JP2005333832A - METHOD FOR DETECTING WHEAT STARCH SYNTHASE II(wSSII) GENE-MUTATED WHEAT, METHOD FOR CLASSIFYING THE SAME, AND wSSII GENE MUTANT - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To solve problems in selecting SGP-1 protein-deleted wheat, and develop a technique of quickly and easily detecting the SGP-1 protein-deleted wheat. <P>SOLUTION: A method for detecting, in a plurality of specific DNA sequences in a wheat wSSII gene, (1) deletion and/or substitution of bases at specific positions on the above DNA sequences, (2) insertion of one or more bases in between specific positions on the above DNA sequences or (3) deletion of bases at specific positions on the above DNA sequences, is provided. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for detecting wheat Starch Synthase II (wSS II) gene mutant wheat, a method for classifying wheat wSS II gene type, and a wSS II gene variant.

It is known that starch synthase II (SS II) is involved in starch synthesis in plants. The wheat wSS II gene (wheat starch synthase II (wSS II)) identified as a gene with high homology to maize SS II is also known as Starh Granule Protein-1 as reported in Zhongyi Li et al. It encodes a protein called (SGP-1). In addition, the genome structure of wheat is an extraneous hexaploid consisting of A, B, and D genomes, and wSS II sits on the short arm of chromosome 7 of each genome (wSS II-A1, wSS II-B1, wSS II-D1). Wheat starch is composed of amylose with a linear chain of glucose with α-1,4 bonds and amylopectin with a branched structure with α-1,6 bonds, and amylose and amylopectin are about 3: 7. Included as a percentage. wSS II is considered to be involved in the synthesis of amylopectin among starches. Yamamori et al. (Non-patent Document 2) selected a line lacking SGP-1 protein (SGP-A1, SGP-B1, SGP-D1) from many wheat lines, , We succeeded in creating a strain lacking all three of these proteins. In this wheat line, the shape of the starch grains was changed, and in the case of the wild type, the gelatinization temperature was around 60 to 70 degrees, but the mutant did not have a clear temperature peak. In addition, the length distribution of the side chain of amylopectin was found to be significantly different from that of the wild type. From the above, Yamamori et al., SGP-1 protein is involved in the synthesis of the side chain of amylopectin, and deletion of this gene changes the chain length of the side chain of amylopectin, resulting in the shape and characteristics of starch granules. Is going to change greatly. There is no actual application of this SGP-1 protein-deficient wheat to food, but the nature of starch, which accounts for about 70% of the wheat endosperm part that becomes flour, has changed significantly. There is a high possibility of flour with processing characteristics.
However, the method of producing SGP-1 protein-deficient wheat, as shown by Yamamori et al., Was complicated by purifying starch granules from seeds and analyzing proteins bound to them by two-dimensional electrophoresis. In addition, in this method, in order to analyze seeds that have been formed by mating, it was necessary to further cultivate seeds and analyze the next generation seeds. This is because it is necessary to grind seeds in order to analyze proteins contained in seeds, and germination cannot be performed from ground seeds.
Therefore, enormous effort and time were required to determine the presence or absence of SGP-1 protein deficiency in many seeds.

Zhongyi Li ,. Xiusheng Chu, Gregory Mouille, Liuling Yan, Behjat Kosar-Hashemi, Sandra Hey, Johnathan Napier, Peter Shewry, Bryan Clarke, Rudi Appels, Matthew K. Morell, and Sadequr Rahman (1999) Plant Physiology Vol. 120: 1147-1155 M. Yamamori, S. Fujita, K. Hayakawa, J. Matsuki, T. Yasui (2000) Theor. Appl. Genet 101: 21-29

  An object of the present invention is to solve the above-mentioned problems in selecting SGP-1 protein-deficient wheat, and to develop and provide a technique for detecting SGP-1 protein-deficient wheat quickly and easily. If this wheat can be detected at the DNA level, this technique will be an effective means for developing a variety that has this characteristic and is practical at the breeding site. Furthermore, the presence or absence of SGP-1 protein-deficient wheat contained in the measurement target can be determined by determining the type of wSS II gene deletion in all stages from the raw material stage to the processing stage of wheat.

  As a result of intensive studies to solve the above problems, it was possible to obtain mutant sequences having greatly different sequences in the wild-type wSS II gene region encoding SGP-1 protein. One of the wheat varieties, Chinese Spring A genome-derived wild-type wSS II-A1 gene region, the mutant wSS II-A1 gene region is 8 bases with a different sequence of 289 base pairs in the region around the start codon It turns out that it is mutated. In gene expression, the region around the start codon plays an important role in both transcription from DNA to mRNA and translation from mRNA to protein, and this region is mutated. This suggests that the II-A1 gene is not expressed. It was also clarified that mutated wSS II-B1 gene region had 175 base pairs in the C-terminal region inserted into wild-type wSS II-B1 gene region derived from B genome. By inserting this sequence, a stop codon appears on the way at the protein level, and a protein shorter than the wild-type wSS II protein is synthesized. For this reason, it was suggested that the original function of this protein cannot be exhibited. It was found that the mutant wSS II-D1 gene region with respect to the wild-type wSS II-D1 gene region derived from D genome had a deletion of 63 base pairs in the N-terminal region. The deleted region sandwiches the boundary between exon and intron, and splicing does not occur well due to this deletion, so there is a high possibility that a stop codon will be inserted in the middle. In the vicinity of this region, there is a binding site for ADP-glucose, which is a substrate for starch synthesis. Due to the deletion, the three-dimensional structure of the protein may change, and the binding of ADP-glucose may be reduced. In any case, it is clear that normal proteins are not synthesized. In the vicinity of this region, there is a binding site for ADP-glucose, which is a substrate for starch synthesis. Deletion of 63 base pairs in the mutant may change the conformation of the protein and reduce ADP-glucose binding.

