JP2003284598A - Method for detection of partially glutinous wheat - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for detection of existence of partially glutinous wheat usable in a stock stage, a processing stage of foods and to provide a method for early stage discrimination/selection of partially glutinous wheat in DNA level in breeding wheat. <P>SOLUTION: The method detects the presence of the partially glutinous wheat in the objects to be measured or whether the objects are partially glutinous wheat or not by carrying out a PCR using a primer which detects each position of specific sequences of genes described in the 1) to 3). 1) A group of primers detecting deficiency of 19 bp occurred in a variant of Kanto 107 type Wx-A1 gene, 2) a group of primers simultaneously amplifying parts which generate polymorphism between wild type three Wx genes, and 3) a group of primers detecting deficiency of 576 bp existing between 3' UTR (un-translated region) generated in variant of Hakuka (Baihou) type Wx-D1 gene. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for detecting partial glutinous wheat, and in particular, it is used for detecting whether or not glutinous wheat is contained in food at any stage from the raw material stage to the processing stage of food. The present invention relates to a method for detecting the presence of partial glutinous wheat. Further, the present invention relates to a method for early identification and selection of partial glutinous wheat at the DNA level in wheat breeding.

[0002]

BACKGROUND OF THE INVENTION There are two types of cereal starch, linear amylose and branched-chain amylopectin, and many cereal grains such as rice and barley contain "ammonose" and "amylopectin". There are "glutinous" ones that do not contain amylose or have very low amylose content. Wheat was not known to be "sticky" in nature, but "sticky" wheat was bred for the first time in Japan. The wx protein is present in the Urutic one, whereas the Wx protein is not present in the waxy one.
Ordinary wheat ( Triticum aestivum)
L. ) Is a hexaploid having 3 Wx genes,
-A1, Wx-B1, Wx-D1 to chromosome arms 7AS, 4
Have on AL and 7DS. Japanese Patent No. 3170595 describes three Wx genes (Wx-A1,
A method for individually confirming the presence or absence of expression of Wx-B1, Wx-D1) using a two-dimensional electrophoresis method is described. This method, after performing isoelectric focusing as the first dimension,
This is a method of performing two-dimensional SDS-PAGE by changing the electrophoretic direction by 90 °, separating Wx proteins in the two-dimensional direction, and analyzing electrophoretic patterns to individually detect the presence or absence of expression of three kinds of Wx proteins. Further, in the same patent publication, 2 of the Wx gene is
A method for producing glutinous wheat using a wheat mutant lacking seed expression for mating is described.

Japanese Patent Laid-Open Publication No. 9-191819 discloses a bread prepared from grain flour containing 0.5 to 30% by weight of waxy wheat flour, which is eaten after being refrigerated or frozen and then heated and thawed. It also describes breads whose texture does not deteriorate. "Development of new breed of wheat and quality control technology" In 2000, Agricultural, Forestry and Fisheries Technology Council Secretariat, Agricultural Research Center, Variety breeding aiming at establishment of 21st century land-use industry to improve food self-sufficiency Comprehensive development of stable production technology,
The Promotion Council material, pages 156 to 157, describes an efficient selection method by PCR of partial waxy mutants (partial waxy wheat) lacking one or two Wx proteins in the study of the present inventors. . In this method, by using the Wx-D1 mutation as a primer, as expected,
It was described that it could be detected as a product of 0.6 kbp with respect to about 1.2 kbp, but "However, the reproducibility of amplification of the same product by PCR is low, and it is considered that proper primer selection is necessary ( FIG. 3) ”is described.
As described above, although various attempts have been made to detect partial glutinous wheat at the DNA level, high reproducibility,
No good method has been obtained.

[0004]

DISCLOSURE OF THE INVENTION The object of the present invention is to solve the above-mentioned problems of the prior art and to make partial glutinous wheat DN
An attempt is made to provide a highly reproducible and excellent method of detecting at the A level. If a highly reproducible detection method is obtained, the presence of partial glutinous wheat can be detected at any stage from the raw material stage to the processing stage of foods. In addition, at wheat breeding sites, partial glutinous wheat can be identified and selected early at the DNA level.

