No. 3; and (d) The 73rd base of a polynucleotide consisting of the base sequence shown in Sequence ID No. 4; The process includes determining at least one of the following as a molecular marker for quercetin content in onion plants.

[0007] A method for producing quercetin-rich onion seed plants according to one aspect of the present invention includes a crossbreeding step of crossbreeding onion seed plants with each other, and a discrimination step of identifying quercetin-rich onion seed plants from the onion seed plants obtained in the crossbreeding step or their progeny lines by the method described in any one of claims 1 to 3.

[0008] An onion plant with a high quercetin content according to one aspect of the present invention can be obtained by the production method according to one aspect of the present invention described above.

[0009] In one aspect of the present invention, the molecular marker for quercetin content in onion seed plants is either the base itself (SNP) corresponding to the bases listed below (a) to (d), or a sequence of polynucleotides containing said base: (a) The 41st base of a polynucleotide consisting of the base sequence shown in Sequence ID No. 1; (b) The 55th base of a polynucleotide consisting of the base sequence shown in Sequence ID No. 2; (c) The 106th base of a polynucleotide consisting of the base sequence shown in Sequence ID No. 3; and (d) The 73rd base of a polynucleotide consisting of the base sequence shown in Sequence ID No. 4; That is the case. [Effects of the Invention]

[0010] According to one aspect of the present invention, a technique for determining the degree of quercetin content in individual onions can be provided. [Brief explanation of the drawing]

[0011] [Figure 1] This is a diagram showing the chain map created in Example 1. [Figure 2] This figure shows the results of the QTL analysis in Example 1. [Figure 3] This figure shows the quercetin content for each genotype of each marker, as measured in Example 1. [Figure 4] This figure shows the sequence analysis results in Example 1. [Figure 5] This figure shows the quercetin content for each genotype of CHR6_176431815, as measured in Example 2. [Figure 6] This figure shows the results of agarose gel electrophoresis in Example 3. [Figure 7] This figure shows the results of agarose gel electrophoresis in Example 4. [Modes for carrying out the invention]

[0012] The present invention will now be described in detail. All references cited herein are incorporated herein by reference. Unless otherwise specified herein, "A to B" representing a numerical range means "greater than or equal to A (including A and greater than A), and less than or equal to B (including B and less than B)."

[0013] As used herein, the term “polynucleotide” is interchangeable with “nucleic acid” or “nucleic acid molecule,” and refers to a polymer of nucleotides. Here, nucleic acid may exist in the form of DNA (e.g., cDNA or genomic DNA) or RNA (e.g., mRNA). DNA or RNA may be double-stranded or single-stranded. Single-stranded DNA or RNA may be a coding strand (sense strand) or a non-coding strand (antisense strand). As used herein, base notation shall be in the single-letter notation as defined by IUPAC and IUB, as appropriate.

[0014] One aspect of the present invention relates to a technique for determining the level of quercetin content in onion species plants. One aspect of the present invention can be used to determine individuals with a high quercetin content in the bulbs of onion species plants. One aspect of the present invention determines an onion species plant individual based on the quercetin content by determining the genotype of a gene involved in the determination of quercetin content present in the genome of the onion species plant. One aspect of the present invention can also be used to determine a variety of onions with a high quercetin content. In one aspect of the present invention, the concept of quercetin content includes the degree of content indicating that the amount of quercetin contained in an onion species plant individual is relatively large or small compared to other onion species plant individuals.

[0015] In one aspect of the present invention, onion species plants include onions (Allium cepa) and shallots, which are variants of onions. The onions can be yellow onions and red onions containing quercetin. Examples of onion varieties include "Kuer Gold", "Keltama", "Sarasa Gold", "Sarasa Red", "Turbo", "Kitamomiji 2000", "Momiji No. 3", "Okhotsk 222", etc.

[0016] Onion species plants can be candidate plants for breeding materials or plants obtained in the breeding process. Candidate plants for breeding materials include, for example, parent plants used for crossing and plants used for molecular breeding using genetic recombination techniques. Plants obtained in the breeding process include, for example, plants obtained by intra-specific crossing of plants belonging to the above-mentioned varieties of onion species plants and their progeny lines. Also, onion species plants may be plants obtained by inter-specific crossing, such as crossing between onion species plants belonging to one variety and onion species plants belonging to another variety, and their progeny lines. Furthermore, onion species plants may be plants obtained by inter-specific crossing between varieties known to have a high quercetin content and their progeny lines. Also, onion species plants may be plants obtained by intra-specific crossing between individuals known to have a high quercetin content and their progeny lines.

[0017] In this specification, a plant may be part or all of a plant body. Examples of part of a plant body include propagation materials (seeds, bulbs, etc.), scale leaves, flowers, roots, and the like.

[0018] Quercetin contained in onion plants is a type of polyphenol and is classified as a flavonol among flavonoids. Quercetin has the effect of maintaining cognitive function and preventing dementia through human intervention tests and has attracted attention as a useful component. Thus, onion plants with high quercetin content have high commercial value. Therefore, it would be very beneficial if onion plants with high quercetin content could be easily selected.

