WO2016072182A1

WO2016072182A1 - Inoperative-type glucoraphasatin synthase gene and use of same

Info

Publication number: WO2016072182A1
Application number: PCT/JP2015/077956
Authority: WO
Inventors: 智博柿崎; 正彦石田; 隆由小原; 伸子吹野
Original assignee: 国立研究開発法人農業・食品産業技術総合研究機構
Priority date: 2014-11-07
Filing date: 2015-10-01
Publication date: 2016-05-12
Also published as: JP2016086761A; JP6499817B2

Abstract

Provided is a technique with which it is possible to produce a glucoraphasatin-deficient daikon line in a short time at high accuracy, as pertains to a technique for fundamentally overcoming the problems of deterioration of flavor, occurrence of radish odor, and deterioration of quality such as yellowing in processed daikon foods and the like. The present invention pertains to an inoperative-type glucoraphasatin synthase gene having (A) the characteristic of having a mutation associated with complete or partial deletion of the 2-oxoglutarate-iron(II)-dependent oxygenase domain in an encoded protein within an exon constituting a glucoraphasatin synthase gene, and (B) the characteristic of having the glucoraphasatin of the root portion deleted or substantially deleted when the glucoraphasatin synthase gene locus in the daikon genome is a homo-type of this inoperative-type gene.

Description

Function deficient glucorafasatin synthase gene and use thereof

The present invention relates to an invention relating to a radish-deficient glucorafasatin synthase gene. The present invention also relates to an invention relating to a technique for producing a radish line lacking glucorafasatin using the function-deficient gene. The present invention also relates to an invention relating to a radish line having the function-deficient gene in a homo form at the glucorafasatin synthase locus in the genome.

Glucosinolate is a general term for sulfur-containing compounds that are also called mustard oil glycosides, and is a secondary metabolite that is characteristically contained in cruciferous and related family plants. More than 120 glucosinolates with different compound structures have been reported for Brassicaceae plants.
In radish (Raphanus sativus), a kind of cruciferous plant, its main glucosinolate is a substance called “glucorafasatin”. 90% or more of the total amount of glucosinolate contained in the radish radish is occupied by glucorafasatin.
Glucorafasatin is a glucosinolate characteristic of radish, and glucorafasatin is the dominant glucosinolate component in the same cruciferous plant as Raphanus to which radish belongs (for example, Brassica, a related genus). No plants have been found to contain.

Although glucorafasatin contained in radish is an almost tasteless and odorless compound itself, the endogenous enzyme myrosinase acts to become a spicy ingredient peculiar to radish called 4-methylthio-3-butenyl isothiocyanate. “4-Methylthio-3-butenyl isothiocyanate” is a kind of isothiocyanate, but since it is an unstable compound, it has the property of being chemically decomposed in a short time. The pungent taste of radish processed products such as radish grated disappears in a short time, and the problem that it is difficult to store with reduced flavor deterioration is a phenomenon caused by instability of 4-methylthio-3-butenyl isothiocyanate.
Furthermore, glucorafasatin contained in radish is a serious causative component that causes problems of odor and yellowing peculiar to radish in foods processed from radish.
As shown in Fig. 1, components that cause generation of methyl mercaptan, which is a causative component of the savory odor (sometimes expressed as radish odor, etc.), which is a characteristic odor of Japanese radish, and color unevenness due to yellowing The yellow component TPMP (2- [3- (2-thioxopyrrolidine-3-ylidene) methyl] -tryptophan) is a degradation of 4-methylthio-3-butenylisothiocyanate produced from glucorafasatin It is a phenomenon caused by
Such “degradation of flavor”, “occurrence of abundant odor”, and “occurrence of color unevenness due to yellowing change” are phenomena that are particularly prominent with changes over time such as storage and heat treatment. In particular, in some processed radish foods such as pickled radish, radish grated radish, and dried radish, it is a major cause of significant deterioration in quality.
Currently, about 60% of the Japanese radish production in Japan is used for processing. Therefore, technologies that can prevent flavor deterioration, low bromide, and yellowing prevention in processed radish are This is a technology that is eagerly desired by manufacturers in various fields of the food and beverage industry.

In response to such a demand from the industrial field, the present inventors have developed a glucosinolate composition in order to solve the problems of quality deterioration such as flavor deterioration, generation of abundant odor, and yellowing change in processed radish foods. We worked on the creation of a radish line completely different from the normal radish variety line.
As a result, the present inventors can produce a radish line having a trait that contains a high content of glucosinolates different from glucorafasatin by performing artificial breeding operations focusing on a specific variety line. (Patent document 1). Specifically, according to the method described in Patent Document 1, the present inventors have a high content of “glucoraphanin” (* a precursor of sulforaphane, which is an anticancer active ingredient) in seeds, sprouts and the like, A number of lines, such as “Glucerucin”, containing a large amount of “Glucerucin” were produced in the beetroot, and one of those lines, “Anno No. 5”, was registered as a radish variety “Daikom Intermediate Mother Farm No. 5” (variety registration No. 22662).

JP 2012-110238 A (Production Method of Japanese Radish Lines Highly Containing Glucoraphanin)

However, the method for obtaining a radish line having a desired glucosinolate composition described in Patent Document 1 is a method of selecting mutant individuals from a specific variety lineage group in which the glucosinolate composition is maintained in the wild type. Therefore, there was a problem that the production efficiency was low.
In addition, in the method described in Patent Document 1, it was necessary to perform analysis using high-performance liquid chromatography (HPLC) or the like using a tissue site where the glucosinolate composition is to be measured in order to determine the phenotype. . However, in order to collect a sufficient amount of sample for use in HPLC analysis or the like, a cultivation period of several weeks is required, so that there is a problem that rapid analysis is difficult. In addition, since information obtained by HPLC analysis or the like is merely phenotypic information, genotype information that enables efficient breeding could not be obtained in principle.
In addition, when the resulting production line (especially the intermediate mother line) is used as a mating parent as a trait donor and crossed with a variety line having other useful traits, There was a problem that enormous effort and time were required for training for the progeny of the next generation.

In view of the above-mentioned problems of the prior art, the present invention relates to a technology that fundamentally solves the problem of quality deterioration such as flavor deterioration, generation of abundant odor, and yellowing change in radish processed foods, etc. It is an object of the present invention to provide a technique capable of producing a radish system having a high accuracy in a short period of time.

In view of the above-mentioned problems, the inventors of the present invention have developed a glucosinolate composition of Japanese radish using a line (NR154E line, MR050E line) that has been developed by the present inventors and exhibits excellent glucorafasatin deficiency. We focused on the viewpoint of identifying a gene that controls glucorafasatin deficiency, which is considered to be one of the causes of the change.
However, in order to identify the glucorafasatin deficient gene, there has been a technical problem that makes it difficult to apply molecular biological techniques due to the peculiar circumstances of this case that the object is a radish plant.

i) First, information on genes involved in glucorafasatin metabolism in plants was completely unknown. Therefore, as a means for solving this problem, there is a means of gene isolation methods (PCR method using degenerate sequence primers, screening method using hybridization, etc.) based on homologous sequences of genes of other plants. It was in a situation where it could not be used at all.
ii) In addition, due to recent advances in gene analysis technologies such as next-generation sequencers, radish draft genome sequences have been released as a database, and the foundation for comprehensive analysis is being improved (Raphanus sativus Genome DataBase (http: / /radish.kazusa.or.jp/)), however, the annotation information of genes for which functional analysis has not been performed has not been published in the database.
Since the predicted gene estimation function on the genome sequence published in the database is merely information on homology search information of other known organisms and information estimated from domain prediction, “analysis of gene function” The existence of “a novel radish gene that has not been performed” could not be found on the database.
Of course, since the sequence information of the mutant gene of each mutant strain is not registered, the causative gene of each mutant could not be found on the database.

As described above, it was impossible to isolate and identify a gene involved in glucorafasatin metabolism (a new gene of radish) by means of using known gene sequence information.
Therefore, the present inventors conducted extensive research and conducted a huge amount of experiments. First, a linkage marker (SNP, SSR) between a glucorafasatin-deficient strain called NR154E strain originally created by the present inventors and a glucorafasatin-containing strain (wild type) was identified, and the linkage between both strains was identified. A marker map was created. Then, by repeating the linkage analysis and the high-accuracy linkage analysis by the positional cloning method using the linkage map, the operation of narrowing the locus region of the target gene was repeated, and the locus region of 23.8 kbp was identified.
As a result of detailed analysis such as sequence prediction, quantitative expression analysis, gain of function gene transfer test, etc., the causative gene governing glucorafasatin deficiency in the locus region is identified. did. This gene was found to be a gene encoding glucorafasatin synthase in wild type radish.
Moreover, since the insertion sequence that induces the functional defect is a mutated sequence in the target gene, it was shown that it can be used as an excellent selection DNA marker that cannot be separated from the target trait.

The present invention has been conceived on the basis of the above findings, and specifically relates to the following function-deficient glucorafasatin synthase gene and an invention relating to the use thereof.
[1]
A function-deficient glucorafasatin synthase gene having the characteristics described in (A) and (B) below;
(A) a feature having a mutation in the exon constituting the gene described in (a1) below, which is accompanied by a deletion of all or part of the 2-oxoglutarate-iron (II) -dependent oxygenase domain in the encoded protein;
(A1) A gene encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence showing 95% or more sequence homology with the amino acid sequence, and is involved in the synthesis reaction from glucoerucin to glucorafasatin A gene encoding a protein to be
(B) The characteristic that the glucorafasatin content of the radish root is 3 μmol / g or less when the glucorafasatin synthase gene locus in the radish genome is homozygous for the function-deficient glucorafasatin synthase gene.
[2]
Furthermore, the function-deficient glucorafasatin synthase gene according to [1], which has the characteristics described in (C) below;
(C) The function-deficient glucorafasatin synthase gene according to [1] above, wherein the mutation described in (A) is a mutation accompanied by insertion of a base sequence of at least 1 kbp.
[3]
The function-deficient glucorafasatin synthase gene according to [1] or [2] above, wherein the function-deficient glucorafasatin synthase gene is a gene comprising the base sequence represented by SEQ ID NO: 7 or 9.
[4]
A method for determining the genotype of a glucorafasatin synthase locus in radish, comprising detecting the presence or absence of the mutation described in (A) above in any one of [1] to [3].
[5]
A genotyping kit for a glucorafasatin synthase gene locus in radish, comprising the oligonucleotide primer described in the following (D) and (E);
(D) A base sequence included in the function-deficient glucorafasatin synthase gene according to any one of [1] to [3] above or a region on the same chromosome as the gene, or a base sequence included in a complementary sequence thereof An oligonucleotide primer comprising a base sequence consisting of at least 12 bases designed to detect the presence of the mutation described in (A) above,
(E) at least 12 contained in the base sequence constituting the function-deficient glucorafasatin synthase gene according to any one of [1] to [3] above or a region on the same chromosome as the gene or a complementary sequence thereof An oligonucleotide primer comprising a base sequence composed of bases and a base sequence at a position capable of forming a primer pair with the primer described in (D) above.
[6]
The genotype determination kit according to [5], wherein the primer described in (D) is the oligonucleotide primer described in (d1) or (d2) below;
(D1) when the mutation described in (A) is an insertion mutation, an oligonucleotide primer comprising a base sequence constituting the mutation or a complementary sequence thereof,
(D2) An oligonucleotide primer comprising a base sequence containing the mutation described in (A) above in its sequence.
[7]
Furthermore, the genotyping kit according to [5] or [6] above, which comprises the oligonucleotide primer described in (F) and (G) below;
(F) The glucorafasatin synthase gene according to any one of (1) to [3] above or a base sequence constituting the same chromosomal region as that gene or a complementary sequence thereof An oligonucleotide primer comprising a base sequence consisting of at least 12 bases designed at a position capable of detecting the absence of the mutation described in (A) above,
(G) glucorafasatin synthase gene according to any one of the above [1] to [3] or a nucleotide sequence constituting the same chromosomal region as the gene or a complementary sequence thereof An oligonucleotide primer comprising a base sequence consisting of at least 12 bases and a base sequence at a position capable of forming a primer pair with the primer described in (F) above.
[8]
A kit for genotyping of the glucorafasatin synthase gene locus in radish, comprising the oligonucleotide probe described in (H) or (I) below;
(H) a base sequence comprising at least 12 bases contained in a base sequence constituting the function-deficient glucorafasatin synthase gene according to any one of [1] to [3] or a complementary sequence thereof, When the mutation described in (A) is an insertion mutation, an oligonucleotide probe comprising a base sequence constituting the mutation or a complementary sequence thereof,
(I) A base sequence comprising at least 12 bases included in the base sequence constituting the function-deficient glucorafasatin synthase gene according to any one of [1] to [3] or a complementary sequence thereof, An oligonucleotide probe comprising a base sequence comprising the mutation described in (A) in its sequence.
[9]
The genotyping kit according to any one of [5] to [8] above, having the characteristics described in (J), (K), and (L) below;
(J) the function-deficient glucorafasatin synthase gene is a function-deficient glucorafasatin synthase gene comprising the base sequence described in SEQ ID NO: 7 or 9,
(K) The above-mentioned glucorafasatin synthase gene is a glucorafasatin synthase gene comprising the base sequence set forth in SEQ ID NO: 2,
(L) The region on the chromosome is composed of a base sequence consisting of the first to 2971st bases in SEQ ID NO: 1, and the 4759th to 7424th bases in SEQ ID NO: 1 A feature that is a region composed of a base sequence consisting of
[10]
The function-deficient glucorafasatin synthase gene according to any one of the above [1] to [3] has a homotype at the glucorafasatin synthase locus in the genome, and glucorafasatin relative to the dry weight of the bean radish A radish line having a content of 3 μmol / g or less.
[11]
The radish line according to [10] obtained by self-breeding the radish according to any one of (M) to (O) below or crossing with a desired radish and then selecting from a progeny population;
(M) Japanese radish in which the function-deficient glucorafasatin synthase gene according to any one of [1] to [3] is introduced into the glucorafasatin synthase gene locus in the genome,
(N) A radish obtained by introducing a function-deficient mutation into a glucorafasatin synthase gene in the genome, wherein the function according to any one of [1] to [3] Japanese radish having a deficient glucorafasatin synthase gene,
(O) A radish obtained from a progeny population of radish according to (M) or (N) above, wherein the function-deficient glucorafasatin synthase gene according to any one of [1] to [3] At the glucorafasatin synthase locus in the genome.
[12]
The radish system according to [10] or [11] above, wherein the function-deficient glucorafasatin synthase gene is a function-deficient glucorafasatin synthase gene comprising the base sequence described in SEQ ID NO: 7 or 9.
[13]
Roots, hypocotyls, hypertrophic roots, leaves, petioles, stems, florets, flowers, seeds, sprout, baby leaves, obtained from the radish plant of any one of [10] to [12] Or seedling.
[14]
A method for producing a glucorafasatin-deficient line in radish, comprising the step described in (P) below;
(P) A radish having the function-deficient glucorafasatin synthase gene according to any one of the above [1] to [3] at the glucorafasatin synthase gene locus in the genome is crossed with a self-breeding operation or desired radish Process.
[15]
The production method according to [14] above, wherein the radish having the function-deficient glucorafasatin synthase gene according to (P) is any of the following radish according to (Q) to (S):
(Q) Japanese radish in which the function-deficient glucorafasatin synthase gene according to any one of [1] to [3] is introduced into the glucorafasatin synthase gene locus in the genome,
(R) A radish obtained by introducing a loss-of-function mutation into a glucorafasatin synthase gene in the genome, wherein the function according to any one of [1] to [3] Japanese radish having a deficient glucorafasatin synthase gene,
(S) A radish obtained from a progeny population of radish according to (Q) or (R) above, wherein the function-deficient glucorafasatin synthase gene according to any of [1] to [3] At the glucorafasatin synthase locus in the genome.
[16]
The production method according to [14] or [15], which is performed using the genotyping method according to [4].
[17]
The production method according to any one of [14] to [16] above, further comprising the step described in (T) below;
(T) A step of selecting a radish individual exhibiting glucorafasatin deficiency from the progeny population obtained by the step described in (P) above using the genotyping method described in [4].