[0005]

[Means for Solving the Problems] That is, the present invention is directed to PCR (Polymerase Chai) using primers capable of detecting the positions of the following specific gene sequences.
n reaction) to detect the presence or absence of partial glutinous wheat in the measurement target or to detect whether the measurement target is partial glutinous wheat. 1) A set of primers capable of detecting a 19 bp deletion (331st to 349th of the gene shown in FIG. 1) that occurs in a mutant type of Wx-A1 gene of Kanto No. 107 type, 2) Three Wx of wild type A portion where polymorphism occurs between genes (1204th to 1235th of the gene shown in FIG. 1,
A set of primers capable of simultaneously amplifying 1240th to 1241st of the gene shown in FIG. 2 and 1248th to 1318th of the gene shown in FIG. 3, and 3) a mutant form of Wx-D1 gene of white fire (bihou) type Existing across 3'UTR (untranslated region)
A set of primers capable of detecting a 76 bp deletion (a region further downstream of the 3 ′ end of the gene shown in FIG. 3).

The method for detecting according to claim 1 or 2, wherein the primers described in 1) to 3) are a set of 10 to 35 mer primers each containing the following sequences. 1) ′ The 256th G to the 263rd C of the gene shown in FIG. 1. 'Antisense side sequence of the 261st A to the 267th A of the gene shown in FIG. 2) '1121st T-112 of the gene shown in FIG.
7th A. 'The antisense sequence of the 1552th C to 1558th G of the gene shown in FIG. 3) '1121st T-112 of the gene shown in FIG.
7th A. 5'- located further 3'downstream of the gene shown in FIG.
Antisense side sequence of GATCATG-3 ′ (SEQ ID NO: 7).

The above-mentioned primers are preferably the following primers. 5'-TCGTGTTCGTCGGGCGCCGAGA
TGG-3 '5'-CCGCCGCTTGTAGCAGTGGAAG
TACC-3'5'-CTGGCCTGTCACTCTCAAGAGC
AACT-3 '5'-CTGACGCTCGCCGTTGACG
A-3 '5'-CTGGCCTGTCACTCTCAAGAGC
AACT-3 '5'-CTGTTTCACCATGATCCGCTCC
CCTT-3 '

There is provided a method in which the measurement target is processed food or a food material, or a detection method in which the measurement target is wheat.

[0009] Western confectionery flour, bread flour, noodle flour, steamed bun flour suitable for microwave reheating, okonomiyaki flour, takoyaki flour, noodle skin flour, or In the production of fried grain flour, wheat used as a raw material is detected by the method according to claim 2 to determine whether it is a partial glutinous wheat, and non-glutinous wheat is mixed with glutinous wheat and / or partial glutinous wheat, Western sweets having desired characteristics or taste, breads, noodles, steamed buns suitable for microwave reheating, okonomiyaki, takoyaki, noodles, or fried foods are obtained, western confectionery flour, bread flour, Noodle powder,
Provided is a method for producing steamed bun powder, okonomiyaki powder, takoyaki powder, noodle skin powder, or fried food powder suitable for microwave oven reheating.

The present invention provides Western confectioneries, breads, noodles, steamed buns, okonomiyaki, takoyaki, noodle skins, or fried foods suitable for reheating in a microwave oven.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention is described in detail below. The present invention uses PCR (Polymeras) using primers capable of detecting the positions of specific gene sequences.
e Chain reaction) to detect the presence or absence of partial glutinous wheat in the measurement target or to detect whether the measurement target is partial glutinous wheat. The measurement target used in the detection method of the present invention may be a raw material, a processing stage, or a processed food. Food includes not only human food but also animal food (feed).

Further, the object to be measured may be any as long as it can be measured at the DNA level,
All wheat of the plant, especially in the growing stage, can be used as the measurement target, and may be seeds, seedlings, seedlings, adults, or a part or all of them. The wheat may be wild type or mutant, or may be a hybrid between wheat and another plant.

According to the method of the present invention, a set of three primers (a total of 6 DNA sequences), each of which has a detectable position at a specific gene sequence and each primer comprises two sets (sense primer and antisense primer) )
By using, it became possible to measure with high reproducibility for the first time, and it became useful as a measuring method. Generally a PC
In the method of detecting whether a specific gene is contained in a sample using R, if a part of the target gene to be detected is known, the information is used to detect the target object to be detected. It is possible to design a primer having high specificity and little cross-reactivity with a hybrid of a near species. However, partial glutinous wheat is a mutation that occurred in the gene of wheat, and therefore, a reproducible primer that can determine whether it is glutinous wheat or not in all kinds of wheat or hybrids with wheat was obtained. Didn't.