[0019] Also, the quercetin content can vary depending on the variety of onion. "Kuer Gold" is a variety with high quercetin content and is suitable for cultivation in Hokkaido. Therefore, the breeding of onion varieties with high quercetin content and suitable for cultivation south of Tohoku is desired. Furthermore, since onions show strong self-incompatibility, the parental lines of the varieties are maintained by mass seeding and are difficult to be completely genetically fixed. Therefore, it is desired to stably and efficiently breed and select varieties with high quercetin content. Also, since the quercetin content of onions is easily affected by cultivation environments such as field unevenness, the variation in quercetin content among individuals of the same variety becomes large. Therefore, it is desired that individuals with high quercetin content can be easily selected.

[0020] 〔Molecular markers related to quercetin content in onion plants〕 The molecular marker according to one aspect of the present invention is a molecular marker related to the quercetin content in onion plants. The molecular marker is either the base itself (SNP) corresponding to the bases of the following (a) to (d) or a continuous polynucleotide containing the base: (a) The 41st base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 1; (b) The 55th base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 2; (c) The 106th base of a polynucleotide consisting of the base sequence shown in Sequence ID No. 3; and (d) The 73rd base of a polynucleotide consisting of the base sequence shown in Sequence ID No. 4; That is the case.

[0021] Molecular markers can be used to identify the gene locus on chromosome 6 of onion plants that is related to quercetin content. A gene locus related to quercetin content refers to a quantitative trait locus or gene region that affects quercetin content. The quantitative trait locus (QTL) generally refers to a chromosomal region involved in the expression of a quantitative trait. QTLs can be defined using molecular markers that indicate a specific locus on a chromosome. Techniques for defining QTLs using such molecular markers are well known in the art.

[0022] Molecular markers according to one aspect of the present invention include, for example, SNP markers, AFLP (Amplified Fragment Length Polymorphism) markers, RFLP markers, microsatellite markers, SCAR markers, and CAPS markers.

[0023] The bases corresponding to the bases described in (a) to (d) above, or the consecutive polynucleotides containing said bases, are the SNP markers described in this embodiment or SNP markers that can be considered identical thereto. The SNP marker may be (i) the base corresponding to the SNP, (ii) a consecutive polynucleotide containing the SNP, or (iii) a consecutive polynucleotide containing two or more SNPs.

[0024] (i: SNP marker) Using a molecular marker according to one aspect of the present invention, the quercetin content in onion plants can be determined.

[0025] Here, SNP refers to a DNA polymorphism in which a single nucleotide variant is observed within a specific region of the DNA base sequence. In the context of SNP markers, SNPs are single nucleotide polymorphisms based on the genome sequence of the onion (Allium cepa). The genome sequence of the onion, the reference plant (Onion genome sequence v1.2), is published in the genome database (https: / / www.oniongenome.wur.nl).

[0026] "(a) to (d) bases" refers to the SNP markers described in this embodiment. "(a) to (d) bases equivalent to the SNP markers described in this embodiment" refers to SNP markers that can be identified with the SNP markers described in this embodiment. "(a) to (d) bases" are mutations at the locus related to quercetin content on chromosome 6. The locus related to quercetin content on chromosome 6 consists of a base sequence derived from a reference onion plant. In other onion plants, there may be regions with different base sequences from the reference base sequence, even in parts other than SNPs. Since the genomic region where this SNP marker is located is conserved among many onion plants, this SNP marker can be identified by methods such as homology searches.

[0027] In other onion species, if there is a locus on chromosome 6 related to quercetin content (i.e., a locus that is highly conserved among plants), then "bases corresponding to (a) to (d)" refers to bases on the locus on chromosome 6 related to quercetin content that have been determined to correspond to (a) to (d) by methods such as homology searches. For example, the polynucleotides described in (a2) to (d2) and (a3) ​​to (d3) below are examples of loci on chromosome 6 related to quercetin content.

[0028] A molecular marker according to one aspect of the present invention is an SNP marker comprising the above-mentioned (a) to (d) SNPs. This SNP marker is a newly identified SNP marker by the present inventors, and those skilled in the art can identify the genomic location of this SNP marker based on the base sequence representing each SNP.

[0029] The SNP in (a) (hereinafter also referred to as SNP(a)) indicates a polymorphism of the 41st base or a corresponding base in the polynucleotide consisting of the base sequence shown in Sequence ID No. 1. The SNP in (b) (hereinafter also referred to as SNP(b)) indicates a polymorphism of the 55th base or a corresponding base in the polynucleotide consisting of the base sequence shown in Sequence ID No. 2. Furthermore, the SNP in (c) (hereinafter also referred to as SNP(c)) indicates a polymorphism of the 106th base or a corresponding base in the polynucleotide consisting of the base sequence shown in Sequence ID No. 3. Furthermore, the SNP in (d) (hereinafter also referred to as SNP(d)) indicates a polymorphism of the 73rd base or a corresponding base in the polynucleotide consisting of the base sequence shown in Sequence ID No. 4. Note that SNP(a), SNP(b), SNP(c), and SNP(d) correspond to CHR6_176431815, CHR6_164337315, CHR6_173524136, and CHR6_171219319, respectively, in the examples described later.