According to the present invention, the function-deficient glucorafasatin synthase gene and its gene sequence information can be effectively used for breeding radish. Thereby, according to the present invention, with respect to problems of quality deterioration such as flavor deterioration, generation of abundant odor, and yellowing change in radish processed foods, etc. It becomes possible to produce the radish system shown in a short period of time with high accuracy.

It is the schematic diagram which showed the degradation pathway regarding glucorafasatin about the common radish variety system | strain whose glucosinolate composition is usually a wild type.

(FIG. 2A) It is the photograph image figure which image | photographed the external appearance shape of the plant body about NR154E system | strain. The symbols in the figure indicate the following. Reference numeral 1: radish. Code 2: A petiole. (FIG. 2B) About the NR154E strain, it is a schematic diagram showing the influence on the degradation pathway due to glucorafasatin deletion.

In Example 1, it is the figure which showed the linkage map (linkage group R1-3) clarified by linkage analysis.

In Example 1, it is the figure which showed the linkage map (linkage group R4-6) clarified by linkage analysis.

In Example 1, it is the figure which showed the linkage map (linkage group R7-9) clarified by linkage analysis.

In the positional cloning method which concerns on Example 1, it is the flowchart which showed typically description of each process. The number indicated by “N” in the figure indicates the number of plants subjected to linkage analysis or high-precision linkage analysis.

In the high precision linkage analysis which concerns on Example 1, it is the schematic diagram which showed the seating area | region of the glucorafasatin deficiency causative gene narrowed down. Numbers 1-7 in the figure indicate candidate genes 1-7. The arrows in the figure indicate genes that are predicted to exist by sequence analysis. The direction of the arrow indicates the direction in which the gene sits.

FIG. 8 is a photographic image obtained by photographing an electrophoresis gel after RT-PCR of gene 3 (glucorafasatin synthase gene, gray arrow in FIG. 7) in expression analysis by RT-PCR according to Example 2. FIG. The abbreviations in the figure indicate the next radish variety lineage name. “TIB”: disease-resistant total fat. “NR154E”: NR154E.

In the sequence analysis which concerns on Example 3, it is the figure which showed the prediction result of the domain structure which exists in gene 3 protein.

(FIG. 10A) In the sequence analysis based on Example 3, it is the figure which showed typically the gene structure of the wild type gene 3 by the exon / intron structure. (FIG. 10B) In the sequence analysis according to Example 3, the gene structure of NR154E type gene 3 (function-deficient gene) is schematically shown by exon / intron structure. (FIG. 10C) In the sequence analysis according to Example 3, the gene structure of MR050E type gene 3 (function-deficient gene) is schematically shown by exon / intron structure.

(FIG. 11A) In the sequence analysis according to Example 3, an insertion site of an insertion sequence of NR154E gene 3 (function deficient gene 3) is shown. In the figure, the one-letter code of English letters at the bottom of the base sequence indicates an amino acid designated by a codon. (FIG. 11B) In the sequence analysis which concerns on Example 3, it is the figure which showed the introduction site | part of the insertion sequence of MR050E type gene 3 (function deficient gene 3). In the figure, the one-letter code of English letters at the bottom of the base sequence indicates an amino acid designated by a codon.

In the sequence analysis which concerns on Example 3, it is the figure which showed the alignment result of the prediction amino acid sequence of the gene 3 from each radish system. The ellipsis in the figure indicates the predicted amino acid sequence of the next protein. “WT”: HAGHN strain. “MR050E”: MR050E system. “NR154E”: NR154E strain. In addition, a region surrounded by a rectangular region in the figure indicates the next domain. “White”: non-haem dioxygenase in morphine synthesis N-terminal domain. “Gray”: 2-oxoglutarate and Fe (II) -dependent oxygenase domain.

FIG. 6 shows the measurement results of the relative expression level of gene 3 in the expression analysis by quantitative RT-PCR according to Example 4. The abbreviations in the figure indicate the next radish variety lineage name. “HAG”: HAGHN. “TIB”: disease-resistant total fat. "MYS": Miyashige Daikon. “KRM”: pungent 199. “NR154E”: NR154E. “MR050E”: MR050E.

(FIG. 14A) In the gene introduction test according to Example 5, it is a view showing a photographic image obtained by photographing a growing plant body (empty vector-introduced Arabidopsis thaliana) before extracting from top view. (FIG. 14B) In the gene introduction test which concerns on Example 5, it is a figure which shows the photograph image which image | photographed the growth plant body (Gene 3 overexpression body Arabidopsis thaliana) before extraction from top view.

FIG. 15A is a diagram showing a glucosinolate profile of a grown plant body (empty vector-introduced Arabidopsis thaliana) by HPLC analysis chromatograph in the gene introduction test according to Example 5. The symbols in the figure indicate the following. Reference numeral 21: peak of glucoerucin. Reference numeral 22: peak of glucorafasatin. (FIG. 15B) In the gene introduction test according to Example 5, a glucosinolate profile of a grown plant body (Gene 3 overexpressing Arabidopsis thaliana) is shown by HPLC analysis chromatograph. The symbols in the figure indicate the following. Reference numeral 21: peak of glucoerucin. Reference numeral 22: peak of glucorafasatin.

It is the schematic diagram which showed the reaction in which glucorafasatin is produced | generated from glucoerucin in wild type radish or gene 3 overexpression body Arabidopsis thaliana.

It is the schematic diagram which showed an example of the design example of the primer set for genotype determination of a glucorafasatin synthetase gene locus. The symbols in the figure indicate the following. Code 31: Genomic DNA constituting the glucorafasatin synthase gene. Code 32: Insertion sequence in a mutant gene of function-deficient type. Reference numeral 33: a primer for detecting a function-deficient gene. Reference numeral 34: a primer for detecting a wild type gene. Symbol 35: common primer.

In the genotyping example which concerns on Example 6, after performing PCR using the primer set for genotyping, it is the photograph image figure which image | photographed the electrophoresis gel.

This application is an application with a priority claim based on Japanese Patent Application No. 2014-226635 filed by the applicant in Japan on November 7, 2014, the entire contents of which are incorporated into this application by reference.

Hereinafter, embodiments of the present invention will be described in detail.
The present invention relates to an invention related to a function-deficient glucorafasatin synthase gene in Japanese radish. The present invention also relates to an invention relating to a technique for producing a radish line lacking glucorafasatin using the function-deficient gene. In addition, the present invention relates to an invention relating to a radish line having the function-deficient gene in a homo form at the genomic glucorafasatin synthase locus.

1. Explanation of Terms Terms used in this specification will be described below.
In the present specification, “radish plant” refers to a plant species belonging to the Brassicaceae genus Raphanus sativus. More specifically, there are over 1000 varieties of varieties, including Japanese radish (R. sativus var. Longipinnatus), radish (R. sativus var. Sativus), black radish (R. sativus var. Niger), etc. It is known to exist. In radish plants, the enlarged roots are mainly edible and used as processed foods. Sprouts, baby leaves and leaves are also edible.
As used herein, “hypertrophic root” refers to the mature root and hypocotyl portion of radish.

In the present specification, “gene” refers to a region in genomic DNA composed of exons and introns, from the most upstream base of the first exon (5 ′ end of ORF: transcription start point) to the top of the last exon. Refers to the region up to the downstream base (3 ′ end of ORF).
In the present specification, the “coding region (CDS)” refers to a region that is translated into a protein in a gene, and refers to a region from the first base of the start codon to the third base of the stop codon in the exon. In eukaryotic genomic DNA, it is often separated by introns.

In the present specification, “glucosinolate” (in this specification, sometimes simply referred to as “GSL”) is a general term for sulfur-containing compounds that are also referred to as mustard oil glycosides. It is a secondary metabolite that is characteristically contained in plants of the family. More than 120 glucosinolates with different compound structures have been reported for Brassicaceae plants. In radish, the main glucosinolate is a substance called “glucorafasatin”. 90% or more of the total amount of glucosinolate contained in radish radish is occupied by glucorafasatin.

In this specification, “Glucoraphasatin” refers to a compound having the chemical formula shown below (Formula 1). Glucorafasatin is a glucosinolate that is characteristically present in radish and is a compound that is hardly present in other plants. Even in the same cruciferous plant as Raphanus to which radish belongs (for example, Brassica which is a related genus), a plant containing glucorafasatin as a dominant glucosinolate component has not been confirmed other than radish.

In the present specification, “wild type radish variety line” refers to a radish variety lineage that exhibits the property of containing glucorafasatin as a main glucosinolate component in a radish plant.

2. Function-deficient glucorafasatin synthase gene The “function-deficient glucorafasatin synthase gene” according to the present invention (sometimes simply referred to as “function-deficient gene” in the present specification) It refers to a mutant gene of the glucorafasatin synthase gene in which a function-deficient mutation exists in the satin synthase gene.

[Wild-type glucorafasatin synthase gene]
The “glucorafasatin synthase gene” (in the present specification, sometimes simply referred to as “wild type gene”) is a novel gene found by the present inventors, and is obtained from glucoercin to glucorafasatin in radish. A gene encoding a protein involved in a synthetic reaction. Specifically, it refers to a gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 5.
The amino acid sequence of the gene is identical to the amino acid sequence described in SEQ ID NO: 5 in varieties such as Miyashige radish inbred line, Nishimachi ideal inbred line, and pungent radish inbred line. It has been confirmed that this is an amino acid sequence.

In addition, the glucorafasatin synthase gene allows gene sequence mutations involving substitution, deletion, insertion, and / or addition of amino acid residues due to differences between cultivar lines in radish species.
Specifically, the allowable range of the variation of the gene sequence is i) a gene encoding a protein comprising an amino acid sequence showing high sequence homology to the amino acid sequence described in SEQ ID NO: 5, and ii) As long as it is a gene encoding a protein consisting of an amino acid sequence that guarantees gene function, mutation to the amino acid sequence shown in SEQ ID NO: 5 is allowed.

Here, i) “high sequence homology” specifically refers to 95% or more, preferably 96% or more, more preferably 97% or more, more preferably, with respect to the amino acid sequence shown in SEQ ID NO: 5. A sequence homology of 98% or more, particularly preferably 99% or more can be mentioned.
Ii) “Guaranteed gene function” means that the encoded protein is guaranteed to be a protein involved in the synthesis reaction from glucoerucin to glucorafasatin.
Specifically, when the gene is expressed at a normal expression level in a radish plant and the encoded protein is normally synthesized in vivo, the radish is a “glucorafasatin-containing” phenotype. Indicates that
Here, “glucorafasatin-containing” specifically means that the content of glucorafasatin with respect to the dry weight of the beetroot exceeds 3 μmol / g, preferably 4 μmol / g or more, more preferably 6 μmol / g. As mentioned above, it refers to the property of exhibiting a phenotype of 10 μmol / g or more.

A “glucorafasatin synthase” that is a protein encoded by the gene is an enzyme protein involved in a reaction for synthesizing glucorafasatin from glucoerucin. The protein functions as an enzyme involved in any of the reactions for producing glucorafasatin from glucoerucin. When the function of the enzyme is lost, glucorafasatin in the radish plant is lost.
The protein has a domain structure of 2-oxoglutarate-iron (II) -dependent oxygenase domain (also referred to as 2-oxoglutarate and Fe (II) -dependent oxygenase domain, 2 OG-Fe (II) oxygenase domain). The domain is a region corresponding to an amino acid sequence consisting of 97 amino acid residues from the 223rd to the 319th in the amino acid sequence shown in SEQ ID NO: 5 (FIGS. 9 and 12).
Proteins having a 2-oxoglutarate-iron (II) -dependent oxygenase domain form a superfamily in plants and are known to be enzymes including various oxidoreductases. The presence of an enzyme having the same activity as rafasatin synthase has not been reported.
In addition, the protein has a non-oxidase domain that is a kind of oxidoreductase domain in a region corresponding to the amino acid sequence consisting of 114 amino acid residues from the 65th to the 178th amino acids in the amino acid sequence shown in SEQ ID NO: 5. There is also a haem dioxygenase in morphine synthesis N-terminal domain (FIGS. 9 and 12).

The genome sequence of the wild-type glucorafasatin synthase gene is not particularly limited as long as it is a gene encoding a protein that is the above-mentioned glucorafasatin synthase. It is. In the Miyaji radish inbred line, the full length of the genome sequence of the wild-type glucorafasatin synthase gene (the most downstream base of the first exon to the most downstream base of the third exon) is represented by SEQ ID NO: 2. It is a base sequence.
In addition, it has been confirmed that the genome structure of the glucorafasatin synthase gene is a structure composed of three exons and two introns in the existing wild type radish variety line (FIG. 10A). It is not limited to those having the same structure as the exon and intron structures.
Note that the number of glucorafasatin synthase loci in the radish genome is recognized as a single locus. In addition, the existence of a gene showing extremely high homology showing functional complementarity and redundancy to the gene has not been confirmed.

In other cruciferous plants other than radish, there is a homologous gene of a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 5. Specifically, Arabidopsis (a related genera of Raphanus to which radish belongs) Arabidopsis thaliana has a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 5 and an amino acid sequence having about 80% sequence homology. Exists.
However, Arabidopsis has no glucorafasatin in the glucosinolate composition. In this regard, Arabidopsis thaliana has a “homologous gene” of a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 5, but the encoded protein of the homologous gene is used in a synthesis reaction from glucoerucin to glucorafasatin. Does not have the function involved.