In the present invention, highly reproducible detection can be achieved only by using the following primers. Suitable primer sets (1 to 3 below) for distinguishing between wild type and mutant type of Wx-A1, Wx-B1, Wx-D1 were prepared. The length of the primer in each set or each set may be the same or different, but 1
The following specific sequences are selected as sequences of 0 to 35 deoxyoligonucleotides. 1) A set of primers (sense and antisense primers) capable of detecting a 19 bp deletion (331st to 349th of the gene shown in FIG. 1) that occurred in a mutant type of Wx-A1 gene of Kanto No. 107 type. As the primers, the 5'-side sense side sequence and the 3'-side antisense side sequence are selected in pairs from the above-mentioned 19 bp deletion. 2) A portion in which polymorphism occurs between three wild-type Wx genes (1204 to 1235 of the gene shown in FIG. 1,
A set of primers capable of simultaneously amplifying 1240th to 1241st of the gene shown in FIG. 2 and 1248th to 1318th of the gene shown in FIG. 3 wild-type Wx
When a primer that can amplify a portion where polymorphism occurs between genes is used, usually, three bands having different sizes are detected. Kanto No. 107 type Wx-B1 mutant is W
Most regions of the x-B1 gene are missing, so
When the above-mentioned primer is used, one of the three bands disappears. From this, it can be seen that although the PCR amplification was performed normally, the wild type Wx-B1 was not present. The primer is selected as a set of a 5′-side sense side sequence and a 3′-side antisense side sequence from the portion where the above polymorphism occurs. 3) Deletion of 576 bp existing across the 3'UTR (untranslated region) (although this region includes a wheat-specific region) generated in the mutant form of Wx-D1 gene of the white-fire (bihou) type (Fig. A set of primers capable of detecting a region further downstream of the 3'end of the gene shown in 3. The primer is 57
A 5'-side sense side sequence and a 3'-side antisense side sequence are selected as a set from a portion where a 6 bp deletion occurs.

Preferably 10 to 35 m containing the following sequences
er primer. 1) A set of a continuous sense DNA containing a sequence 'and a continuous antisense DNA containing a sequence' for detecting the presence or absence of a mutation in the Wx-A1 gene 2) To detect the presence or absence of the Wx-B1 gene Array of '
Containing a continuous sense-side DNA containing the sequence and a continuous antisense-side DNA containing the sequence'3) containing a continuous sense-side DNA and a sequence'containing a sequence'for detecting the presence or absence of a mutation in the Wx-D1 gene Pair with continuous antisense DNA. That is, 1)
'The 256th G to the 263rd C of the gene shown in FIG. 'Antisense side sequence of the 261st A to the 267th A of the gene shown in FIG. 2) '1121st T-112 of the gene shown in FIG.
7th A. 'The 1552th C of the gene shown in Figure 1
˜1558th G antisense sequence. 3) '1121st T-112 of the gene shown in FIG.
7th A. 'An antisense sequence of 5'-GATCATG-3' (SEQ ID NO: 6) located 3'downstream of the gene shown in FIG.

The following primers are more preferred. 5'-TCGTGTTCGTCGGGCGCCGAGA
TGG-3 ′ SEQ ID NO: 1 5′-CCGCGCTTGTAGCAGTGGAAG
TACC-3 ′ SEQ ID NO: 25′-CTGGCCTGCTACCTCAAGAGC
AACT-3 ′ SEQ ID NO: 3 5′-CTGACGTCCATGCCGTTGACG
A-3 'SEQ ID NO: 45'-CTGGCCTGCTACCTCAAGAGC
AACT-3 ′ SEQ ID NO: 3 5′-CTGTTTCACCATGATCCGCTCC
CCTT-3 ′ SEQ ID NO: 5

The method of the present invention is also characterized by using PCR conditions in which the above three primer sets can be used under the same conditions.