[0030] According to one aspect of the present invention, a molecular marker can be used to determine that an onion plant has a high quercetin content when it is homozygous for an allele in which SNP(a) is G, SNP(b) is T, SNP(c) is T, or SNP(d) is C. Alternatively, the molecular marker may be used to determine the quercetin content in an onion plant by analyzing at least two of SNP(a) to (d) as a haplotype block.

[0031] Here, when we say that onion plants have a high quercetin content, we mean that the amount of quercetin contained in onion plants is relatively high compared to other onion plant individuals.

[0032] (ii: Polynucleotides containing SNPs) A molecular marker according to one aspect of the present invention may be a continuous polynucleotide containing SNP(a) (hereinafter also referred to as polynucleotide(a)), a continuous polynucleotide containing SNP(b) (hereinafter also referred to as polynucleotide(b)), a continuous polynucleotide containing SNP(c) (hereinafter also referred to as polynucleotide(c)), or a continuous polynucleotide containing SNP(d) (hereinafter also referred to as polynucleotide(d)).

[0033] Polynucleotide (a) is a polynucleotide consisting of (a1) a region containing SNP(a) in the base sequence shown in Sequence ID No. 1, (a2) a base sequence in which one or more bases are substituted, deleted, added or inserted to the base sequence other than SNP(a) in the base sequence of the polynucleotide of (a1), and which has the function of determining the quercetin content in onion plants, or (a3) ​​a base sequence in which the base sequence of the polynucleotide of (a1) has 90% or more identity with respect to the base sequence other than SNP(a), and which has the function of determining the quercetin content in onion plants.

[0034] Polynucleotide (b) is a polynucleotide consisting of the base sequence of the region containing SNP(b) in the base sequence shown in Sequence ID No. 2 (b1), (b2) a base sequence in which one or more bases are substituted, deleted, added or inserted to the base sequence other than SNP(b) in the base sequence of the polynucleotide of (b1), and which has the function of determining the quercetin content in onion plants, or (b3) a base sequence in which the base sequence of the polynucleotide of (b1) has 90% or more identity with respect to the base sequence other than SNP(b), and which has the function of determining the quercetin content in onion plants.

[0035] Polynucleotide (c) is a polynucleotide consisting of a base sequence containing SNP(c) in the base sequence shown in Sequence ID No. 3 (c1), a base sequence in which one or more bases are substituted, deleted, added or inserted in the base sequence of the polynucleotide of (c1) other than SNP(c), and which has the function of determining the quercetin content in onion plants, or a polynucleotide consisting of a base sequence having 90% or more identity with respect to the base sequence other than SNP(c) in the base sequence of the polynucleotide of (c1), and which has the function of determining the quercetin content in onion plants.

[0036] Polynucleotide (d) is a polynucleotide consisting of a base sequence containing SNP(d) in the base sequence shown in Sequence ID No. 4 (d1), a base sequence in which one or more bases are substituted, deleted, added or inserted in the base sequence of the polynucleotide of (d1) other than SNP(d), and which has the function of determining the quercetin content in onion plants, or a polynucleotide consisting of a base sequence having 90% or more identity with respect to the base sequence other than SNP(d) in the base sequence of the polynucleotide of (d1), and which has the function of determining the quercetin content in onion plants.

[0037] The polynucleotides (a1) to (d1) can be obtained, for example, from quercetin-rich onion plants (e.g., quercetin-rich Quergold) based on the base sequence of the gene locus related to quercetin content on chromosome 6.

[0038] The polynucleotides (a2) to (d2) may have the bases corresponding to SNPs (a) to (d) conserved in the base sequence of the polynucleotides (a1) to (d1), and the remaining base sequence may contain several (e.g., 1 to 10, preferably 1 to 5, more preferably 1, 2, or 3) base modifications (substitutions, deletions, insertions, or additions). The base sequence of such a polynucleotide is obvious to those skilled in the art and can be determined by referring to the onion genome sequence described above, or by sequencing the region near the SNP on the genome of a quercetin-rich onion plant.

[0039] The polynucleotides (a3) ​​to (d3) may have a degree of identity with respect to the other base sequences of the polynucleotides (a1) to (d1), with the bases corresponding to SNPs (a) to (d) being conserved, for example, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Such sequence identity can be determined, for example, by aligning the two base sequences using analysis software such as BLAST or FASTA.

[0040] A plant can be determined to have a high quercetin content if it is homozygous for the allele where the base corresponding to SNP(a) in polynucleotide(a) is G, homozygous for the allele where the base corresponding to SNP(b) in polynucleotide(b) is T, homozygous for the allele where the base corresponding to SNP(c) in polynucleotide(c) is T, or homozygous for the allele where the base corresponding to SNP(d) in polynucleotide(d) is C. Furthermore, the quercetin content in a plant can be determined by analyzing at least two of polynucleotides (a) to (d) as haplotype blocks.