[Characteristics of functional deficient glucorafasatin synthase gene]
The function-deficient glucorafasatin synthase gene according to the present invention is a gene having a mutation that lacks an important function as a gene on the base sequence in the exon constituting the glucorafasatin synthase gene.
Here, whether or not the glucorafasatin synthase gene is a “function-deficient gene” depends on whether the locus of the glucorafasatin synthase gene in radish is a homozygous form of the mutant gene. If glucorafasatin in the body is completely or substantially deleted, it can be determined to be a function-deficient gene.

In the present specification, the determination of whether or not “a state in which glucorafasatin is substantially deleted in the radish plant” is based on the following value. When the glucorafasatin synthase locus in radish is homozygous for the mutant gene of interest, the glucorafasatin content relative to the dry weight of the radish root is 3 μmol / g or less, preferably 2 μmol / g or less. If it is preferably 1 μmol / g or less, more preferably 0.1 μmol / g or less, particularly preferably 0.01 μmol / g or less, it may be determined that glucorafasatin is in a substantially deleted state. it can.
Incidentally, most of the wild radish variety lines have a content of several tens to several hundreds μmol / g.

As another determination index, when the glucorafasatin synthase locus in radish is homozygous for the mutant gene of interest, the glucorafasatin content relative to the dry weight of the enlarged radish is the total glucosinolate. If the amount is 1/10 or less, preferably 1/100 or less, more preferably 1/1000 or less, it can be determined that glucorafasatin is substantially absent.
Further, it is more preferable that the ratio index with respect to the total glucosinolate amount and the index of the glucorafasatin content are combined as a determination index.
Here, the measurement of glucorafasatin and glucosinolate content can be carried out by HPLC analysis or the like by a conventional method.

Most preferably, when the glucorafasatin synthase locus in radish is a homozygous form of the mutant gene, a state in which no glucorafasatin is contained in the hypertrophic root (completely deleted state) The function deficient gene is most preferred.

・ Functional deficiency in protein structure In order for the glucorafasatin synthase gene to become a function deficient gene, the amino acid sequence constituting the coding protein of the gene has a mutation involving substitution, deletion, insertion and / or addition. It is necessary. Specifically, in order for the glucorafasatin synthase gene to become a “function-deficient gene”, the encoded protein of the gene is all or part of the “2 oxoglutarate-iron (II) -dependent oxygenase domain” It is preferable that the mutation be accompanied by deletion of.
Here, the “mutation that partially deletes” means a deletion of 1 amino acid residue or more, preferably 5 amino acid residues or more, more preferably 10 amino acid residues or more, out of 97 amino acid residues constituting the domain. Mutations with loss can be mentioned. In particular, 16 amino acid residues or more, preferably 32 amino acid residues or more, more preferably 48 amino acid residues or more, still more preferably 64 amino acid residues or more, particularly preferably 72 amino acid residues constituting the C-terminal side of the domain. In the case of a mutation involving deletion of more than one group, it is more suitable as a function-deficient mutation of the gene.
In the present invention, the mutation is most preferably a mutation that deletes the entire domain. The “mutation that deletes all” means a mutation that involves deletion of the entire domain of the domain consisting of 97 amino acid residues constituting the domain.

The site where the mutation is present is not particularly limited as long as it is a mutation involving the above-mentioned “domain deletion”, but is preferably a mutation in the first exon or the second exon, more preferably a mutation in the first exon. It is preferable that
Specifically, mutations involving the “domain deletion” include i) mutation due to substitution, insertion, or deletion accompanied by the appearance of a stop codon in the base sequence within the coding region, and ii) insertion of the base sequence. Examples of the mutation include a mutation accompanied by the appearance of a stop codon on the inserted nucleotide sequence, and iii) a mutation caused by insertion or deletion accompanied by the appearance of a stop codon due to a frame shift.

-Gene function deficiency due to reduced gene expression The function-deficient glucorafasatin synthase gene according to the present invention has its structural function deficiency even if it is assumed that the structural protein deficiency is somewhat insufficient. When the “gene expression level” is greatly reduced, it is recognized as a function-deficient gene.
For example, although there is a mutation in the coding region, even if it is assumed that the lack of enzyme function on the amino acid sequence is somewhat insufficient, if the gene expression level is significantly reduced, Glucorafasatin in the radish plant is completely or substantially deleted. That is, the mutant gene is a “function-deficient gene” as a glucorafasatin synthase gene. In addition, the state in which glucorafasatin is deleted or substantially deleted in the radish plant refers to the state described in the above paragraph “2. function-deficient glucorafasatin synthase gene”.

A mutant gene having a gene expression level greatly reduced is preferably a mutant gene having an “insertion mutation” in an exon. In this case, it is recognized that the gene expression level tends to decrease as the transcribed mRNA length increases. For example, by having an insertion mutation of at least 1 kbp, the gene expression level is greatly reduced. Examples of the inserted base length include 1 kbp or more, preferably 1.2 kbp or more, more preferably 3 kbp or more, still more preferably 5 kbp or more, particularly preferably 8.8 kbp or more, and further preferably 9 kbp or more.
The upper limit of the inserted sequence is not particularly limited, but if it is strongly mentioned, it can be 1000 kbp or less, preferably 500 kbp or less, more preferably 100 kbp or less, and still more preferably 50 kbp or less.
For example, when the expression level of the glucorafasatin synthase gene is compared with the cultivar “Disease-resistant total fat” (usually wild type which is a general glucosinolate composition), an insertion mutant gene of about 1.2 kbp is homozygous. In the Japanese radish mutant, the gene expression level is drastically reduced to about 1/40.
In particular, a homozygous mutant of an insertion mutant gene of about 9 kbp is about 1/1000 or less, and gene expression hardly occurs. The mutant is close to the null mutant.

-Range of the number of mutations The function-deficient glucorafasatin synthase gene according to the present invention allows the presence of mutations other than the mutations accompanied by the above-mentioned "function deficiency", but the upper limit of the number of mutations in the gene For, the range of the number of mutations can be defined as follows:

The range of the “number of mutations” includes the number of mutations present in the “region corresponding to the coding region” of the gene consisting of the base sequence described in SEQ ID NO: 2 when compared with the base sequence described in SEQ ID NO: 2. Can be defined to be less than a certain number. Specifically, the number of mutations due to substitution, insertion, and / or deletion present in the “region corresponding to the coding region” in the mutant gene is the coding region in the gene comprising the base sequence described in SEQ ID NO: 2 , 5% or less, preferably 4% or less, more preferably 3% or less, even more preferably 2% or less, and particularly preferably 1% or less.

Here, the “number of mutations” in this specification is a number indicating the number of mutations (that is, the number of mutations introduced). For example, if a 100 bp insertion sequence is introduced by “one time” insertion mutation, the number of mutations is “1”. On the other hand, if the 1 bp substitution mutation is independently introduced at “10 sites”, the number of mutations is “10”.
For example, since the number of bases in the coding region (SEQ ID NO: 4) in the base sequence shown in SEQ ID NO: 2 is 1119 bp, the number corresponding to 5% thereof is about “56”. Even if it is assumed that all of the mutations are mutations involving amino acid mutations, about 85% of the amino acid sequence shown in SEQ ID NO: 5 (amino acid sequence constituting wild-type glucorafasatin synthase) It is included in the range of amino acid sequences having high sequence homology.

In addition, the range of the number of mutations of the function-deficient glucorafasatin synthase gene according to the present invention is defined as described above. You can also.
Specifically, when compared with the base sequence described in SEQ ID NO: 2, the number of mutations due to substitution, insertion, deletion, and / or addition present in the “entire region” of the mutant gene is SEQ ID NO: 20% or less, preferably 15% or less, more preferably 10% or less, still more preferably 5% or less, particularly preferably 2% or less, more preferably 1% or less of the number of bases constituting the base sequence described in 2, Can be defined as a number. Here, the “number of mutations” has the same meaning as described above, and indicates the number of mutations (that is, the number of mutations introduced).

-Specific examples of function-deficient genes Examples of the function-deficient glucorafasatin synthase genes as described above include the function-deficient mutant genes described in the examples described later.
Specifically, the glucorafasatin synthase gene (NR154E type gene) including the base sequence set forth in SEQ ID NO: 7 can be exemplified. The function-deficient gene is an insertion sequence of about 9 kbp (from the 558th position in SEQ ID NO: 7) between the 557th base and the 558th base of the wild-type glucorafasatin synthase gene (SEQ ID NO: 2). This is a mutated gene having a mutation introduced in the base sequence consisting of the bases up to the 9410th position and having a stop codon appearing in the inframe. The encoded protein has a structure in which the 2oxoglutarate-iron (II) -dependent oxygenase domain is completely deleted.
Moreover, the glucorafasatin synthase gene (MR050E type gene) containing the base sequence of sequence number 9 can also be mentioned. The function-deficient gene is an approximately 1.2 kbp insertion sequence (1244 in SEQ ID NO: 9) between the 1243th base and 1244th base of the wild-type glucorafasatin synthase gene (SEQ ID NO: 2). (Mutant base sequence consisting of the 2nd to 2465th bases) is introduced, and is a mutant gene in which a stop codon appears in an inlay. The encoded protein has a structure in which 16 amino acid residues on the C-terminal side of the 2-oxoglutarate-iron (II) -dependent oxygenase domain are deleted.

3. DNA marker for genotyping In the present invention, the presence of a “mutation causing a functional defect” of the glucorafasatin synthase gene in the radish genome is used as a DNA marker for genotyping the glucorafasatin synthase gene. It becomes possible.
That is, in the present invention, it is possible to determine the “genotype” at the glucorafasatin synthase gene locus with high accuracy by detecting the presence or absence of a mutation that causes a functional defect of the glucorafasatin synthase gene. .

When the function-deficient mutation is used as a DNA marker, it is understood that in principle, a phenomenon that does not match the target genotype by recombination separation does not occur because the mutation is a mutation in the glucorafasatin synthase gene itself. The That is, the genotype of the glucorafasatin synthase gene can be determined with extremely high accuracy.
On the other hand, when using genotypes such as SNP (single nucleotide polymorphism) and SSR (microsatellite polymorphism) which are linkage markers as an index, unless the region in a very close region is used, linkage is performed by recombination separation. The marker type and target genotype may not match. In this case, an accurate genotype cannot always be determined.

Also, in the present invention, by determining the genotype of the glucorafasatin synthase gene, the “phenotype” relating to the glucosinolate composition of an individual having the genotype can be determined with high accuracy. That is, whether a target radish individual belongs to a glucorafasatin-deficient line or an individual belonging to a glucorafasatin-containing line simply by examining the genotype using a general nucleic acid detection technique Can be identified with high accuracy.

The function-deficient mutation can be easily detected by using an existing nucleic acid detection technique. For example, a technique using a PCR method and a technique using a hybridization method can be used. It is also possible to use a polymorphism detection technique such as a real-time PCR method using a probe and a primer in combination, or a dot blot method using a combination of a mutant type and a wild type probe. It is also possible to detect mutations by restriction enzyme fragment length polymorphism, sequencing by a sequencer, and the like.

[Oligonucleotide primer]
In order to detect the function-deficient mutation by a nucleic acid detection technique, a method using a PCR method using an oligonucleotide primer is effective.
In the present specification, an “oligonucleotide primer” is an oligonucleotide synthesized by polymerizing deoxynucleotides, which functions as a starting point for a DNA extension reaction in a PCR reaction and specifically hybridizes to a specific base sequence. It refers to a single-stranded DNA designed to be composed of sequences.

As the oligonucleotide primer, a primer comprising at least 12 bases in succession of a base sequence constituting a glucorafasatin synthase gene (function-deficient gene, wild type gene) or a base sequence included in its complementary sequence is suitable. is there. As a suitable base length (base: mer), the base sequence contained in the base sequence or its complementary sequence is continuously at least 12 bases, preferably 15 bases or more, more preferably 20 bases or more, further preferably A primer containing 25 or more bases is preferred.
The upper limit of the base length of the oligonucleotide primer is not particularly limited as long as it does not deviate from the range of functioning as a primer. For example, the base length of the entire primer length is 200 bases or less, preferably 100 bases or less. Preferably, those having 50 bases or less can be mentioned.
In addition, in order to increase sequence specificity and reduce non-specific PCR amplification, it is preferable to design an appropriate primer having a long base length after matching the Tm value with the primer forming the primer pair. It is.
The 5 ′ end of the oligonucleotide primer may be added with a restriction enzyme site for use in vector insertion or the like or a modified base sequence for introducing various vectors. Moreover, the thing of the form which couple | bonded the fluorescent substance, the labeling substance, etc. may be sufficient.
Moreover, it is preferable that the 3 ′ end of the oligonucleotide primer is a nucleotide sequence that completely matches the nucleotide sequence constituting the function-deficient gene or its complementary sequence.

-Primer set for detecting a function-deficient gene In the present invention, a genotype having a function-deficient gene is determined by designing a primer set that can specifically detect the presence of the function-deficient glucorafasatin synthase gene. Is possible. Here, as a primer set capable of detecting the presence of a function-deficient gene, “a base sequence including a function-deficient mutation” or “a base sequence constituting a function-deficient mutation” can be specifically amplified by a PCR reaction. This refers to a primer set consisting of a primer pair (

primers

1 and 2 for detecting a defective gene).

As a primer set for detecting the function-deficient mutation using the PCR method, it is necessary to design “function-deficient gene detection primer 1”. The function-deficient gene detection primer 1 is an oligonucleotide primer including a base sequence capable of detecting the “presence” of the function-deficient mutation.
As long as the conditions described in (1) or (2) below are satisfied, the designable position of the primer 1 for detecting a defective gene is on a genomic DNA region on the same chromosome as the functional defective glucorafasatin synthase gene. If so, it is possible to design a base sequence in any region (a base sequence constituting the region or a complementary sequence thereof). That is, it is possible to design not only the region of the function-deficient glucorafasatin synthase gene but also the promoter and spacer region around it.

Specific examples of the primer 1 for detecting a function-deficient gene include (1) a primer containing a “base sequence specific to a function-deficient gene” present in the function-deficient gene. That is, the primer is a primer that does not hybridize to the wild type gene. In this case, PCR is performed on the function-deficient gene by performing PCR with the function-deficient gene detection primer 2 (described later), but the PCR amplification product is not obtained for the wild-type gene. Set. For example, in the case of i) an insertion mutation, a primer containing a base sequence on the insertion mutation or a complementary sequence thereof can be mentioned. In addition, in the case of ii) substitution, deletion, or insertion mutation, a primer including a base sequence containing the mutation in the sequence (preferably a base sequence having a mutation site at the 3 ′ end) can be mentioned. That is, these primers are primers that do not hybridize to the wild type gene.
In addition, as a primer 1 for detecting a function-deficient gene, (2) when there is a function-deficient mutation due to insertion or deletion in a function-deficient gene, a primer designed at “one of the positions sandwiching the mutation” Can be mentioned. In this case, PCR amplification products having different base lengths are obtained for the function-deficient gene and the wild-type gene by performing PCR with the primer 2 for detecting the function-deficient gene. For example, iii) a primer containing a base sequence on the 5 ′ side of the mutation in the case of an insertion or deletion mutation can be mentioned.