The PCR method is not particularly limited, and various known improvement methods can be used. For example, in addition to a primer pair, a template (test) DNA, T
ris-HCl, KCl, MgCl2, each dNTP, T
Reagents such as aq DNA polymerase are mixed to prepare a PCR reaction solution. One cycle of PCR consists of three steps: heat denaturation, primer annealing, and DNA synthesis reaction by DNA polymerase. Since each step requires a different reaction temperature and reaction time, it should be within an appropriate range depending on the nucleotide sequence of the DNA region to be amplified and its length. The for such operations
The rmal cycler is commercially available. The following formula Tm (° C.) = 4 × (G + C) + 2 × (A + T) obtained from the GC content and the length of the sequence is used as an index of the annealing temperature.

The detection sensitivity may be further improved by examining suitable PCR conditions such as Taq DNA polymerase and MgCl2 concentration and the number of reaction cycles, or by using nested PCR.

The PCR reaction product may be identified by an immunoreaction or by any method,
If a clear band is observed in the electrophoretic image using a positive control or a negative control if necessary, it can be confirmed that the detection substance (partial sticky wheat) is present in the test substance (food).

The presence or absence of 3Wx protein in wheat is shown in Table 1 below. Partial mochi wheats are types 2 to 7 shown in Table 1, type 1 is non-mochi wheat, and type 8 is mochi wheat. The apparent amylose content measured by an auto analyzer is also shown.

Although the object to be measured is not limited, the method of the present invention can be applied to strong wheat, medium strength (semi-strong) wheat, thin wheat,
It can be detected with high reproducibility if it is used for measurement of edible single grain wheat.

According to the method of the present invention, Western confectioneries, breads, noodles, steamed buns suitable for reheating in a microwave oven, okonomiyaki, takoyaki, noodles, or the presence or absence of partial sticky wheat in wheat used as a raw material for fried foods. Judgment is made, and based on the information, JP-A-9-191818 and JP-A-9-
191819, JP-A-9-191842,
JP-A-10-66511 and JP-A-10-6652.
No. 7, JP-A-10-66529, JP-A-10-66529.
The grain powder described in Japanese Patent No. 66530 can be produced. If it can be determined by the method of the present invention that the raw material wheat is non-mochi wheat, partial mochi wheat, or mochi wheat, from that information, the flour for Western confectionery is 1 to 30% by weight, preferably 1 to 20% by weight. It can be designed to be flour. If it can be determined by the method of the present invention that the raw material wheat is non-mochi wheat, partial mochi wheat, or mochi wheat, from that information, the bread flour is 0.5 to 30% by weight, preferably 1 to 20% by weight. It can be designed to be waxy flour.
If the wheat of the raw material can be determined by the method of the present invention to be non-mochi wheat, partial mochi wheat, or mochi wheat, from that information, flour for noodles is 5 to 70% by weight, preferably 10 to 60% by weight, more preferably It can be designed such that 20-50% by weight is waxy flour. If it can be determined by the method of the present invention that the raw material wheat is non-mochi wheat, partial mochi wheat, or mochi wheat, from that information, the steamed bun flour suitable for microwave oven reheating is 1 to 80% by weight, preferably 10% by weight. ~ 70
It can be designed such that the wt% is waxy flour. If it can be determined by the method of the present invention that the raw material wheat is non-mochi wheat, partial mochi wheat, or mochi wheat, then 1-50% by weight, preferably 5-30% by weight of okonomiyaki and takoyaki flour is obtained from that information. It can be designed to be natural flour. If the raw material wheat can be determined by the method of the present invention to be non-mochi wheat, partial mochi wheat, mochi wheat, from the information,
10 to 90% by weight of the flour for noodle skin, preferably 30
It can be designed such that ~ 70% by weight is waxy flour. If it can be determined by the method of the present invention that the raw material wheat is non-mochi wheat, partial mochi wheat, or mochi wheat, from that information, the flour for frying is 1 to 80% by weight, preferably 2 to 70% by weight of the waxy wheat flour. Can be designed to be. Other information such as the apparent amylose content measured by an autoanalyzer may be further used for the design, but from the information of the partial glutinous wheat obtained from the present invention, the trial mixing ratio is determined, and each of the prototypes is tested. The mixing ratio can be determined by manufacturing and feeding back information such as taste. As wheat flour other than wheat, rye flour, starch and the like may be further used. In each secondary processed product, if the content of waxy wheat flour in the grain is less than the appropriate range, the effects described in the respective publications cannot be obtained, and if it exceeds the appropriate range, the characteristics of the original food become too weak. Is not preferable. Western confectionery flour, bread flour, noodle flour, steamed bun flour suitable for microwave reheating, okonomiyaki flour, takoyaki flour, noodle skin flour, or fried food grain The powder may be produced by separately producing (milling) non-glutinous wheat, partial glutinous wheat, glutinous wheat flour and other cereal flour, and mixing them.
Partial glutinous wheat, glutinous wheat and other grains may be mixed and then milled.