[0041] (iii: Polynucleotides containing three SNPs) A molecular marker according to one aspect of the present invention may be a consecutive polynucleotide (hereinafter also referred to as polynucleotide(e)) containing at least two SNP(a) to SNP(d). Polynucleotide(e) contains the region between at least two sites of SNP(a) to SNP(d) together with at least two sites of SNP(a) to SNP(d). Polynucleotide(e) can be obtained, for example, by referencing the region between at least two sites of the corresponding SNP(a) to SNP(d) in a quercetin-rich onion plant. The base sequence of polynucleotide(e) is at least partially identical to the base sequence of a quercetin-rich onion plant.

[0042] The method for determining the quercetin content in onion seed plants using a molecular marker according to one aspect of the present invention is not particularly limited, and for example, known SNP analysis methods for detecting SNPs can be used. Such known SNP analysis methods include methods for SNP analysis by detecting SNPs in PCR-amplified fragments of onion seed plant subjects. Onion seed plant subjects may be leaves, stems, roots, etc. of seedlings, or bulbs. Furthermore, one aspect of the method for determining the quercetin content in onion seed plants using a molecular marker according to one aspect of the present invention is the method for determining the quercetin content in onion seed plants according to one aspect of the present invention, which will be described later.

[0043] [Onion varieties with high quercetin content] A quercetin-rich onion plant according to one aspect of the present invention is a plant obtained by the production method described later. A quercetin-rich onion plant has the above-mentioned SNP identified by the above-mentioned molecular marker and is a plant with a high quercetin content.

[0044] A quercetin-rich onion plant according to one aspect of the present invention can be obtained by crossbreeding onion plants with each other and identifying the quercetin-rich onion plant from the resulting plants and their descendants using the molecular markers described above, as shown in the production method described later. A quercetin-rich onion plant genetically modified to have the above-mentioned SNP is also included in the scope of the present invention.

[0045] [Method for determining the degree of quercetin content in onion seed plants] A method for determining the degree of quercetin content in an onion seed plant according to one aspect of the present invention is to determine in an onion seed plant whether the base itself (SNP) corresponding to the bases (a) to (d) below, or a sequence of polynucleotides contained in said base: (a) The 41st base of a polynucleotide consisting of the base sequence shown in Sequence ID No. 1; (b) The 55th base of a polynucleotide consisting of the base sequence shown in Sequence ID No. 2; (c) The 106th base of a polynucleotide consisting of the base sequence shown in Sequence ID No. 3; and (d) The 73rd base of a polynucleotide consisting of the base sequence shown in Sequence ID No. 4; The process includes determining at least one of the following as a molecular marker for quercetin content in onion plants.

[0046] A method for determining the quercetin content in onion seed plants according to one aspect of the present invention involves determining the quercetin content in a test onion seed plant using the molecular markers described above. A method for determining the quercetin content in onion seed plants according to one aspect of the present invention involves identifying one of SNP(a) to SNP(d) on the genome of a test onion seed plant using one of the molecular markers described above, thereby determining whether the test onion seed plant has a high quercetin content.

[0047] In a method for determining the quercetin content of an onion seed plant according to one aspect of the present invention, the onion seed plant being tested is either a candidate plant for breeding material or a plant obtained in the breeding process. The onion seed plant being tested may be, for example, a hybrid plant of a high-quercetin onion seed plant and another high-quercetin onion seed plant, a hybrid plant of a high-quercetin onion seed plant and another low-quercetin onion seed plant, a hybrid plant of a low-quercetin onion seed plant and another low-quercetin onion seed plant, and their offspring lines. The onion seed plant being tested may also be a hybrid plant obtained by crossing onion seed plants with unknown quercetin content and their offspring lines.

[0048] A method for determining the quercetin content in an onion seed plant according to one aspect of the present invention is to determine that the onion seed plant has a high quercetin content when at least one of the following conditions is met: (a') it is homozygous for an allele in which the 41st base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 1 is G; (b') it is homozygous for an allele in which the 55th base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 2 is T; (c') it is homozygous for an allele in which the 106th base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 3 is T; and (d') it is homozygous for an allele in which the 73rd base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 4 is C.

[0049] Details of the molecular marker used in the method for determining the quercetin content in onion seed plants according to one aspect of the present invention should be referenced to the description of the molecular marker according to one aspect of the present invention described above.

[0050] In a method for determining the quercetin content in onion seed plants according to one aspect of the present invention, the method for examining the quercetin content in onion seed plants using molecular markers is not particularly limited, and known SNP analysis methods can be used. For example, a method of SNP analysis can be used by detecting SNPs in PCR-amplified fragments of onion seed plants as a subject. In a method for determining the quercetin content in onion seed plants according to one aspect of the present invention, the region in the DNA of the onion seed plant may be amplified using a primer set that amplifies the region containing the molecular marker. For example, such a primer set is a primer set that amplifies a region containing any of SNP(a) to SNP(d). That is, a primer set that amplifies a region containing any of SNP(a) to SNP(d) can be a determination kit for determining onion seed plants with high quercetin content.

[0051] Amplification of the aforementioned region in the DNA of an onion seed plant subject can be performed by polymerase chain reaction (PCR) using DNA extracted from the onion seed plant subject as a template and primers that amplify the region containing the SNP. Then, the base (genotype) of the SNP in the obtained amplified fragment is determined, and the quercetin content in the fruit of the onion seed plant is determined based on data showing the relationship between the determined base (genotype) and the quercetin content in the onion seed plant. Conventional known methods can be used to determine the genotype of the amplified fragment, for example, DNA sequencing analysis, agarose gel electrophoresis, and TaqMan analysis using real-time PCR.