It should be noted that the region for designing the function-deficient gene detection primer 1 is preferably designed in the 5 ′ side region close to the boundary between the insertion sequence and the original gene sequence in the case of i). It is. In the case of ii) above, it is preferable to design the mutation so that the mutation is contained on the 3 'side (preferably at the 3' end) in the primer sequence. In the case of (iii) above, it is preferable to design in the 5 'region adjacent to the function-deficient mutation.

In order to detect the function-deficient mutation by the PCR method, it is necessary to design “a primer 2 for detecting a function-deficient gene”. The function-deficient gene detection primer 2 is an oligonucleotide primer comprising the above-described function-deficient gene detection primer 1 and a base sequence in a position capable of forming a primer pair in the PCR reaction.
The region capable of designing the function-deficient gene detection primer 2 is a radish genome region that forms a primer pair with the above-described function-deficient gene detection primer 1 and is capable of PCR amplification. Alternatively, it is possible to design a region on the same chromosome as the gene (a base sequence constituting the region or a complementary sequence thereof).
In consideration of the technical point of PCR, it is preferable that the base length of the amplification product is a region suitable for PCR amplification as a region where the function-deficient gene detection primer 2 can be designed. Specifically, it is preferable to design the function-deficient gene detection primer 2 at a position where the base length (base pair: bp) of the PCR amplification product is 5 kbp or less. The base length is preferably 30 bp to 5 kbp, preferably 50 bp to 3 kbp, more preferably 75 bp to 2 kbp, and still more preferably 100 bp to 1.5 kbp. In addition, when the amplification length is designed to be too long, it is difficult to obtain a PCR amplification product, which is not preferable.

Specifically, regions capable of designing the function-deficient

gene detection primers

1 and 2 include SEQ ID NOs: 1, 2, or 7 so as to satisfy the above conditions when detecting mutations in the NR154E type gene. It can be designed on the described base sequence or its complementary sequence. In addition, when detecting a mutation in the MR050E type gene, an oligonucleotide primer can be designed on the base sequence described in SEQ ID NO: 1, 2, or 9 or its complementary sequence so as to satisfy the above conditions.

-Primer set for wild-type gene detection Further, in the present invention, by further designing a primer set capable of specifically detecting the presence of the wild-type glucorafasatin synthase gene, it is possible to determine the genotype in more detail. Become. Here, as a primer capable of detecting a wild type gene, a primer pair (

primers

1 and 2 for detecting a wild type gene) that can specifically amplify a base sequence constituting a wild type glucorafasatin synthase gene by PCR reaction. ), And a primer set.

As a primer set for specifically detecting the wild type gene using the PCR method, it is necessary to design “Primer 1 for detecting wild type gene”. The wild-type gene detection primer 1 is an oligonucleotide primer including a base sequence capable of detecting “absence” of the function-deficient mutation.
As long as the designable position of the wild-type gene detection primer 1 satisfies the conditions described in (3) or (4) below, if it is on a genomic DNA region on the same chromosome as the glucorafasatin synthase gene, It is possible to design a nucleotide sequence in any region (a nucleotide sequence constituting the region or a complementary sequence thereof). That is, it is possible to design not only the region of the wild-type glucorafasatin synthase gene but also the promoter and spacer region around it.

Specific examples of the primer 1 for detecting a wild type gene include (3) a primer containing a base sequence specific to a wild type that does not exist in a function-deficient gene. In this case, the primer set is such that PCR amplification is performed for the wild-type gene but no PCR amplification product is obtained for the function-deficient gene. That is, by performing PCR with the wild-type gene detection primer 2 (described later), PCR amplification is performed for the wild-type gene, but no PCR amplification product is obtained for the function-deficient gene. Become. For example, when the function-deficient mutation is present in a function-deficient gene due to substitution, deletion, or insertion, a base sequence having a wild-type site corresponding to the function-deficient mutation (preferably, a wild-type corresponding to the function-deficient mutation) And a primer including a base sequence having a type site at the 3 ′ end. That is, the primer does not hybridize with a base sequence having a function-deficient mutation.
In addition, the wild-type gene detection primer 1 was designed at one of the positions sandwiching the corresponding wild-type site when there is a function-deficient mutation due to insertion or deletion in the function-deficient gene (4) A primer can be mentioned. In this case, by performing PCR with the wild-type gene detection primer 2, a primer set is obtained in which PCR amplification products having different base lengths are obtained between the wild-type gene and the function-deficient gene. For example, when an insertion or deletion mutation is present in the function-deficient gene, a primer including a base sequence on the 5 ′ side of the wild-type site corresponding to the function-deficient mutation can be exemplified.

In the case of (3) above, the region for designing the wild-type gene detection primer 1 is preferably the 3 ′ side (preferably the 3 ′ end) of the primer sequence corresponding to the mutation. It is preferable to design so as to be included in In the case of (4) above, it is preferable to design in the 5 'side region close to the function-deficient mutation.

In order to distinguish and detect the wild-type gene from the function-deficient gene by the PCR method, it is necessary to design the “wild-type gene detection primer 2”. The wild-type gene detection primer 2 is an oligonucleotide primer including the above-described wild-type gene detection primer 1 and a base sequence at a position where a primer pair can be formed in the PCR reaction.
The region where the wild type gene detection primer 2 can be designed is a wild type gene or the gene thereof as long as it is a radish genome region capable of PCR amplification by forming a primer pair with the wild type gene detection primer 1 described above. A region on the same chromosome as (a base sequence constituting the region or a complementary sequence thereof) can be used.

Considering the technical point of PCR, it is preferable that the base length of the amplification product is a region suitable for PCR amplification as a region where the wild-type gene detection primer 2 can be designed. Specifically, it is preferable to design the wild-type gene detection primer 2 at a position where the base length (base pair: bp) of the PCR amplification product is 5 kbp or less. The base length is preferably 30 bp to 5 kbp, preferably 50 bp to 3 kbp, more preferably 75 bp to 2 kbp, and still more preferably 100 bp to 1.5 kbp. If the amplification length is designed to be too long, it is difficult to obtain a PCR amplification product, which is not preferable.

Specifically, the region where the wild-type

gene detection primers

1 and 2 can be designed may be designed on the base sequence described in SEQ ID NO: 1 or 2 or its complementary sequence so as to satisfy the above conditions. it can.

-Genotyping by PCR method In order to perform genotyping by PCR method, it is necessary to prepare DNA in the target radish plant as a sample that can be amplified by PCR reaction. Depending on the design conditions of the primer set, it is possible to use a crushed sample, an elution sample, etc., but DNA extraction is preferably performed using a conventional method or a commercially available kit to prepare a sample suitable for PCR reaction It is desirable. Note that since the PCR method is a detection method with extremely high sensitivity, even a very small amount of sample to be prepared (for example, a part of a sprout immediately after germination) can be sufficiently subjected to analysis. The tissue of the radish plant used for sample preparation can be any tissue containing DNA.

In the present invention, by performing a PCR reaction using the above-mentioned “function-deficient

gene detection primers

1 and 2”, the presence of “function-deficient mutation” in the glucorafasatin synthase gene of the target radish is detected. It becomes possible to detect the genotype based on this result.
Specifically, when a PCR amplification product specific for a function-deficient gene is obtained using the primer set described in (1) or (2) above, the genotype of the glucorafasatin synthase locus of the target radish Can be determined to be a “genotype having a function-deficient gene”. That is, in this case, the genotype of the target radish can be determined to be “function-deficient gene homotype” or “heterotype” (function-deficient gene / heterotype of wild-type gene).
In the case of using the primer set described in (2) above, if the base length of the amplification product is appropriate, the “function-deficient gene homotype” and “heterotype” are discriminated by the difference in base length. Is also possible.
Since the radish individuals of these genotypes are individuals having a function-deficient glucorafasatin synthase gene in their genome, they are effective as breeding materials and gene resources for creating glucorafasatin deficient lines. Can be used.

Furthermore, in the present invention, by using “wild type

gene detection primers

1 and 2”, it is possible to distinguish between “function deficient gene homotype” and “heterotype” and more reliably detect them. Become.
Specifically, when a PCR amplification product specific for the wild-type gene is obtained using the primer set described in (3) or (4) above, the genotype of the glucorafasatin synthase gene locus of the target radish is , “A genotype having a wild type gene”. That is, in this case, the genotype of the target radish can be determined to be “wild type gene homotype” or “heterotype”.
Therefore, in the present invention, by using both of the two primer sets of “function-deficient

gene detection primers

1 and 2” and “wild-type

gene detection primers

1 and 2”, “function-deficient gene homotype” It is possible to distinguish genotypes with high accuracy by distinguishing three types of genotypes, “heterotype” and “wild type gene homotype”.

Here, the PCR reaction described above can be performed by a normal PCR method. A method of performing a PCR reaction by binding a labeling substance to dNTP is also possible. After the PCR reaction, it is easily detected whether PCR amplification products are obtained by the usual methods (intercalation method, fluorescence detection method, luminescence detection method, color detection method, antibody detection method, RI detection method, etc.) It is possible. As a simple method, it can be easily detected using ethidium bromide, a fluorescent substance, or the like. It is also preferable to use a technique using a real-time PCR apparatus.

In the present invention, when PCR is carried out using two pairs of primer sets of “function-deficient

gene detection primers

1 and 2” and “wild-type

gene detection primers

1 and 2”, specifically, PCR can be performed by this method.
First, i) as the first method, there can be mentioned a method in which two reaction solutions are prepared for each primer set and PCR reaction is separately performed. In this case, the genotype can be determined by electrophoresis of each reaction solution and integrating the amplification patterns.
In addition, ii) as the second method, there can be mentioned a method in which the two pairs of primer sets are mixed to prepare one reaction solution, and one PCR is performed. In this case, the genotype can be determined from the amplification pattern after electrophoresis in one lane. Therefore, a result can be obtained only by preparing one sample for each sample, which is effective when the number of samples is very large. However, in this method, it is necessary to be able to distinguish a difference between two types of amplification products between an amplification product derived from a wild-type gene and an amplification product derived from a function-deficient gene. For example, it is necessary to design the primer sets so that the base lengths of the amplification products are different, or to add a labeling substance or the like that can distinguish the two to the primers.
Examples of genotypes that can be detected by each primer set are shown in Table 1.

In addition, as an application of the design example of two pairs of primer sets, two pairs of primers are designed by designing the primer set so that the functional deficient gene detection primer 2 and the wild type gene detection primer 2 are shared. There can be mentioned an embodiment in which the set is composed of only three kinds of primers. Specific embodiments in this case include “function-deficient gene detection primer 1”, “primer for common use (primer for function-deficient gene detection and primer for wild-type gene detection that forms a primer pair with the primer”) 2), and “wild type gene detection primer 1” that forms a primer pair with the common primer.

[Oligonucleotide probe]
In the present invention, a method using a technique using an oligonucleotide probe is also effective as a means for detecting the function-deficient mutation.
In this specification, an “oligonucleotide probe” is an oligonucleotide synthesized by polymerizing deoxynucleotides or nucleotides, and is a single-stranded nucleic acid composed of a base sequence that specifically hybridizes to a specific base sequence. It is what you point to. Either DNA or RNA can be used as the nucleic acid molecular species. In addition, nucleic acid analogs such as morpholino oligos can be used.

In order to distinguish and detect the function-deficient gene from the wild-type gene by the hybridization method, it is necessary to design a probe that can specifically detect the presence of the function-deficient mutation.
Here, as a probe for detecting the function-deficient mutation (“function-deficient gene detection probe”), a base sequence constituting a function-deficient glucorafasatin synthase gene or a base sequence included in its complementary sequence Thus, a probe including a base sequence capable of specifically detecting the function-deficient mutation can be exemplified.
Specifically, in the case of i) an insertion mutation, a probe including a base sequence on the insertion mutation or a complementary sequence thereof can be mentioned. In the case of this embodiment, in order to avoid mishybridization with the wild-type gene and ensure the specificity of the detection signal, it preferably does not include the base sequence of the region commonly present in the wild-type gene or its complementary sequence A probe is preferred. In this embodiment, the probe specifically hybridizes to a function-deficient gene but does not hybridize to a wild-type gene.
As another embodiment of the probe, there can be mentioned a probe containing ii) a base sequence containing the mutation site in the case of substitution, deletion, or insertion mutation. In the case of this embodiment, the probe is easy to hybridize to a function-deficient gene but difficult to hybridize to a wild-type gene.

As a means for avoiding mishybridization between the function-deficient gene detection probe and the wild-type gene and improving the detection sensitivity, use of a masking probe can be mentioned. Here, the “masking probe” refers to a probe including a base sequence specific to a wild-type gene or a complementary sequence thereof, and including a base sequence including a wild-type site corresponding to the function-deficient mutation. Is.
Since the masking probe has a higher affinity for hybridizing to the wild-type gene than the function-deficient gene detection probe, the function-deficient gene is present in the coexistence of the function-deficient gene detection probe and the masking probe. It becomes possible to remarkably suppress the detection probe from being mishybridized to the wild type gene. In particular, in the case of the probe described in ii) above, the use of a masking probe is very suitable.

The oligonucleotide probe varies depending on the use of the probe, but at least a base sequence that constitutes a glucorafasatin synthase gene (function-deficient gene, wild-type gene) or a complementary sequence thereof is continuously at least. A probe having 12 bases is preferred. As a suitable base length (base: mer), the base sequence contained in the base sequence or its complementary sequence is continuously at least 12 bases, preferably 15 bases or more, more preferably 20 bases or more, further preferably A probe containing 25 bases or more is preferred.
The upper limit of the base length of the oligonucleotide probe may be the upper limit of the base length itself of the inserted sequence. For example, it may be 1000 bases or less, preferably 500 bases or less, more preferably 100 bases or less. it can.
If a difference of one to several bases is detected by dot blot method, TaqMan (R) probe method, microarray method, etc., it is preferable to use a chain with a short upper limit value to ensure detection sensitivity. is there. In this case, the upper limit is preferably a short probe of about 200 bases or less, preferably 100 bases or less, more preferably about 50 bases or less.
In addition, the 5 ′ end or 3 ′ end of the oligonucleotide probe can be suitably used by binding a fluorescent substance for detection, a labeling substance or the like. Further, at the time of probe synthesis, dNTP or NTP bound with a labeling substance can be incorporated and used as a probe constituent base.