Western confectionery flour, bread flour, noodle flour, steamed bun flour suitable for microwave oven reheating, okonomiyaki flour, and takoyaki grain produced by the production method of the present invention. Powder, noodle powder for noodles, or foods produced by using fried food flour, because they have the characteristics described in their respective publications, in the production of frozen foods to eat by thawing in a microwave oven etc. Especially excellent. DNA is stable to heat and can be detected in minute amounts in processed foods. In addition, since it can be used at any stage from the raw material stage to the processing stage of food, flour for Western confectionery, flour for breads, flour for noodles, steamed bun flour suitable for microwave reheating, okonomiyaki Not only raw materials for flour, takoyaki flour, noodle skin flour, or fried food flour, the presence or absence of partial waxy wheat can be detected by the method of the present invention at each stage of the manufacturing process of foods, From the information, it is possible to design a flour that gives foods having a better taste.

[0025]

EXAMPLES The present invention will be specifically described below, but the present invention is not limited thereto. (1) Extraction of DNA The surface of wheat seeds was washed with 1% Triton X (Wako Pure Chemical Industries), rinsed with distilled water, thoroughly dried, and then finely pulverized with a multi-beads shocker (Yasui Kikai). Next, 1 to 1.5 g of the crushed sample is added to Dneasy P
DNA was extracted using the ant Maxi kit (Qiagen). Regarding processed foods, high-moisture content is 1 g after freeze-drying for 24 hours, and low-moisture content is 1 g as it is from Genomic Ti.
DNA was extracted using p20 / G (Qiagen). After the concentration of the extracted DNA is determined by measuring the absorbance, it is diluted to 10 ng / μl with pure water, and the diluted P
It was used as a template (test) DNA sample solution for CR.

(2) Detection of Wheat by PCR and Electrophoresis Image The PCR reaction solution was prepared as follows. Ie PCR
Buffer solution (PCR buffer II, Applied Biosystems), 200 μmol / l; dNTP, 1.
5 mmol / l; MgCl ₂ , 0.5 μmol / l; sense and antisense primers, and 0.6
25 units Taq DNA polymerase (Ampl
iTaq Gold, Upright Biosystems)
DNA sample solution adjusted to 10 ng / μl in a solution containing 2.
5 μl was added to bring the total volume to 25 μl. However, when the concentration of the extracted DNA is 10 ng / μl or less, or in processed foods containing many additives, the absorbance OD of the obtained DNA
The value of 260 / OD280 is 1.7 or less, and there are many impurities D
If the purity of NA is low, the extracted DNA stock solution or 10
2.5 μl to 17.8 μl PCR for ng / μl dilution
It was added to the reaction solution, and the amount was adjusted to 25 μl with pure water according to the amount. Ge is used as the PCR amplification device.
Using neAmp PCR System 9600 (Applied Biosystems), reaction conditions were set as follows. First, keep the temperature at 95 ° C for 5 minutes to start the reaction,
Next, 95 ° C for 30 seconds, 65 ° C for 30 seconds, 72 ° C 2
PCR was amplified for 32 cycles, with one minute as one cycle. Finally, as a termination reaction, the temperature was maintained at 72 ° C for 7 minutes and then stored at 4 ° C, and the obtained reaction solution was used as a PCR amplification reaction solution. PCR amplification reaction solution contains ethidium bromide 4
% Electrophoresis on agarose gel, positive control (DNA extracted from complete glutinous wheat seeds) and negative control (DN extracted from non-glutinous wheat seeds)
The validity of PCR was judged based on the presence or absence of the amplification band of A), and the presence or absence of the partial sticky wheat in the sample was determined by confirming the optimal size DNA amplification band by each primer.