[0052] The primer set used in PCR is not particularly limited as long as it can amplify the DNA fragment of the region containing the target SNP, and the primer set may be designed to shorten the length of the amplified fragment. For example, the primer set may be designed so that the length of the primer amplified fragment is preferably 200 base pairs (bp) or less, 160 bp or less, 150 bp or less, 140 bp or less, 130 bp or less, 120 bp or less, or 100 bp or less. The primer set includes a forward primer and a reverse primer. The length of these primers may be, for example, 15 bp or more, 16 bp or more, 17 bp or more, 18 bp or more, 19 bp or more, 20 bp or more, or 25 bp or more, and may be 50 base pairs (bp) or less, 40 bp or less, or 30 bp or less.

[0053] The primer set that amplifies the region containing SNP(a) is a diagnostic kit for identifying onion plants with high quercetin content. The primer set that amplifies the region containing SNP(a) includes either the following primer set (I) or (II): (I) A primer set comprising a first primer containing 15 or more consecutive bases in the nucleotide sequence shown in Sequence ID No. 5, and a second primer containing 15 or more consecutive bases in the nucleotide sequence shown in Sequence ID No. 6; (II) A primer set comprising a third primer containing 15 or more consecutive bases in the nucleotide sequence shown in Sequence ID No. 7, a fourth primer containing 15 or more consecutive bases in the nucleotide sequence shown in Sequence ID No. 8, and a fifth primer containing 15 or more consecutive bases in the nucleotide sequence shown in Sequence ID No. 9.

[0054] By using the primer set (I) or (II) described above, a PCR amplification product consisting of the nucleotide sequence shown in Sequence ID No. 1 can be obtained in onion plants with high quercetin content, and the nucleotide corresponding to SNP(a) can be detected. Furthermore, by using the primer set (II) described above, the allele locus in the region containing SNP(a) can be specifically amplified, and the genotype can be determined by agarose electrophoresis.

[0055] The primer set (III) for amplifying the region containing SNP(b) is, for example, a primer set consisting of a sixth primer containing 15 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 10, and a seventh primer containing 15 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 11. Using this primer set, a PCR amplification product consisting of the nucleotide sequence shown in SEQ ID NO: 2 can be obtained in onion plants with high quercetin content, and the base corresponding to SNP(b) can be detected.

[0056] The primer set (IV) for amplifying the region containing SNP(c) is, for example, a primer set consisting of an eighth primer containing 15 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 12, and a ninth primer containing 15 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 13. Using this primer set, a PCR amplification product consisting of the nucleotide sequence shown in SEQ ID NO: 3 can be obtained in onion plants with high quercetin content, and the base corresponding to SNP(c) can be detected.

[0057] The primer set (V) for amplifying the region containing SNP(d) is, for example, a primer set consisting of a 10th primer containing 15 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 14, and an 11th primer containing 15 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 15. Using this primer set, a PCR amplification product consisting of the nucleotide sequence shown in SEQ ID NO: 4 can be obtained in onion plants with high quercetin content, and the base corresponding to SNP(d) can be detected.

[0058] PCR in SNP analysis may be either singleplex PCR, which amplifies DNA fragments in a reaction system containing a single primer set, or multiplex PCR, which amplifies genes in a reaction system containing multiple primer sets. In the case of multiplex PCR, primer sets labeled with fluorescent substances having different wavelengths (e.g., NED, 6-FAM, VIC, PET) may be mixed.

[0059] The reaction conditions for PCR can be appropriately set depending on the type of DNA polymerase and PCR instrument used, the length of the amplification fragment, etc. As for the cycle conditions, a 3-step PCR method consisting of three steps—denaturation, annealing, and extension—as one cycle, and a 2-step PCR method consisting of two steps—denaturation and annealing / extension—as one cycle, can be applied. An example of reaction conditions in 3-step PCR is to perform a denaturation step of 40-60 seconds at 90-100°C (e.g., 30 seconds at 94°C), an annealing step of 10-60 seconds (e.g., 30 seconds), and an extension step of 10-90 seconds at 65-80°C (e.g., 60 seconds at 72°C) for 10-40 cycles (e.g., 15 cycles). One example of an annealing temperature is to gradually decrease it in predetermined cycles from an initial annealing temperature of 55-70°C (e.g., 60°C) to a final annealing temperature of 40-60°C (e.g., 45°C) (for example, decreasing by 1°C per cycle). Depending on the state of the template DNA, the PCR reaction conditions may be adjusted to stably detect SNPs.

[0060] For SNP analysis, real-time PCR such as the TaqMan®-PCR method, which performs amplification by PCR and identification of SNP markers, may be used. That is, a TaqMan® probe may be used to detect SNPs contained in the amplified fragment amplified using a primer set. By using the real-time PCR method, a highly efficient discrimination method can be provided. As a method for identifying SNPs in the amplified fragment amplified by PCR, the base sequence of the amplified fragment may be determined using an automated DNA sequencer or the like for analysis.