-Genotyping using oligonucleotide probes In order to perform genotyping using probes, it is necessary to prepare a sample capable of PCR reaction using DNA in the target radish plant as a template.
Depending on the hybridization conditions, it is possible to use a disrupted sample or an elution sample. Specifically, a DNA sample is extracted using a conventional method or a commercially available kit to obtain a sample suitable for the hybridization reaction. It is desirable to prepare. Note that since the hybridization method is a detection method with extremely high sensitivity, even a trace amount of sample to be prepared (for example, a part of a sprout immediately after germination) can be sufficiently subjected to analysis. The tissue of the radish plant used for sample preparation can be any tissue containing DNA.
As a signal detection method, is a specific hybridization signal obtained by a conventional method (intercalation method, fluorescence detection method, luminescence detection method, color detection method, antibody detection method, RI detection method, etc.)? Can be easily detected. Preferably, a method in which a fluorescent substance or a labeling substance is bound to the probe itself and the signal of the hybridized probe is specifically detected is suitable.

In the present invention, when a signal specific to a function-deficient gene is obtained using the probe for detecting a function-deficient gene, the genotype of the glucorafasatin synthase locus of the target radish is “function-deficient type”. It can be determined that the genotype has a gene. That is, in this case, the genotype of the target radish can be determined to be “function-deficient gene homotype” or “heterotype”.
Since the radish individuals of these genotypes have the function-deficient glucorafasatin synthase gene in the genome, they are effective as breeding materials and genetic resources for creating glucorafasatin deficient lines. Can be used.

[Use of proximity region linkage marker]
In the present invention, a linkage marker (SNP, SSR, etc.) present in a very close region that cannot be separated from the glucorafasatin synthase gene is used to detect the presence or absence of a mutation in the glucorafasatin synthase gene. It is possible to use. That is, for the adjacent region linkage marker, the presence or absence of “function-deficient mutation” of the locus can be indirectly detected with relatively high accuracy, and the genotype of the glucorafasatin synthase locus can be determined. It becomes possible.

Specifically, such a proximity region is within 200 kbp, preferably within 100 kbp, more preferably within 75 kbp, and even more preferably within 50 kbp adjacent to each of the 3 ′ side and 5 ′ side of the glucorafasatin synthase gene. Particularly preferred is a region within 20 kbp, more preferably within 10 kbp. If such a linked marker is located in a region very close to the glucorafasatin synthase gene locus, separation from the glucorafasatin synthase gene by recombination is extremely difficult. The presence or absence of can be determined. This is a finding that is supported by the fact that in order to use a nearby linkage marker by the positional cloning method, a high-precision linkage analysis using a large-scale segregated population is required.
The detection of the linked marker type existing in the adjacent region can be performed using a technique using a PCR method or a technique using a hybridization method. It is also possible to use a polymorphism detection technique such as a real-time PCR method using a probe and a primer in combination, or a dot blot method using a combination of a mutant type and a wild type probe. It is also possible to detect the marker type by restriction enzyme fragment length polymorphism, base sequence determination by a sequencer, and the like.

As an example of the proximity region linkage marker, the linkage marker described in Example 1 (6) can be used in the case of the NR154E strain described later or a strain derived from the strain. However, in the case of detecting a loss-of-function gene in another line other than the NR154E line (for example, MR050E line), it is necessary to search for and use a nearby linked marker specific to each mutant line. Become.

[System identification]
In the present invention, by determining the genotype of the glucorafasatin synthase gene locus, it is possible to identify the phenotype related to glucorafasatin-containing or deficiency using the determined genotype information. Become. Here, since the genotype uses the mutation of the glucorafasatin synthase gene itself as an index, unlike a method using a normal linkage marker (excluding the above-mentioned proximity region linkage marker), the glucorafasatin deletion It is possible to identify and select a phenotype exhibiting sex with high accuracy.
Of course, when the function-deficient mutation is used as a DNA marker, in principle, more accurate identification and selection are possible than the above-mentioned nearby linked marker.

Since glucorafasatin deficiency is due to a recessive gene, the relationship between genotype and phenotype is as follows.
Specifically, when the genotype is a genotype indicating “wild type gene homotype” and “heterotype”, the radish individual lacks glucorafasatin in the gene responsible for other glucorafasatin metabolism As long as there is no mutation to cause, the phenotype is a phenotype exhibiting glucorafasatin content.
On the other hand, when the genotype is a “function-deficient gene homotype”, it becomes a phenotype exhibiting glucorafasatin deficiency.

As described above, in the present invention, by performing genotype determination using the above primer set and / or probe, a “glucorafasatin deficient strain” resulting from the genotype is identified with high accuracy. It becomes possible.

[Genotyping kit, strain identification kit]
In the present invention, the kit containing the oligonucleotide primer set and / or the oligonucleotide probe can be used as a genotype determination kit for the glucorafasatin synthase gene locus. The kit may be a product kit including a PCR reaction reagent, a hybridization reaction reagent, a detection reagent, and the like.
The oligonucleotide primer set and / or oligonucleotide probe can be fixed to a cellulose carrier such as a membrane or filter paper, a base carrier such as a glass chip, a column carrier such as a synthetic resin, etc., and genotype determination can be easily performed. It can also be a product form.
Further, the genotyping kit may be a product form of a glucorafasatin-deficient strain identification kit as a strain identification kit.

4). In radish genomes with homozygous dysfunctional genes, individuals with the glucorafasatin synthase locus at the glucorafasatin synthase locus as “homotypes” have an glucosinolate composition that lacks glucorafasatin It becomes a radish showing.
Here, “glucorafasatin deficiency” refers to the property that glucorafasatin is deleted or substantially deleted in a radish plant. Specifically, it refers to the property of the glucorafasatin content and / or glucosinolate composition described in the above paragraph “2. Function-deficient glucorafasatin synthase gene”.
In addition, in individuals having a combination of the function-deficient gene and the wild-type gene at the locus (function-deficient gene / heterotype individual of the wild-type gene), an individual exhibiting “glucorafasatin deficiency” In other words, it becomes an individual exhibiting a “glucorafasatin-containing” phenotype, which is usually a wild-type glucosinolate composition.

Here, the “radish plant” that can be homozygous for the function-deficient gene at the glucorafasatin synthase locus is a plant species belonging to the general Raphanus sativus. , Any cultivar line can be mentioned. Specific examples include Japanese radish (R. sativus var. Longipinnatus), Japanese radish (R. sativus var. Sativus), black radish (R. sativus var. Niger), and the like. More specifically, a variety line belonging to Japanese radish (R. sativus var. Longipinnatus) can be mentioned.
Note that the number of glucorafasatin synthase loci in the radish genome is recognized as a single locus. In addition, the existence of a gene showing extremely high homology showing functional complementarity and redundancy to the gene has not been confirmed.
In addition, normal radish is diploid, but if it is a cultivar line in which chromosome polyploidization (for example, double diploidization, tetraploidization, etc.) has occurred, all genes at the gene locus function. By making it a deficient gene (being a homozygous gene), it becomes a radish showing a glucorafasatin deficient phenotype.

[Difficulty of creating homozygous strains with deficient genes]
The causative gene that induces the glucorafasatin deficiency discovered by the present inventors is a positional cloning method (linkage analysis and high-precision linkage analysis) using the NR154E strain, quantitative expression analysis, gain-of-function gene transfer test, It is a causative gene that induces glucorafasatin deficiency, identified by gene sequence analysis. The wild type gene of the causative gene is a gene encoding an enzyme involved in the synthesis reaction of glucorafasatin.

In the NR154E line, the breeding “Nishimachi Ideal” (generally showing a wild-type glucosinolate composition) is used as an original population, and artificial self-propagation and selection of excellent individuals using glucosinolate composition as an index is repeated. This is one of the created lines. Specifically, it is a radish system having a homozygous gene containing the nucleotide sequence set forth in SEQ ID NO: 7 at the glucorafasatin synthase locus in the genome.
The original cultivar “Nishimachi Ideal” is a variety that normally contains wild-type glucorafasatin, and is not known to exhibit glucorafasatin deficiency. The NR154E strain is an indicator of self-breeding operations and glucorafasatin content from within the population of individuals who have a homozygous mutant gene of the function deficiency that has occurred in an artificially cultivated population of the variety “Nishimachi Ideal” It is the system obtained for the first time by selecting as.

Here, “self-breeding operation” of Japanese radish is an artificial isolation operation to pollinate self-pollen within a certain period of time before self-incompatibility before flowering. It is a step of performing an operation of selecting individuals exhibiting a deletion property. In addition, Japanese radish self-pollination is a phenomenon that is unlikely to occur under free mating conditions.
In addition, the phenotype of “glucorafasatin deficiency” is a trait related to the glucosinolate composition related to the endogenous component that cannot be distinguished from the outline of the plant body. It is necessary to carry out by HPLC analysis or the like. That is, the expression type cannot be determined by looking at the form of radish (shape, color, etc.).
As is clear from these points, in order to fix the trait called “glucorafasatin deficiency” within a population, after measuring the glucosinolate content of each individual by HPLC analysis or the like, It is recognized that it is necessary to perform selfing and selection operations.

In order to start research related to the present invention, the present inventors have targeted radish gene resources "more than 650 varieties" that are conserved all over the world. Although exhaustive search was conducted, no radish cultivar line was found in which the trait indicating “glucorafasatin deficiency” was fixed in the population.
The reason why the glucorafasatin-deficient line (specifically, the group including the radish line having the homologous function-deficient gene) was not found in the conventional variety lines is considered as follows. That is, in radish, i) it is difficult for the recessive mutation according to the present invention to occur in a normal state, and ii) the recessive mutation type gene may have been deceived and disappeared before spreading in the population. .

[Mechanism to induce glucorafasatin deficiency]
The glucorafasatin-deficient radish line according to the present invention has a glucorafasatin-deficiency by having a homozygous function-deficient gene at the glucorafasatin synthase locus (action mechanism). It is a radish system.
For example, the NR154E strain is a radish strain having a homozygous gene having a function-deficient mutation in the first exon of the glucorafasatin synthase gene (a gene containing the base sequence described in SEQ ID NO: 7). The MR050E strain is a radish strain having a homozygous gene having a function-deficient mutation in the third exon of the glucorafasatin synthase gene (a gene containing the base sequence described in SEQ ID NO: 9).

Here, in principle, a plurality of genetic factors involved in glucorafasatin metabolism (synthesis, degradation, etc.) in radish are considered to exist in addition to the glucorafasatin synthase gene according to the present invention. Specifically, in addition to the gene, a gene involved in the synthesis and production of glucorafasatin, a gene that regulates the expression of the glucorafasatin synthase gene, a gene involved in the degradation reaction of glucorafasatin, glucorafafa Presence of genes involved in the production of secondary metabolites from satin is presumed.
If any of these genes has a mutation that lacks or overacts its function, it is presumed that the phenotype lacks glucorafasatin.
Although not an example of glucorafasatin metabolism, Arabidopsis thaliana, a related genus plant, may have a Myb transcription factor that is a transcriptional regulatory factor that controls the overall biosynthesis of an aliphatic glucosinolate. Known (Hirai et al., 2007 PNAS, 104: 6478-6483).
The above findings indicate that not all radish strains exhibiting “glucorafasatin deficiency” may be induced by the functional deficiency of the glucorafasatin synthase gene according to the present invention. That is, the phenotype of “glucorafasatin deficiency” may be induced by a functional defect or functional abnormality of another gene different from the glucorafasatin synthase gene, and is completely different from the present invention. The existence of “mechanism” is also assumed.

[Method for producing glucorafasatin deficient strain]
In order to create a radish line that lacks glucorafasatin, it is necessary to create a radish that has the function-deficient gene in a “homotype” at the glucorafasatin synthase locus. For this purpose, it is preferable to use a breeding technique using a radish having the loss-of-function gene at the locus as a “gene donor”.
Here, “radish having the function-deficient gene at the gene locus” used as a gene donor (mating parent) means any individual as long as it is a radish having the function-deficient gene at the gene locus. Can be used. Further, not only individuals having a function-deficient gene in a homo form but also individuals having a hetero form (function-deficient gene / wild type gene hetero form) can be suitably used as a gene donor.

Specifically, as a radish that can be used as a “donor of a function-deficient gene (mating parent)”, i) use of “radish introduced with the function-deficient gene” at the locus it can. Here, as a means for introducing the function-deficient gene into the gene locus, any gene introducing means can be exemplified. For example, radish in which the function-deficient gene is introduced into the gene locus can be exemplified by homologous recombination techniques using gene introduction such as Agrobacterium, electroporation, particle gun, cell fusion and the like. Moreover, radish in which the function-deficient gene has been introduced into the gene locus by an artificial mating technique can be mentioned.
In addition, ii) a radish in which a function-deficient mutation is introduced into a wild-type glucorafasatin synthase gene present at the locus can be used, and the radish can be used as a gene donor. Here, as the introduction of the function-deficient mutation into the gene, any mutation can be mentioned. For example, mutation introduction accompanied by introduction of an insertion sequence via a transposon, retrotransposon, plant virus or the like can be mentioned. In addition, mutation can be introduced by accelerating mutation by performing irradiation treatment on seeds, heavy ion beam treatment, treatment with a solution containing a mutagen substance, and the like. In addition, mutation introduction by gene editing techniques (ZFN, CRISPR, etc.) can be mentioned.
An individual having a function-deficient gene obtained by such mutagenesis can be suitably used as a gene donor.
In addition, iii) when there is a radish having the function-deficient gene at the gene locus already created, the radish can be used as a gene donor.
Iv) A radish belonging to a progeny line derived from any one of the above radish (i) to iii) (a line obtained by self-breeding and / or mating) and having a function-deficient gene at the locus Japanese radish can also be used as a gene donor.

On the other hand, as the other mating parent (desired radish), normally, i) an individual belonging to a radish variety line having a desired trait (desired radish individual) is used as a receptor for the function-deficient gene. be able to.
Here, “desired traits” can include all traits that are advantageous for cultivation characteristics and quality, processed radish products, and the like. For example, traits that improve the quality of enlarged radish (for example, size of enlarged radish, shape of enlarged radish, density of soft tissue of enlarged radish, etc.), traits related to environmental resistance (eg, cold resistance, heat resistance, etc.), disease resistance Traits related to sex, traits related to growth (for example, early or late cultivation period, plant hormone synthesis system, etc.), traits related to reproduction (eg, flowering control, self-incompatibility, cytoplasmic male sterility, etc.), traits related to pigments ( For example, anthocyanin composition, anthocyanin content, etc.) can be mentioned.
As the other mating parent (desired radish), ii) radish having the function-deficient gene can be used as it is. In this case, an operation is performed in which two individuals (same strains or other strains) that are radish having the function-deficient gene are pollinated by cross breeding. Moreover, iii) If desired, it is also possible to obtain a desired Japanese radish by performing crossing by self-pollination (self-breeding operation) without using the other crossing parent.