(3) Detection of partial glutinous wheat mutant type A PCR fragment obtained by performing PCR of the varieties shown in Table 2 under the above conditions using the following three primer sets was
Wild type or mutant type was determined at each gene position and shown in Table 2. The results were applied to Table 1 to determine the type of glutinous wheat and shown in Table 2. Separately, the results of confirming the presence of each of the three Wx proteins by two-dimensional electrophoresis (2D-PAGE) described in Japanese Patent No. 3170595 are shown in Table 2 together. 2D
-The type of glutinous wheat determined by PAGE and the glutinous wheat type determined by the method of the present invention were in perfect agreement. It has been found that the method of the present invention can detect not only Japanese wheat varieties but also partial glutinous wheat variants existing in Australian, American and Canadian varieties. From this, it was inferred that the origins of the mutations occurring in the partial wheat mulberry in Japan and those countries were the same. 1) Set 5'-TCGTGTTCGTCGGGCGCCG
AGATGG-3 '5'-CCGCCGCTTGTAGCAGTGGAAG
TACC-3 '2) Set 5'-CTGGCCTGCTACCTCAAG
AGCAACT-3'5'-CTGACGTCCATGCCGTTGACG
A-3 '3) Set 5'-CTGGCCTGTCACCTCAAG
AGCAACT-3'5'-CTGTTTCACCATGATCCGCTCC
CCTT-3 '

[0028]

(4) Detection of partial glutinous wheat from processed food by PCR Using the above-mentioned primers, partial glutinous wheat was detected from processed food which is thought to contain wheat as a raw material. The samples used were cake mix, donut mix, spaghetti, cereal, frozen udon, bread, biscuits, and castella.
easy Plant Maxi kit or Gen
D using omnic Tip20 / G (Qiagen)
NA was extracted and subjected to PCR. As a result, in some frozen udon, the presence of partial type 3 glutinous wheat was confirmed.

The method according to the present invention, which is capable of detecting three kinds of proteins at the DNA level with high reproducibility, has been able to detect a partial glutinous wheat with good reproducibility. It can also be detected in processed foods in which proteins may be denatured by the heating step.

[0031]

EFFECTS OF THE INVENTION Since DNA has higher heat resistance than protein, it often remains in processed foods. Therefore, it is considered that the method for detecting partial glutinous wheat by PCR using the specific gene sequence of the present invention as an index is extremely useful particularly in processed foods. The measurement target is D
Any substance can be used as long as it can be measured at the NA level, and wheat can be used as a measurement target at all stages of the plant, especially at all stages of growth, and seeds, seedlings, seedlings,
Since it is possible to determine whether an adult, and some of these wild type, mutant, hybrids with other plants, etc. are partial glutinous wheats, it is possible to determine early in the wheat breeding field at a low amylose wheat DNA level. It can be used for selection.

[0032]

[Sequence list]

[0033]

[0034]

[0035]

[0036]

[0037]

[Brief description of drawings]

FIG. 1 is a view showing a DNA sequence of Wx-A1a gene.

FIG. 2 shows the DNA sequence of Wx-B1a gene.

FIG. 3 shows the DNA sequence of Wx-D1a gene.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 15/09 C12N 15/00 A (72) Inventor Patricia Lin Vrintin Saskatchewan, Canada S7H5J 7 Saskatoon Edinburgh Wraith (No address) (72) Inventor Katsushi Hayakawa 5-3-1, Tsurugaoka, Oi-cho, Iruma-gun, Saitama Nisshin Flour Milling Co., Ltd. Headquarters R & D / Quality Control Headquarters Basic Research Laboratory F-term (reference) CA04 CA05 CA09 HA14 4B032 DB01 DG02 4B036 LF12 LF13 LF14 LF15 LH22 4B046 LA01 LA09 LG29 4B063 QA01 QA12 QA18 QQ43 QQ67 QR08 QR32 QR38 QR42 QR55 QR62 QR82 QS16 QS25 QS34 QS39 QX01

Claims (8)