[0061] The method for extracting DNA to be amplified by PCR from onion seed plants is not particularly limited, and known DNA extraction methods can be used. Alternatively, commercially available DNA extraction kits may be used to extract the DNA. Depending on the type of sample and the amount of impurities, appropriate pretreatment may be performed before the DNA extraction step. Furthermore, the DNA extracted from the sample may be washed or purified as needed for use as a template in the PCR reaction. In addition, restriction enzyme digestion fragments obtained by digesting the DNA extracted from the sample with two types of restriction enzymes may be amplified by PCR.

[0062] Furthermore, in a method for determining the quercetin content in onion seed plants according to one aspect of the present invention, gene polymorphisms in a linkage disequilibrium state with SNP(a) to SNP(d) may be analyzed to identify SNP(a) to SNP(d). The above linkage disequilibrium state is, for example, a linkage disequilibrium state in which the linkage disequilibrium coefficient is 0.9 or higher.

[0063] According to a method for determining the quercetin content in onion seed plants according to one aspect of the present invention, it is possible to determine whether or not a test onion seed plant has a high quercetin content using a molecular marker. Therefore, onion seed plants and their progeny lines with high quercetin content can be selected based on the determination result.

[0064] According to one aspect of the present invention, a method for determining the quercetin content in onion seedlings makes it possible to select onion seedlings with high quercetin content at the seedling stage, significantly shortening the breeding selection period. Furthermore, by planting and growing only the seedlings with high quercetin content selected at the seedling stage, the efficiency of field utilization can be improved.

[0065] [Manufacturing method for producing onion seeds with high quercetin content] A manufacturing method according to one aspect of the present invention is a method for producing onion seed plants with a high quercetin content, comprising a crossbreeding step of crossbreeding onion seed plants with each other, and a discrimination step of determining the quercetin content of onion seed plants according to one aspect of the present invention from onion seed plants or their descendant lines obtained by the crossbreeding step.

[0066] Therefore, the description of a molecular marker according to one aspect of the present invention, an onion seed plant with a high quercetin content, and a method for determining the quercetin content in an onion seed plant will be incorporated into the description of a method for producing an onion seed plant with a high quercetin content.

[0067] In the hybridization process, the onion seed plants used as parent plants may include, for example, onion seed plants with high quercetin content, onion seed plants with low quercetin content, onion seed plants with unknown quercetin content, and their offspring lines.

[0068] Furthermore, in the crossbreeding process, the onion seed plants used as parent plants may be onion seed plants with a high quercetin content according to one aspect of the present invention. Alternatively, the onion seed plants used in the crossbreeding process may be onion seed plants with a high quercetin content selected by a method for determining the quercetin content in onion seed plants according to one aspect of the present invention. In other words, the manufacturing method according to one aspect of the present invention may further include a selection step before the crossbreeding step in which onion seed plants with a high quercetin content are identified from test onion seed plants by a method for determining the quercetin content in onion seed plants according to one aspect of the present invention.

[0069] The discrimination step involves identifying onion seed plants with a high quercetin content from onion seed plants obtained through the crossbreeding step or from their progeny lines, using a method for determining the quercetin content in onion seed plants according to one aspect of the present invention.

[0070] According to one aspect of the present invention, a manufacturing method can be used to determine whether or not an onion seed plant has a high quercetin content using a molecular marker, and to produce selected onion seed plants with a high quercetin content based on the determination result.

[0071] The present invention is not limited to the embodiments described above, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0072] Examples of the present invention are described below. [Examples]

[0073] (Example 1: Next-generation sequencing analysis) The following experiment was conducted in 2019. First, individuals (F1) grown from a cross between two different onion varieties (OMP-3 and Turbo) were self-pollinated, and a hybrid progeny population (F2, 184 individuals) was cultivated in the field. In addition, leaf blade samples were collected from each F2 individual during cultivation and used as specimens for DNA extraction and next-generation sequencing analysis.

[0074] The acquired samples were subjected to PCR reactions using a 167 primer set that performs target-specific amplification. The resulting amplified products were then further subjected to PCR to add sequences for next-generation sequencing analysis.

[0075] PCR was performed as follows. First, as a primary PCR, multiplex PCR was performed for each Primer Plex (each containing approximately 40 primer sets) which was created by dividing a 167-primer set into four parts. The composition of the primary PCR is shown in Table 1, and the PCR conditions are shown in Table 2.

[0076] [Table 1]

[0077] [Table 2]

[0078] The primary PCR products of four Primer Plex samples were combined and purified using AMPure XP Reagent. The purified template was diluted 100-fold and used for secondary PCR. Secondary PCR yielded sequences in which adapter sequences for next-generation sequencing analysis and barcode sequences for individual identification were added to the primary PCR products. The composition of the secondary PCR is shown in Table 3, and the conditions are shown in Table 4.

[0079] [Table 3]

[0080] [Table 4]

[0081] The sequences of 184 secondary PCR products were analyzed together using a next-generation sequencer (Thermo Fisher Scientific, Ion Torrent Proton system). The obtained sequence data were classified for each individual using Torrent Suite ver. 5 software (Thermo Fisher Scientific) and mapped to reference sequences. Subsequently, polymorphism information (marker genotypes) within each amplification region was extracted using Torrent Variant Caller software.