After performing the above-mentioned mating operation, it is possible to obtain an individual having a desired excellent trait and having the function-deficient gene in a homo form by performing self-breeding operation and selection operation as necessary. . Further, if necessary, a radish individual having further excellent traits can be obtained by repeatedly performing mating with a desired radish individual, self-breeding operation, and selection operation.
In the present invention, a radish line (or cultivar) showing glucorafasatin deficiency can be produced by obtaining a population in which the glucorafasatin deficiency and a desired trait are genetically fixed. .

In the present invention, in the “selection operation” from the progeny population after mating, the genotype of the glucorafasatin synthase gene locus can be determined with high accuracy by using the genotyping method according to the present invention. It becomes possible. This makes it possible to select a desired individual relating to glucorafasatin with high accuracy, and to efficiently produce a glucorafasatin deficient line in a short period of time.

[Plant of glucorafasatin-deficient strain]
The plant obtained from the radish of the glucorafasatin deficient strain according to the present invention can be used very effectively in various fields such as agriculture and food. By using the plant body, in various fields such as radish as agricultural products, radish processed foods (salmon, pickles, radish grated, dried radish, tsuma, etc.), various products (beverages, pigments, etc.) using radish, It becomes possible to fundamentally solve the quality degradation problems such as “flavor deterioration”, “generation of scented odor”, and “occurrence of color unevenness due to yellowing”.
Here, examples of the “plant obtained from radish” include all parts and tissues constituting the radish plant and plants belonging to all growth stages. Specific examples include roots (including lateral roots and undeveloped roots), hypocotyls, leaves, petioles, stems, florets, flowers, seeds, sprout, baby leaves, seedlings, and the like.
In consideration of the use of crops, processed foods, and the like, it is possible to suitably use a radish (part consisting of root and hypocotyl) portion. Sprouts (plants consisting of cotyledons, hypocotyls, and roots immediately after germination) can also be suitably used as fresh food. Baby leaves and leaves can also be used as food.
In addition, seeds and seedlings (preferably seedlings up to about 40 days after germination) can be suitably used for cultivation and breeding.

Hereinafter, the present invention will be described with reference to examples, but the scope of the present invention is not limited thereto.

[Example 1] “Identification of loci by positional cloning”
Linkage analysis by positional cloning and high-precision linkage analysis were performed to identify the locus responsible for the glucorafasatin-deficient trait.

(1) "Glucorafasatin deficient radish line"
In order to identify the causal locus of glucorafasatin deficiency by the positional cloning method, the “NR154E” strain whose glucosinolate composition phenotype exhibits glucorafasatin deficiency was used (FIGS. 2A and 2B).
The NR154E strain is one of a plurality of glucorafasatin-deficient strains independently created by the present inventors. Specifically, the line is a good individual with an index of artificial self-propagation and glucosinolate composition, using the cultivar “Nishimachi Ideal” (generally showing wild-type glucosinolate composition) as the original population. By repeating selection, it is one of the created lines.
The “NR154E” line does not contain glucorafasatin, but instead has a high glucoerucin content, as compared to other normal wild type cultivar lines. Moreover, the property that the total amount of glucosinolate itself is low is shown.

In order to confirm the excellent glucorafasatin deficiency of the “NR154E” strain, the glucosinolate content per dry weight of the radish (the enlarged root and hypocotyl) part was measured. The measurement involves freeze-powdering the radish root with liquid nitrogen, then desulfurizing glucosinolate extracted with 70% methanol and subjecting it to HPLC analysis, and quantifying the glucosinolate composition in terms of sinigrin as an internal standard. I went there.
As a result, as shown in Table 2, no glucorafasatin was detected in the “NR154E” line, indicating that the line exhibits extremely superior properties in terms of glucorafasatin deficiency. (* The “MR050E” line in Table 2 is also a glucorafasatin-deficient line produced by the present inventors, but is a line with a large total amount of glucosinolates originating from a different source from the NR154E line.)

In addition, the present inventors have comprehensively searched for cultivars that show glucorafasatin deficiency for radish gene resources “650 cultivars or more” conserved all over the world. It was not possible to find a breed line in which the glucorafasatin-deficient trait was fixed.

(2) “Separation ratio analysis”
Analysis of the segregation ratio of the backcross (BC ₁ ) population suggested that the glucorafasatin deficiency was due to a single factor recessive gene.

(3) “Identification of causal loci (linkage analysis)”
DNA was extracted from individuals of both “NR154E” and “HAGHN” strains, 2880 randomly selected gene regions were amplified by PCR, and their nucleotide sequences were determined to identify polymorphisms between both strains did.
Of the obtained polymorphisms, a labeled oligonucleotide probe having a nucleotide sequence containing NR154E type SNP was designed in 131 SNP regions. In addition, an unlabeled oligonucleotide probe comprising a nucleotide sequence of SNP HAGHN type (allelic locus) was also designed. A solution containing two kinds of oligonucleotide probes in the relationship of these confrontations was mixed.
A probe solution was similarly prepared for each of the 131 SNPs, and a probe solution for SNP detection corresponding to each of the 131 SNP regions was prepared. In addition, primers that specifically amplify 49 sites were identified.

In order to secure a population for linkage analysis, the “NR154E” line that is deficient in glucorafasatin (mutant type) and the radish inbred line “HAGHN” that is glucorafasatin-containing (usually wild type) by mating, gluco Lafayette satin deletion trait gene responsible (hereinafter, simply referred to as the causative gene.) were grown separated F ₂ population to identify.
DNA was extracted from _{F 2} population 96 individuals relevant breeding, the SNP region of the 131 locations was amplified by PCR, and fixed by spotting the PCR amplification products obtained from each individual for each SNP on the nylon membrane. (That is, 131 nylon membranes spotted with PCR products for each SNP were prepared.)
Using a probe solution for SNP detection corresponding to each nylon membrane (PCR amplification product of each SNP), a dot blot hybridization method (Dot-blot hybridization method; Shirasawa et. Al., 2006 Theor. Appl. Genet. 113: P147-155) by performing, to determine the SNP type 131 locations in _{F 2} population 96 individuals each.
The recombination value between each marker was calculated from the genotype information using JoinMap4.0 (Kyazma), and a linkage map was constructed. In the linkage map, SNPs used for mapping of the causative gene according to the present example are shown in Table 3 and FIGS.

Using this leaf 1g of F ₂ population 96 individuals, by measuring the glucoamylase Lafayette satin content was determined the phenotype of each individual. The measurement of glucorafasatin content was performed by HPLC analysis according to the method described in Ishida et al. (Ishida et al., 2011 Breeding Science 61: 208-211).
Here, as described in (2) above, since it is suggested that the glucorafasatin deficiency trait is due to a single factor recessive gene, the genotype of the causative gene was assumed as follows.
That is, i) It was assumed that the genotype of an individual exhibiting glucorafasatin deficiency was “NR154E homotype”. In addition, ii) the genotype of an individual showing glucorafasatin-containing properties was assumed to be “HAGHN homotype” or “heterotype”.

The recombination titer between the causative gene and each marker was calculated using the predicted genotype of the causative gene in each individual and the information on each marker genotype. Based on the value of the recombination value, the locus region of the causative gene was positioned on the linkage map.
As a result, it was shown that the locus region of the causative gene exists between the SNP markers “CL4624” and “CL6024” (FIG. 6).

(4) “Restriction of causal loci (linkage analysis)”
Using a nearby linkage marker (SNP, SSR, etc.), the causal locus was further narrowed down.
The causative gene Zajo in the vicinity SNP region to determine the _{F 2} population 1358 individuals SNP type of the "CL4624""CL6024", were selected two 44 individuals recombination has occurred between the SNP.
Next, in the region sandwiched between the two SNPs, a DNA marker that can distinguish between the “NR154E” line and the “HAGHN” line was newly searched and identified. The search for the DNA marker and the determination of the DNA marker type were performed according to the procedure for determining the SNP type described in (3) above. As a result of the search, new SNPs and SSRs were identified.
For the 44 individuals selected, the glucorafasatin content in the true leaves was measured in the same manner as in the method described in (3) above, and the phenotype and the genotype of the causative gene were determined. Then, the recombination value between the causative gene and each of the newly found DNA markers was calculated and compared with the recombination value to further narrow down the locus region.
As a result, it became clear that the locus region of the causative gene can be narrowed down between the SSR marker “4D01_NED” and the SNP marker “6C04_post” (about 1 cM) (FIG. 6).

(5) "Genome contig sequence"
Next, using the base sequence and Scaffold sequence (Rsa1.0_00457.1) of the “Aokubi Sh” strain, which is a glucorafasatin-containing (wild-type) strain, the SSR markers “4D01_NED” and SNP described above are used. Screening using the marker “6C04_post” was performed. The radish genome sequence was obtained from Raphanus sativus Genome DataBase (http://radish.kazusa.or.jp/).
As a result, it was clarified that the causative gene sits within a region covered by one Scaffold sequence analyzed by the “Aokubi Sh” strain and a two-clone BAC library (FIG. 6).
From these base sequence information, a genomic contig sequence (genomic sequence of the wild type “Aokubi Sh” strain) that physically covers the locus range of the target causative gene was created.

(6) “High precision linkage analysis”
In order to isolate the causative gene by map-based cloning by high-precision linkage analysis, it is necessary to calculate the recombination titer using a large number of segregated populations. Therefore, self-breeding operation was performed for F ₂ individuals in which the causal locus is “heterotype”, and an F ₃ population consisting of 3840 individuals was bred. Development of F ₃ populations was performed using a 128 hole cell trays, DNA was extracted from each plant.
Paying attention to the SSR markers “sca8159_A02” and “node11_F03” at the position where the causative gene is sandwiched, 21 individuals with recombination between these two markers were selected. The obtained recombinant individuals were transplanted into 9 cm pots.
About the selected individual | organism | solid, the glucorafasatin content in a main leaf was measured similarly to the method as described in said (3), and the phenotype and the genotype of the causative gene were determined.

Next, in order to narrow the locus region of the causative gene, the recombination value between the causative gene and each DNA marker identified in (4) above is calculated with high accuracy and compared with the recombination value. The riding area was further narrowed down.
As a result, it was finally clarified that the causal gene sits within the region of about 23.8 kbp sandwiched between the SSR markers “sca8159_D07” and “ssG02” (FIG. 6).

[Example 2] “Estimation of a causative gene for glucorafasatin deficiency”
In the locus region clarified by the high-accuracy linkage analysis, a glucorafasatin deficient causative gene was estimated from genes existing in the locus region.

(1) “Sequence prediction”
Regarding the locus region of the causative gene identified in Example 1, all genes included in the region were estimated. In order to estimate the gene sequence, sequence analysis was performed by Augustus (http://augustus.gobics.de/) and GENSCAN (http://genes.mit.edu/GENSCAN.html), which are gene prediction programs.
As a result, it was estimated that seven genes (genes 1 to 7) exist in the locus region narrowed down by high-precision linkage analysis (FIG. 7). That is, the analysis showed that the candidates for the glucorafasatin-deficient causal gene could be narrowed down to seven candidate genes.

(2) “Estimation of candidate genes by expression analysis”
Expression analysis was performed on the seven candidate genes (genes 1 to 7) present in the locus region obtained by the high-accuracy linkage analysis to narrow down the candidates for the causative gene.
Total RNA (total RNA) was extracted from the hypertrophic root and true leaves of the “NR154E” strain, which is a glucorafasatin-deficient strain, using an RNeasy plant mini kit (QIAGEN). Using the obtained RNA as a template, cDNA (complementary strand DNA) was synthesized using PrimeScript RT reagent Kit (TaKaRa bio). As a control sample, a glucorafasatin-containing cultivar (wild type), “disease-resistant total fat”, was used to extract total RNA and synthesize cDNA.
RT-PCR was performed using each synthesized cDNA as a template to detect the gene expression of each of genes 1-7. The PCR reaction uses SYBR (R) Premix Ex Taq (TM) (Tli RNaseH Plus) (TaKaRa bio), and is shown in Table 4 at 95 ° C. for 5 seconds and 60 ° C. for 30 seconds using the template cDNA. PCR reaction of 30 cycles or 35 cycles was performed using primers.
The obtained PCR product is electrophoresed on an agarose gel and the degree of amplification of the PCR product between samples is detected, so that the relative expression level between “NR154E” and “total disease resistance” in each gene is detected. A significant difference was detected. The results are shown in Table 5. In the table, “+” indicates a sample in which PCR amplification was clearly confirmed, and “−” indicates a sample in which amplification was not clearly confirmed. Moreover, when a clear difference was detected in the amount of amplification between both systems, it was indicated by “*”.

As a result, it became clear that there was a marked difference in gene expression of gene 3 between the “NR154E” line and “disease resistant fat” (Table 5). Specifically, as shown in the gel photograph of FIG. 8, the gene expression of gene 3 is “Glullafasatin-deficient line” “NR154E” line of “Glucurafasatin-containing varieties (wild type)” ”Was found to be significantly reduced. The said result was a result which shows the same expression pattern in both a radish root part and a true leaf.
On the other hand, in the other six genes (

genes

1, 2, 4 to 7), no clear difference in expression pattern was detected between the “NR154E” line and “disease-resistant fat” (Table 5).

From these results, it was suggested that the gene 3 is normally expressed in the “disease resistant total fat” which is a glucorafasatin-containing variety (wild type). On the other hand, in the “NR154E” line, which is a glucorafasatin-deficient line, the gene was not expressed, suggesting the possibility that the gene function was deleted.
Based on the results of the gene expression analysis, among the seven genes (genes 1 to 7) existing in the locus region narrowed down by high-precision linkage analysis, “gene 3” is the gene responsible for the deficiency of glucorafasatin. It was suggested that there is.

[Example 3] "Gene structure of the causative gene for glucorafasatin deficiency"
The gene structure of “Gene 3”, which was estimated to be a glucorafasatin-deficient gene in the above example, was analyzed in detail.

(1) “Structure of wild-type gene 3”
DNA was extracted from the true leaf of the glucorafasatin-containing strain “HAGHN” (wild type), and PCR was performed using a primer created based on the Scaffold sequence (Rsa1.0_00457.1) of “Aokubi Sh”. The genomic sequence of gene 3 was isolated and the base sequence of the genome was determined.

In addition, total RNA is extracted from the true leaves of “HAGHN” (wild type) using RNeasy Plant (QIAGEN), and mRNA having a CAP structure is extracted using First Choice RLM-RACE Kit (Life Technologies). Specific selection was performed, and the cDNA sequence was isolated by the 5′RACE method. Using a sequencer (3730xl DNA Analyzer, Applied Biosystems), determine the 5'-end cDNA sequence and compare it with the genome sequence to obtain a "transcription start point" (5 'end of ORF, first base of first exon) Made a decision.
As a result, it was speculated that the “transcription start point” of gene 3 is located 29 bp upstream from the first base of the translation initiation codon.