[Claims] 1. A method for detecting the presence or absence of partial glutinous wheat in a measurement target by carrying out PCR using a primer capable of detecting the position of each of the gene sequences described in 1) to 3) below. 1) A set of primers capable of detecting a 19 bp deletion (331st to 349th of the gene shown in FIG. 1) that occurs in a mutant type of Wx-A1 gene of Kanto No. 107 type, 2) Three Wx of wild type A portion where polymorphism occurs between genes (1204th to 1235th of the gene shown in FIG. 1,
A set of primers capable of simultaneously amplifying 1240th to 1241st of the gene shown in FIG. 2 and 1248th to 1318th of the gene shown in FIG. 3, and 3) a mutant form of Wx-D1 gene of white fire (bihou) type Existing across 3'UTR (untranslated region)
A set of primers capable of detecting a 76 bp deletion (a region further downstream of the 3 ′ end of the gene shown in FIG. 3). 2. A method for detecting whether or not the object to be measured is partial glutinous wheat by carrying out PCR using a primer capable of detecting the position of each of the gene sequences described in 1) to 3) below. 1) A set of primers capable of detecting a 19 bp deletion (331st to 349th of the gene shown in FIG. 1) that occurs in a mutant type of Wx-A1 gene of Kanto No. 107 type, 2) Three Wx of wild type A portion where polymorphism occurs between genes (1204th to 1235th of the gene shown in FIG. 1,
A set of primers capable of simultaneously amplifying 1240th to 1241st of the gene shown in FIG. 2 and 1248th to 1318th of the gene shown in FIG. 3, and 3) a mutant form of Wx-D1 gene of white fire (bihou) type Existing across 3'UTR (untranslated region)
A set of primers capable of detecting a 76 bp deletion (a region further downstream of the 3 ′ end of the gene shown in FIG. 3). 3. The method for detecting according to claim 1, wherein the primers described in 1) to 3) are a set of 10 to 35 mer primers each containing the following sequences. 1) ′ The 256th G to the 263rd C of the gene shown in FIG. 1. 'Antisense side sequence of the 261st A to the 267th A of the gene shown in FIG. 2) '1121st T-112 of the gene shown in FIG.
7th A. 'The antisense sequence of the 1552th C to 1558th G of the gene shown in FIG. 3) '1121st T-112 of the gene shown in FIG.
7th A. 5'- located further 3'downstream of the gene shown in FIG.
Antisense side sequence of GATCATG-3 '. 4. Each of the following primers is
The method according to claim 3, which is a primer. 5'-TCGTGTTCGTCGGGCGCCGAGA
TGG-3 '5'-CCGCCGCTTGTAGCAGTGGAAG
TACC-3'5'-CTGGCCTGTCACTCTCAAGAGC
AACT-3 '5'-CTGACGCTCGCCGTTGACG
A-3 '5'-CTGGCCTGTCACTCTCAAGAGC
AACT-3 '5'-CTGTTTCACCATGATCCGCTCC
CCTT-3 ' 5. The detection method according to claim 1, wherein the measurement object is a processed food or food material. 6. The object to be measured is wheat.
5. The method for detecting according to any one of 4 to 4. 7. A grain powder for Western confectionery, a grain flour for breads, a grain flour for noodles, a grain flour for steamed buns suitable for reheating in a microwave oven, a grain flour for okonomiyaki, a grain flour for takoyaki, and a grain flour for noodles. Or in the production of fried grain flour, the wheat used as a raw material is detected by the method according to claim 2 to determine whether it is a partial glutinous wheat, and non-glutinous wheat is mixed with glutinous wheat and / or partial glutinous wheat. , Western confectionery of desired characteristics or taste, breads, noodles, steamed buns suitable for reheating in a microwave oven, okonomiyaki, takoyaki, noodle skin, or fried foods, Western confectionery flour, bread grain Powder, cereal powder for noodles,
A method for producing steamed bun powder, okonomiyaki powder, takoyaki powder, noodle skin powder, or fried food powder suitable for reheating in a microwave oven. 8. Western confectioneries, breads, noodles, steamed buns, okonomiyaki, takoyaki, noodle skins, or fried foods suitable for reheating in a microwave oven, which are produced by the method according to claim 7.