[0082] For each individual onion bulb, quercetin-3,4'-diglucoside and quercetin-4'-glucoside, the main quercetin glycosides contained in the bulb, were quantitatively analyzed by high-performance liquid chromatography (HPLC). From each individual, the edible part of the bulb was peeled, freeze-dried, and powdered using a mill mixer to obtain the sample for quercetin extraction. The freeze-dried powder was weighed into a 15 ml tube and extracted by shaking with 80% methanol. After extraction, the mixture was centrifuged, and the supernatant was used as the quercetin extract. This extract was mixed in equal volumes with 1 mg / 100 ml of quercetin dihydrate (Fujifilm Wako Pure Chemical Industries, Ltd., Osaka Prefecture), which was used as an internal standard, and prepared as the HPLC analysis sample.

[0083] Details of the HPLC procedure are described below. A Shimadzu SCL-10A HPLC system equipped with a Mightysil RP-18 GP Aqua column (4.6 × 150 mm, Kanto Chemical), an SPD-10AV UV detector (wavelength: 360 nm, Shimadzu Corporation), an LC-10AD pump (Shimadzu Corporation), and a CTO-10A column oven (Shimadzu Corporation) was used. 10 μl of the analytical sample was injected into the HPLC. A linear gradient analysis was performed by adding solvent B (methanol containing 2% acetic acid) to solvent A (Milli-Q water containing 2% acetic acid) from 20% to 88% over 17 minutes. Then, all components contained in the sample were eluted and separated by stabilizing solvent B at 20% in solvent A for 10 minutes at 30°C at a flow rate of 1.0 ml / min. Quercetin-3,4'-diglucoside and quercetin-4'-glucoside were calculated using a calibration curve for the internal standard quercetin dihydrate. The combined value of quercetin-3,4'-diglucoside and quercetin-4'-glucoside was quantified as the quercetin content.

[0084] Based on the marker genotype determined for each individual, a linkage map was constructed using the software Join map 4.0 (Figure 1). As shown in Figure 1, each marker was classified by linkage group, and the order of the markers within each linkage group was estimated.

[0085] After rearranging the marker genotypes of each individual according to the obtained linkage map, QTL analysis was performed using the R / qtl software based on the quercetin content of each individual. The results are shown in Figure 2. Based on Figure 2, CHR6_176431815 (s1179403) was selected as the optimal marker with the highest contribution rate. CHR6_164337315 (s38962), CHR6_173524136 (s454861), and CHR6_171219319 (s615552) were selected as candidate markers in the vicinity of CHR6_176431815. Details of the four selected markers are shown in Table 5. In Table 5, the number following "CHR6_" in the marker name represents the position of each SNP on chromosome 6 of onion.

[0086] [Table 5] The 184 individuals used in the analysis were classified into three groups based on genotype for each of the four selected markers, and Tukey's HSD test was performed based on quercetin content. The results are shown in Figure 3.

[0087] As shown in Figure 3, in CHR6_176431815, individuals with homozygous genotype G had significantly higher quercetin content compared to individuals with homozygous genotype A and individuals with heterozygous genotype GA. Regarding nearby markers, in CHR6_164337315, individuals with homozygous genotype T had significantly higher quercetin content compared to individuals with homozygous genotype C and individuals with heterozygous genotype TC. In CHR6_173524136, individuals with homozygous genotype T had significantly higher quercetin content compared to individuals with homozygous genotype C and individuals with heterozygous genotype TC. In CHR6_171219319, individuals with homozygous genotype C had significantly higher quercetin content compared to individuals with homozygous genotype T or individuals with heterozygous genotype CT.

[0088] (Example 2: Sanger sequencing analysis) The following experiment was conducted in 2020. The same F1 individuals as in Example 1 were self-pollinated again, and the resulting hybrid progeny (F2, 192 individuals) were cultivated in the field. Samples for sequencing reactions were collected from each F2 individual in the same manner as in Example 1.

[0089] The acquired samples were analyzed by Sanger sequencing using primers 1 and 2, shown in Table 6, as a primer set. CHR6_176431815, selected in Example 1, was amplified using the primer set of primers 1 and 2. Waveform analysis was performed on the amplified products to determine the genotype of each individual. The results of the waveform analysis are shown in Figure 4.

[0090] [Table 6] The quercetin content of each individual was quantified using the same method as in Example 1. The 192 individuals used for analysis were then classified into three groups according to the genotype of CHR6_176431815, and Tukey's HSD test based on quercetin content was performed in the same manner as in Example 1. The results are shown in Figure 5. As shown in Figure 5, individuals with homozygous genotype G had significantly higher quercetin content compared to individuals with homozygous genotype A and individuals with heterozygous genotype GA.

[0091] (Example 3: Analysis by agarose gel electrophoresis) For CHR6_176431815 analyzed in Examples 1 and 2, primers were designed to allow genotype determination by agarose gel electrophoresis. Primers 3-5, listed in Table 6, were used. PCR reactions were performed using primer pairs of primers 3 and 4, and primer pairs of primers 3 and 5. The composition of the PCR solution is shown in Table 7, and the PCR conditions are shown in Table 8. Taq DNA polymerase used was NEB's Taq DNA Polymerase with ThermoPol Buffer (M0267X).