Similarly, 3′RACE is performed using RNA from the true leaf of “HAGHN” (wild type), the 3 ′ end cDNA sequence is determined, and compared with the genome sequence to compare the “ORF 3 ′ end” ( Determination of the most downstream base of the third exon, which is the end of the open reading frame, was performed.
As a result, it was speculated that the “ORF 3 ′ end” of gene 3 was located at 334 bp from the third base of the stop codon.

As described above, when the obtained genome sequence information, transcription start point and ORF end information are combined, the wild type gene 3 (gene derived from “HAGHN”) is a gene having a total length of 1787 bp consisting of the nucleotide sequence set forth in SEQ ID NO: 2. It became clear that. It was revealed that the gene structure on the genome sequence was a genome structure containing 3 exons and 2 introns (FIG. 10A).
Here, the full length of the wild type gene 3 was the base sequence (full length 1787 bp) from the most upstream base (ORF 5 ′ end) of the first exon to the most downstream base (ORF 3 ′ end) of the third exon ( Table 6). Further, the genomic DNA sequence from the “HAGHN” (wild type) matches the gene 3 sequence of the Scaffold sequence of “Aokubi Sh” (base sequence consisting of bases from 2972 to 4758 in SEQ ID NO: 1). It was confirmed that the sequence was.

When full-length cDNA was isolated from the glucorafasatin-containing strain “HAGHN” (wild type) based on the genomic sequence of the wild-type gene 3, the cDNA sequence of gene 3 has the nucleotide sequence described in SEQ ID NO: 3. It became clear that it was an array. The structure was DNA of 1482 bp in total length from 5′UTR to 3′UTR (Table 7).
The coding region (CDS: first base of start codon to third base of stop codon) was 1119 bp DNA consisting of the base sequence of SEQ ID NO: 4 (Table 7).

Next, when the amino acid sequence encoded by the wild type gene 3 was predicted using amino acid sequence prediction software (GENETYX ver.12.0.1, manufactured by GENETYX), a total length of 372aa consisting of the amino acid sequence set forth in SEQ ID NO: 5 It was shown to be a protein.
As a result of homology search using the NCBI database, it was found that there was no preceding registered sequence, and the gene completely encodes a novel protein. In addition, when domain prediction analysis (NCBI, Conserved Domain Search Service (CD Service), http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) was performed, 2oxoglutarate-iron ( II) It was shown to have a dependent oxygenase domain (2-oxoglutarate and Fe (II) -dependent oxygenase domain, also referred to as 2 OG-Fe (II) oxygenase domain) (FIG. 9, Table 8).
Here, the protein which has the said enzyme domain forms the superfamily in a plant, and is an enzyme group containing various oxidoreductases.
From the above findings, the translation product of gene 3 was recognized as an enzyme protein that functions as a kind of oxidoreductase. There were no other genes encoding enzymes in the other six candidate genes (

genes

1, 2, 4 to 6) in the advanced linkage analysis.

(2) “Structure of mutant gene 3”
i) NR154E mutant gene 3
Primers were prepared from the sequence of the wild-type gene 3 and the nucleotide sequence of gene 3 of the “NR154E” strain, which is a glucorafasatin-deficient strain, was determined.
As a result, it was revealed that gene 3 from the NR154E line was a mutant gene in which an insertion sequence of about 9 kbp was introduced into a region corresponding to the first exon of wild-type gene 3 (FIG. 10B).
Specifically, an insertion sequence of 8853 bp (consisting of the 558th to 9410th bases in SEQ ID NO: 7) between the 557th base and the 558th base of the wild type gene 3 (SEQ ID NO: 2) It was revealed that this was a mutant gene (NR154E type gene 3) having a mutation introduced in the base sequence and a stop codon introduced in-frame (FIG. 11A). The genome sequence related to the mutant gene 3 is shown in SEQ ID NO: 7.
The predicted amino acid sequence was estimated to be a protein with a total length of 176aa consisting of the amino acid sequence set forth in SEQ ID NO: 8, but had a structure in which the C-terminal 184aa of the wild type gene 3 protein was deleted (FIG. 12). . In particular, it was speculated that the protein function as an enzyme was lost due to the complete deletion of the 2oxoglutarate-iron (II) -dependent oxygenase domain.
The result was that there was a loss-of-function insertion mutation of gene 3 in the NR154E strain. That is, this result was recognized as a result supporting that gene 3 is a causative gene for glucorafasatin deficiency.

ii) Structure of MR050E type gene 3 The “MR050E” strain is one of the glucorafasatin-deficient strains created separately by the inventors from the NR154E strain. Therefore, the base sequence of gene 3 was also analyzed for the MR050E strain, and the presence or absence of gene 3 mutation was confirmed.
As a result, it was revealed that gene 3 from the MR050E strain is a mutant gene in which an insertion sequence of about 1.2 kbp was introduced into a region corresponding to the third exon of wild-type gene 3 (FIG. 10C). ).
Specifically, between the 1243th base and the 1244th base of the wild-type gene 3 (SEQ ID NO: 2), a 1222 bp insertion sequence (from the 1244th to 2465th base in SEQ ID NO: 9) It was revealed that this is a mutant gene (MR050E type gene 3) having a mutation introduced therein and a stop codon introduced in an inlay (FIG. 11B). The genome sequence related to the mutant gene 3 is shown in SEQ ID NO: 9.
The predicted amino acid sequence was estimated to be a protein with a total length of 303aa consisting of the amino acid sequence shown in SEQ ID NO: 10, but had a structure in which the C-terminal side 69aa of the wild-type gene 3 protein was deleted (FIG. 12). . In particular, since the C-terminal 16aa of the 2-oxoglutarate-iron (II) -dependent oxygenase domain was deleted, it was assumed that the protein function as an enzyme was greatly deleted.

As apparent from the results, the MR050E strain was shown to be a glucorafasatin-deficient strain created by introducing an in-frame insertion mutation into the same gene as the NR154E strain. However, the mutation of gene 3 possessed by MR050E strain (MR050E type mutation) was different from the mutation of NR154E strain (NR154E mutation).
The above results were recognized as supporting results that the glucorafasatin-deficient phenotype could be created by the loss of function of gene 3.

[Example 4] "Quantitative expression analysis of gene 3"
For gene 3, which was estimated to be the causative gene according to the above example, the gene expression level comparing the glucorafasatin-deficient line with the wild-type variety line was analyzed in detail by quantitative RT-PCR.

Total RNA was extracted from the true leaves of each radish variety shown in Table 11 using RNeasy plant mini kit (manufactured by QIAGEN). Using the obtained RNA as a template, cDNA (complementary strand DNA) was synthesized using PrimeScript RT reagent Kit (TaKaRa bio).
Quantitative RT-PCR of the glucorafasatin synthase gene was performed by real-time PCR method using the obtained cDNA from each true leaf (sample). As an amplification primer for the glucorafasatin synthase gene, the primer set shown in Table 10 was used, which targeted the common sequence of 3′UTR from the latter half of the third exon. Moreover, the actin gene (Zou et al. 2013 PLos One 8: e53541.) Was adopted as an internal standard gene, and the primer set shown in Table 10 was used as its amplification primer.
Real-time PCR analysis was performed using Thermal Cycler Dice® Real Time System II (TaKaRa bio) as an analyzer. The PCR reaction uses SYBR (R) Premix Ex Taq (TM) (Tli RNaseH Plus) (TaKaRa bio), and the cycle of 95 ° C. for 5 seconds and 60 ° C. for 30 seconds using the template cDNA and the primer set. A PCR reaction was performed in which the reaction was performed for 35 cycles. The fluorescence intensity of the amplification product during the PCR reaction was measured over time in real time using the above analyzer.
It is possible to obtain a value obtained by dividing the measurement target "fluorescence intensity measurement value (average value) of glucorafasatin synthase gene" by "fluorescence intensity measurement value (average value) of internal standard gene" and compare between samples. It was calculated as the relative expression level of the glucorafasatin synthase gene. The results are shown in Table 11 and FIG. In the table, the values in parentheses are values based on the value of the variety “Disease-resistant total fat”.

As a result, the gene expression level in the true leaf was “Gene 3” in both the “NR154E” and “MR050E” lines, which are glucorafasatin-deficient lines, as compared to the glucorafasatin-containing variety line (wild type). It became clear that the expression level of was significantly decreased.
Specifically, it was shown that in the “MR050E” line, only 1/40 of the gene is expressed as compared to the “HAGHN” line which is a wild type. In particular, the “NR154E” line showed that the gene was expressed in a trace amount of less than 1/1000 compared to the wild-type “HAGHN” line.
On the other hand, there is no significant difference in the expression level of the glucorafasatin synthase gene among the four cultivar lines, “HAGHN”, “disease-resistant fat”, “Miyashige radish”, and “pungency 199”, which are wild-type cultivars. The gene was found to be normally expressed and functioning.

The results show that in the glucorafasatin-deficient strain, the function of “gene 3” is greatly lost in terms of gene expression in addition to protein structural deletion. It was.

[Example 5] "Functional acquisition type gene transfer test"
With respect to “gene 3”, which was estimated to be a glucorafasatin-deficient causative gene in the above-described Examples, a function acquisition type gene introduction test into Arabidopsis thaliana was performed.

(1) Significance of gain-of-function gene transfer test in Arabidopsis
Here, “Arabidopsis thaliana” is a plant belonging to Arabidopsis, a genus closely related to Raphanus to which radish belongs. As a result of homology search using the NCBI database for gene 3, the genome sequence of Arabidopsis thaliana has a gene having a 2-oxoglutarate-iron (II) -dependent oxygenase domain that shows sequence similarity to gene 3 It has been shown that there are multiple genes belonging to the gene family (The Arabidopsis Information Resource (TAIR), http://www.arabidopsis.org/).
However, “glucorafasatin” is a glucosinolate characteristic of radish plants, and Arabidopsis thaliana has a completely different glucosinolate composition in that it does not contain any glucorafasatin. (Table 12).
In Arabidopsis thaliana, the entire genome sequence has been decoded (Nature 2000 Dec.14; 408 (6814): p796-815).
As is clear from the above findings, there is no “gene having a function equivalent to gene 3” in the Arabidopsis genome sequence.
Therefore, an overexpressing body in which “gene 3” is introduced into Arabidopsis thaliana is prepared, and if glucorafasatin is detected in Arabidopsis which should not originally contain glucorafasatin, “gene 3” can be used for glucorafasatin synthesis. Evidence that the gene is involved.

(2) “Forced expression construct”
Total RNA was extracted from the true leaf of “HAGHN”, a glucorafasatin-containing strain (wild type), using RNeasy plant mini kit (QIAGEN) and cDNA using PrimeScript RT reagent Kit (TaKaRa bio). Was synthesized. Using the cDNA as a template, the full-length cDNA of wild-type gene 3 was amplified by RT-PCR using the primers shown in Table 13.
The obtained full-length cDNA was ligated downstream of the CaMV35S promoter, and then modified from pPZP202 (Hajdukiewicz P. et al., 1994 Plant Mol. Biol, 25: p989-994) pZK3B (Kuroda et al., 2010). Biosci. Biotechnol. Biochem., 74: 2348-2351) was prepared, and plasmid DNA of the construct was prepared.
As a control, an empty vector plasmid DNA was prepared in the same manner except that no cDNA was incorporated.

(3) “Preparation of overexpressed gene 3”
The prepared plasmid DNA was transformed into Agrobacterium GV3101 and introduced into Arabidopsis thaliana (Col-0 line) by the floral dip method (Floral-dip method) using the Agrobacterium suspension to obtain seeds.
The resulting seeds After screening of the transformants was grown in kanamycin presence of 50 mg / mL, the transgene by T ₂ generation progeny assay were selected lines is fixed to homozygous. The transformed plants (gene 3 overexpressing body (experimental subject) and empty vector introduced body (control)) were grown in a chamber at 22 ° C. for 12 hours. In addition, the difference in the growth state of the plant body was not seen between the gene 3 forced expression body (experiment subject) and the empty vector introduction body (control) (FIG. 14A, FIG. 14B).

(4) "Analysis of glucosinolate composition"
About the growing individual | organism | solid, the glucosinolate composition was analyzed by the HPLC analysis from the plant body just before drawing. The analysis method was performed according to the method described in Example 1 (1). The results of glucosinolate profiling after HPCL analysis are shown in FIGS. 15A and 15B. In the figure, the peak at an elution time of 13.897 minutes represents “glucoercin” (symbol 21), and the peak at an elution time of 14.655 minutes represents “glucorafasatin” (symbol 21).
Table 14 shows the peak area values (relative values) for the peaks of glucoerucine (symbol 21) and glucorafasatin (symbol 22).

As a result, as shown in FIG. 15A, FIG. 15B, and Table 14, in the empty vector introduced body (control), the peak of glucoerucin (symbol 21) was confirmed, but the peak of glucorafasatin (symbol 22) was completely absent. It was not confirmed.
On the other hand, in the gene 3 overexpressing body (experimental object), it was confirmed that the peak of glucoerucine (symbol 21) was reduced and the peak of glucorafasatin (symbol 22) was generated.

(5) “Identification of causative gene”
From the above results, the following important findings were obtained regarding the glucorafasatin deficient causative gene in Japanese radish.
i) In a normal wild-type Arabidopsis thaliana plant, since it does not contain glucorafasatin, it was shown that "gene 3" is a "gene involved in glucorafasatin synthesis".
ii) In the “gene 3 overexpressing body”, the glucoerucin content was decreased and the production of glucorafasatin was confirmed instead. The molecular structure of glucoerucine is in the relationship of becoming the molecular structure of glucorafasatin when the 3rd and 4th carbons are double-bonded (oxidized). From the results of amino acid sequence prediction, it is recognized that the coding protein of gene 3 is not a protein (transcription factor or the like) that regulates gene expression but a kind of oxidoreductase.
From the above findings, it was recognized that the protein encoded by “gene 3” is an enzyme involved in any reaction for synthesizing glucorafasatin from glucoerucin (FIG. 16). That is, the gene 3 was recognized as a “glucorafasatin synthase gene”.
iii) Wild-type Arabidopsis thaliana exhibits a glucorafasatin-deficient phenotype in terms of glucosinolate composition. Therefore, the gene introduction test (gain of function) in this example can be regarded as a function complementation test in a radish glucorafasatin-deficient line.
Therefore, in this example, the result that glucorafasatin that should not originally exist was detected from the gene 3 overexpressing Arabidopsis thaliana. It was recognized that
iv) The findings regarding the identification of the causative gene were supported by the fact that a loss of function was introduced into the gene 3 in the MR050E strain created separately from the NR154E strain. It was.

From the above results, it was identified that gene 3 is the causative gene of the glucorafasatin deficient causative gene in Japanese radish. Further, gene 3 was identified as a gene (glucorafasatin synthase gene) encoding an enzyme involved in the reaction for producing glucorafasatin from glucoerucin.
In addition, considering that “glucorafasatin” does not exist in wild-type Arabidopsis thaliana, the gene function of gene 3 is similar to that of Arabidopsis thaliana (eg, At1g03410, The Arabidopsis Information Resource (TAIR), http: / (/www.arabidopsis.org/) was shown to be a feature that does not exist.

[Example 6] “Use as a selected DNA marker”
A PCR primer set capable of genotype determination at the glucorafasatin synthase gene locus was designed using the insertion base sequence of the function-deficient glucorafasatin synthase gene.

(1) "Primer set design"
As a PCR primer set using the nucleotide sequence of the glucorafasatin synthase gene, a primer set consisting of the following three primers was designed (Table 15, FIG. 17).
First, i) As a first primer, a “function-deficient gene detection primer” was designed on the insertion sequence into the first exon of the glucorafasatin synthase gene in the NR154E strain (FIG. 17). Specifically, a primer consisting of the base sequence described in SEQ ID NO: 47 (reverse primer shown in FIG. 17, symbol 33) was designed.
In addition, as a second primer, a “wild type gene detection primer” was designed downstream of the site where the above insertion sequence was inserted. Specifically, a primer having the base sequence set forth in SEQ ID NO: 48 (reverse primer shown in FIG. 17, symbol 34) was designed.
In addition, as a third primer, a “common primer” to be an amplification pair with the primers described in i) and ii) above was designed on the first exon of the glucorafasatin synthase gene. Specifically, a primer consisting of the base sequence set forth in SEQ ID NO: 49 (forward primer indicated by reference numeral 35 in FIG. 17) was designed.

(2) “Genotyping by PCR amplification pattern”
DNA was extracted from the leaves of lines or F ₁ shown in Table 17, PCR was carried out using a primer set containing all primers above three. The PCR reaction was performed under the conditions described in Table 16. The results are shown in Table 17 and FIG.
As a result, it was confirmed that from the “NR154E” strain, which is a glucorafasatin-deficient strain, an amplification of 588 bp corresponding to the amplification length of the function-deficient gene detection primer and the common primer was obtained. The amplification was found to be derived from the NR154E strain mutant gene.
On the other hand, from the wild type “HAGHN” line, amplification of 714 bp corresponding to the amplification lengths of the wild type gene detection primer and the common primer was obtained. The amplification was observed to be derived from the wild type gene of the HAGHN line.
Also, from both the F ₁ individuals, two amplification corresponding to the mutant gene and the wild-type gene was confirmed.
From the above results, it was shown that the genotype of the glucorafasatin synthase locus can be identified from the obtained amplification pattern by PCR using the primer set. In particular, it was shown that "heterotype", which is a genotype that does not appear in the phenotype, can be detected.

(3) “Correlation between segregated population and phenotype”
It was confirmed whether the marker genotype and the phenotype correlate for the segregated population classified using the created primer set.
For the “NR154E” line which is a glucorafasatin-deficient line, backcrossing with the glucorafasatin-containing (wild-type) lines shown in Tables 18 to 20 is performed twice, followed by self-breeding operations twice. _{A 2} F ₂ population was obtained.
For each individual of the BC ₂ F ₂ population, a PCR reaction was performed in the same manner as in the operation described in (2) above, and the genotype of the glucorafasatin synthase locus was estimated from the PCR amplification pattern, and 3 populations ( Functionally deficient gene homotype, wild type gene homotype, and heterotype).
Moreover, for each individual in BC ₂ F ₂ population, a gluco Lafayette satin content in hypertrophic roots were measured to confirm the match between genotype and phenotype was classified by a selectable marker. In addition, the measuring method of glucorafasatin content was performed like the method as described in Example 1 (3). The results are shown in Tables 18-20.

As a result, the genotype and phenotype of the segregated population obtained by classification using the above primer set in the population that had been backcrossed and self-bred with any glucorafasatin-containing (wild type) strain Confirmed to do.
That is, the group in which the genotype was classified as a “function-deficient gene homotype” according to the PCR amplification pattern of the selection marker showed a remarkably low glucorafasatin content. On the other hand, the group in which the genotype was classified into “wild type gene homotype” or “heterotype” showed a high glucorafasatin content.
Moreover, since the variation of the standard error of the glucorafasatin content was remarkably small in all of these classified groups, it was shown that the phenotype discrimination accuracy by the selected DNA marker was extremely high.

[Knowledge from Examples 1 to 6]
As mentioned above, the knowledge from the experimental results according to the above-described example has been organized and shown.

(1) “Glucorafasatin deficiency causative gene”
From the above Examples 1 to 5, the following findings were obtained regarding the glucorafasatin deficient causative gene.
i) From the linkage analysis by the positional cloning method and the high-accuracy linkage analysis, the locus region of the gene responsible for glucorafasatin deficiency was identified. The presence of seven candidate genes was predicted in the locus region. As a result of RT-PCR expression analysis, it was estimated that “gene 3” was a candidate gene.
ii) In both the NR154E and MR050E strains, which are glucorafasatin-deficient strains, the gene 3 coding protein is a mutated protein in which a stop codon has been inserted in an inlay, and the important structure of the protein It has been shown that all or part of the 2-oxoglutarate-iron (II) -dependent oxygenase domain is deleted.
In addition, the expression level of mutant gene 3 in both lines was significantly lower than that in the wild type line, indicating that the gene function was lost in terms of gene expression level.
iii) The gene 3 gain-of-function gene transfer test into Arabidopsis thaliana has identified the gene responsible for glucorafasatin deficiency in radish as gene 3. Gene 3 was identified as a glucorafasatin synthase gene.

(2) “About DNA markers”
Moreover, the following knowledge was obtained from Example 6 about using the insertion sequence in the genomic sequence of the glucorafasatin synthase gene as a DNA marker.
i) It was shown that the genotype (wild type, mutant type, hetero type) of the glucorafasatin synthase locus can be easily determined by the PCR amplification pattern.
ii) Unlike ordinary linkage markers (SNP, SSR, etc.), since the mutation of the glucorafasatin synthase gene itself is used as an index, by using the genotype of the glucorafasatin synthase gene, glucorafasatin It was shown that the phenotype showing the deletion property can be identified and selected with high accuracy.
iii) Conventionally, in order to perform backcross after the BC ₁ generation of recessive homogenes, it was necessary to select individuals in which the genes are heterozygous. Therefore, in order to discriminate heterogeneous genotypes, it was necessary to carry out selfing operations on each individual and to examine the phenotypic segregation ratio of the progeny population. It was shown that by using the selected DNA marker, the “heterotype” genotype can be easily discriminated only by examining the PCR amplification pattern in each individual without growing a progeny population.
iv) This example is an example of detecting a mutant gene of the NR154E strain. However, in principle, if it is possible to design a primer that can identify a mutant sequence, all of the function-deficient genes can be detected. It was recognized that it could be applied.

The present invention is expected to be a technology that can be used effectively in seed and seedling manufacturers and public test sites that are developing radish variety lines. In addition, it is expected to be a technology that can be effectively used by radish producers and manufacturers in various fields that handle radish processed foods and drinks.

1. 1. radish Petiole

21. 21. Peak showing glucoerucine Peak showing glucorafasatin

31. Glucorafasatin synthase gene (genomic DNA)
32. Insertion sequence in a deficient gene 33. Primer for detection of function-deficient gene 34. Primer for detecting wild type gene 35. Common primer

Claims

A function-deficient glucorafasatin synthase gene having the characteristics described in (A) and (B) below;
(A) a feature having a mutation in the exon constituting the gene described in (a1) below, which is accompanied by a deletion of all or part of the 2-oxoglutarate-iron (II) -dependent oxygenase domain in the encoded protein;
(A1) A gene encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence showing 95% or more sequence homology with the amino acid sequence, and is involved in the synthesis reaction from glucoerucin to glucorafasatin A gene encoding a protein to be
(B) The characteristic that the glucorafasatin content of the radish root is 3 μmol / g or less when the glucorafasatin synthase gene locus in the radish genome is homozygous for the function-deficient glucorafasatin synthase gene.
Furthermore, the function-deficient glucorafasatin synthase gene according to claim 1, which has the characteristics described in (C) below;
(C) The function-deficient glucorafasatin synthase gene according to claim 1, wherein the mutation described in (A) is a mutation accompanied by insertion of a base sequence of at least 1 kbp.
The function-deficient glucorafasatin synthase gene according to claim 1 or 2, wherein the function-deficient glucorafasatin synthase gene is a gene comprising the base sequence represented by SEQ ID NO: 7 or 9.
A method for determining the genotype of a glucorafasatin synthase locus in radish, comprising detecting the presence or absence of the mutation according to (A) in any one of claims 1 to 3.
A genotyping kit for a glucorafasatin synthase gene locus in radish, comprising the oligonucleotide primer described in the following (D) and (E);
(D) a base sequence included in a base sequence constituting the function-deficient glucorafasatin synthase gene according to any one of claims 1 to 3 or a region on the same chromosome as the gene or a complementary sequence thereof, An oligonucleotide primer comprising a base sequence consisting of at least 12 bases designed at a position where the presence of the mutation described in (A) can be detected,
(E) It comprises at least 12 bases included in a base sequence constituting the function-deficient glucorafasatin synthase gene according to any one of claims 1 to 3 or a region on the same chromosome as the gene or a complementary sequence thereof An oligonucleotide primer comprising a base sequence at a position capable of forming a primer pair with the primer described in (D) above.
The genotyping kit according to claim 5, wherein the primer described in (D) is the oligonucleotide primer described in (d1) or (d2) below;
(D1) when the mutation described in (A) is an insertion mutation, an oligonucleotide primer comprising a base sequence constituting the mutation or a complementary sequence thereof,
(D2) An oligonucleotide primer comprising a base sequence containing the mutation described in (A) above in its sequence.
Furthermore, the genotyping kit according to claim 5 or 6, comprising the oligonucleotide primer described in (F) and (G) below;
(F) a base sequence constituting a glucorafasatin synthase gene according to any one of claims 1 to 3 or a region on the same chromosome as the gene, or a base sequence included in a complementary sequence thereof. An oligonucleotide primer comprising a base sequence consisting of at least 12 bases designed to detect the absence of the mutation described in (A) above,
(G) at least 12 bases included in the base sequence constituting the glucorafasatin synthase gene according to any one of claims 1 to 3 or a region on the same chromosome as the gene or a complementary sequence thereof An oligonucleotide primer comprising a base sequence comprising: a base sequence at a position capable of forming a primer pair with the primer described in (F) above.
A kit for genotyping of the glucorafasatin synthase gene locus in radish, comprising the oligonucleotide probe described in (H) or (I) below;
(H) a base sequence comprising at least 12 bases contained in a base sequence constituting the function-deficient glucorafasatin synthase gene according to any one of claims 1 to 3 or a complementary sequence thereof, An oligonucleotide probe comprising a base sequence constituting the mutation or a complementary sequence thereof when the mutation described in 1 is an insertion mutation,
(I) A base sequence comprising at least 12 bases contained in a base sequence constituting the function-deficient glucorafasatin synthase gene according to any one of claims 1 to 3 or a complementary sequence thereof, An oligonucleotide probe comprising a nucleotide sequence comprising the mutation described in 1 in its sequence.
The genotyping kit according to any one of claims 5 to 8, which has the characteristics described in the following (J), (K), and (L);
(J) the function-deficient glucorafasatin synthase gene is a function-deficient glucorafasatin synthase gene comprising the base sequence described in SEQ ID NO: 7 or 9,
(K) The above-mentioned glucorafasatin synthase gene is a glucorafasatin synthase gene comprising the base sequence set forth in SEQ ID NO: 2,
(L) The region on the chromosome is composed of a base sequence consisting of the first to 2971st bases in SEQ ID NO: 1, and the 4759th to 7424th bases in SEQ ID NO: 1 A feature that is a region composed of a base sequence consisting of
The function-deficient glucorafasatin synthase gene according to any one of claims 1 to 3 is homozygous at the glucorafasatin synthase gene locus in the genome, and the glucorafasatin content relative to the dry weight of the enlarged radish is 3 μmol A Japanese radish system characterized by being not more than / g.
The radish line according to claim 10, wherein the radish line according to any one of the following (M) to (O) is obtained by self-breeding or mating with a desired radish and then selecting from a progeny population;
(M) Japanese radish in which the function-deficient glucorafasatin synthase gene according to any one of claims 1 to 3 is introduced into the glucorafasatin synthase gene locus in the genome,
(N) A radish in which a function-deficient mutation has been introduced into the glucorafasatin synthase gene in the genome, wherein the function-deficient glucose according to any one of claims 1 to 3 is present at the glucorafasatin synthase gene locus in the genome. Japanese radish having a rafasatin synthase gene,
(O) A radish obtained from a progeny population of radish according to (M) or (N) above, wherein the function-deficient glucorafasatin synthase gene according to any one of claims 1 to 3 is present in a genome. Japanese radish at the glucorafasatin synthase locus.
The radish system according to claim 10 or 11, wherein the function-deficient glucorafasatin synthase gene is a function-deficient glucorafasatin synthase gene comprising the base sequence represented by SEQ ID NO: 7 or 9.
A root, hypocotyl part, hypertrophic root part, leaf, petiole, stem, flower bud, flower, seed, sprout, baby leaf, or seedling obtained from a plant of the radish line according to any one of claims 10 to 12.
A method for producing a glucorafasatin-deficient line in radish, comprising the step described in (P) below;
(P) A step of mating a radish having the function-deficient glucorafasatin synthase gene according to any one of claims 1 to 3 at a glucorafasatin synthase gene locus in a genome with a self-breeding operation or a desired radish.
The production method according to claim 14, wherein the radish having the function-deficient glucorafasatin synthase gene described in (P) is any of the radish described in (Q) to (S) below;
(Q) Japanese radish in which the function-deficient glucorafasatin synthase gene according to any one of claims 1 to 3 is introduced into a glucorafasatin synthase gene locus in the genome,
(R) A radish obtained by introducing a function-deficient mutation into a glucorafasatin synthase gene in the genome, wherein the function-deficient gluco according to any one of claims 1 to 3 is present at the glucorafasatin synthase gene locus in the genome. Japanese radish having a rafasatin synthase gene,
(S) A radish obtained from a progeny population of radish according to (Q) or (R) above, wherein the function-deficient glucorafasatin synthase gene according to any one of claims 1 to 3 is present in the genome. Japanese radish at the glucorafasatin synthase locus.
The production method according to claim 14 or 15, which is carried out using the genotyping method according to claim 4.
The production method according to any one of claims 14 to 16, further comprising the step described in (T) below;
(T) A step of selecting a radish individual exhibiting glucorafasatin deficiency from the progeny population obtained by the step described in (P) above, using the genotyping method according to claim 4.