[0092] [Table 7]

[0093] [Table 8] The allele locus of CHR6_176431815 was specifically amplified by PCR, and the genotype was determined by agarose electrophoresis. Electrophoresis was performed using a 100 bp ladder on both sides of the PCR product produced by the primer pair of each sample, on an agarose gel prepared with a concentration of 4% (w / v) in TBE solution. The results are shown in Figure 6. As shown in Figure 6, it was demonstrated that the three genotypes of CHR6_176431815 can be identified by agarose gel electrophoresis.

[0094] (Example 4: Analysis by agarose gel electrophoresis for each variety) In various onion varieties both domestically and internationally, the CHR6_176431815 allele was specifically amplified, similar to Example 3, and the genotype was determined by agarose electrophoresis. DNA from 96 onion varieties (one arbitrary individual from each variety) shown in Table 9 was used. The results are shown in Figure 7. As shown in Figure 7, the genotype of CHR6_176431815 was identified for each variety.

[0095] [Table 9] JPEG0007847816000010.jpg242152JPEG0007847816000011.jpg181153 [Industrial applicability]

[0096] This invention can be used in fields such as agriculture and plant breeding.

Claims

1. A method for determining the degree of quercetin content in onion seed plants, In onion plants, either a base (SNP) corresponding to any of the bases listed below (a) to (d), or a sequence of polynucleotides containing said base: (a) The 41st base of a polynucleotide consisting of the base sequence shown in Sequence ID No. 1; (b) The 55th base of a polynucleotide consisting of the base sequence shown in Sequence ID No. 2; (c) The 106th base of a polynucleotide consisting of the base sequence shown in Sequence ID No. 3; and (d) The 73rd base of a polynucleotide consisting of the base sequence shown in Sequence ID No. 4; The process includes determining at least one genotype, The above determination step is performed when the genotype is as follows (a') to (d'): (a') Homozygous for alleles in which the base of (a) is G; (b') Homozygous for alleles where the base of (b) is T; (c') homozygous for alleles where the base of (c) is T; and (d') Homozygous for alleles where the base of (d) is C; A method for determining that an onion plant has a high quercetin content when at least one of the following conditions is met.

2. The method according to claim 1, wherein in the determination step, the region in the DNA of the onion seed plant is amplified using a primer set that amplifies a region containing a base corresponding to any of the bases (a) to (d).

3. A method for producing onion seed plants with a high quercetin content, The process of crossbreeding onion seed plants, A discrimination step of identifying onion seed plants with a high quercetin content from onion seed plants or their descendant lines obtained by the aforementioned crossbreeding step, using the method described in claim 1 or 2. A manufacturing method that includes this.

4. A determination kit used for determining the degree of quercetin content in onion seed plants, The method for determining the above is to determine in an onion seed plant whether the base itself (SNP) corresponds to any of the following bases (a) to (d), or a sequence of polynucleotides containing said base: (a) The 41st base of a polynucleotide consisting of the base sequence shown in Sequence ID No. 1; (b) The 55th base of a polynucleotide consisting of the base sequence shown in Sequence ID No. 2; (c) The 106th base of a polynucleotide consisting of the base sequence shown in Sequence ID No. 3; and (d) The 73rd base of a polynucleotide consisting of the base sequence shown in Sequence ID No. 4; The process includes determining at least one genotype, The above determination step is performed when the genotype is as follows (a') to (d'): (a') Homozygous for alleles in which the base of (a) is G; (b') Homozygous for alleles where the base of (b) is T; (c') homozygous for alleles where the base of (c) is T; and (d') Homozygous for alleles where the base of (d) is C; The process of determining that the onion plant has a high quercetin content when at least one of the following conditions is met, A diagnostic kit containing at least one of the primer sets (I) through (V) below: (I) A primer set comprising a first primer containing 15 or more consecutive bases in the nucleotide sequence shown in Sequence ID No. 5, and a second primer containing 15 or more consecutive bases in the nucleotide sequence shown in Sequence ID No. 6; (II) A primer set comprising a third primer containing 15 or more consecutive bases in the nucleotide sequence shown in Sequence ID No. 7, a fourth primer containing 15 or more consecutive bases in the nucleotide sequence shown in Sequence ID No. 8, and a fifth primer containing 15 or more consecutive bases in the nucleotide sequence shown in Sequence ID No. 9; (III) A primer set comprising a sixth primer containing 15 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 10, and a seventh primer containing 15 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 11; (IV) A primer set comprising an eighth primer containing 15 or more consecutive bases in the nucleotide sequence shown in Sequence ID No. 12, and a ninth primer containing 15 or more consecutive bases in the nucleotide sequence shown in Sequence ID No. 13; (V) A primer set comprising a 10th primer containing 15 or more consecutive bases in the nucleotide sequence shown in Sequence ID No. 14, and an 11th primer containing 15 or more consecutive bases in the nucleotide sequence shown in Sequence ID No. 15.

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