Since no large mutant sequences were detected in addition to the above-mentioned mutant portions, it was found in the SGP-1 protein-deficient wheat lines shown by Yamamori et al. It was clear that the starch became characteristic.
Therefore, the present inventors have devised a method for easily detecting wSS II gene mutant wheat (SGP-1 protein-deficient wheat) by detecting the difference between the wild-type and mutant sequences that have been clarified at the DNA level. .
In the present invention, the wSS II gene mutant wheat is reliably and rapidly detected by determining the wild type or mutant type of the wSS II gene region at the DNA level. That is, the present invention relates to (1) deletion and / or substitution of bases at positions 506 to 794 of the DNA sequence of SEQ ID NO: 1, and (2) one between positions 5900 to 5901 of the DNA sequence of SEQ ID NO: 2. Provided is a method for detecting insertion of the above bases or (3) deletion of bases at positions 2355 to 2417 in the DNA sequence of SEQ ID NO: 3.
The present invention also includes (1) deletion and / or substitution of bases at positions 506 to 794 of the DNA sequence of SEQ ID NO: 1, and (2) one between positions 5900 to 5901 of the DNA sequence of SEQ ID NO: 2. Provided is a method for classifying a wSS II gene type by detecting insertion of the above bases and (3) deletion of bases at positions 2355 to 2417 in the DNA sequence of SEQ ID NO: 3.
However, the sequence mutation to be detected does not necessarily need to be completely identical with the corresponding sequence described in SEQ ID NOs: 1 to 3. This is because, in the DNA replication process, substitution, insertion, and deletion of gene sequences that do not ultimately affect the function at the protein level can occur frequently. In the process of actually analyzing the sequence, several sequence variations that do not involve changes at the protein level have been confirmed. What is important is that the sequence region clarified in this study is mutated with respect to the wild type sequence, resulting in a decrease in the expression or function of the protein derived from the wSS II gene, and the original function cannot be performed. It is that.
The present invention also relates to a wSS II-A1 gene mutant in which at least nucleotides at positions 506 to 794 are deleted and / or substituted in the wSS II-A1 gene of SEQ ID NO: 1, and the wSS II-B1 gene of SEQ ID NO: 2. A wSS II-B1 gene variant in which one or more bases are inserted between at least positions 5900-5901 and wSS II-D1 gene of SEQ ID NO: 3 wherein at least positions 2355-2417 are deleted. -Provide a D1 gene variant.

  The present invention enables early identification and selection of wSS II gene-mutated wheat at the DNA level in wheat breeding. In other words, it can be used for selection of wheat lines used for crossing and selection of progeny seeds generated by crossing, and it can be expected that the efficiency and speed of breeding will be greatly improved. In addition, the presence of wSS II gene mutant wheat can be detected at any stage from the raw material stage to the processing stage of food. Furthermore, it is conceivable that the characteristics vary greatly depending on the type of combination of mutants (combination of missing SGP-1 protein). By using the present invention, not only the production of such wheat is facilitated, but if the use for each type can be found, the wheat or the processed product that can be used for the use can be easily determined. Is possible. For example, when it is used as a raw material for bread, it may be unsuitable if it lacks the three proteins SGP-A1, B1, and D1, but it may give excellent processability if only A1 is missing. Can be considered. By using the present invention in such a case, it is possible to select and use a wheat or a processed product thereof suitable for this use by determining the wSS II gene type in which only the A genome has been mutated. .

The method for detecting a wSS II gene mutant wheat of the present invention comprises (1) deletion and / or substitution of bases at positions 506 to 794 of the DNA sequence of SEQ ID NO: 1, and (2) positions 5900 to 5 of the DNA sequence of SEQ ID NO: 2. By detecting one or more base insertions between 5901 or (3) one or more base deletions at positions 2355 to 2417 of the DNA sequence of SEQ ID NO: 3.
The measurement object used in the detection method of the present invention may be a raw material, at any stage of processing, or a processed food. The food includes not only human food but also animal food (feed). In addition, the measurement object may be any object as long as it can be measured at the DNA level, and all wheat in the plant, particularly in the growth stage, can be used as an object to be measured. Seeds, seedlings, seedlings, Adults and some or all of them may be used. Wheat may be either wild type or mutant, and may be a hybrid of wheat and other plants.

Any method known to those skilled in the art may be used as long as the method for detecting a mutation in the gene sequence (1) to (3) can detect the mutation in the gene sequence (1) to (3). For example, PCR method, LAMP method, NASBA method, LCR method, SDA method, RCR method, TMA method and the like can be mentioned.
The PCR method is not particularly limited, and various known improved methods can be used. For example, in addition to a primer pair and template (test) DNA, Tris-HCl, KCl, MgCl ₂ , Reagents such as dNTP and TaqDNA polymerase are mixed to obtain a PCR reaction solution. One cycle of PCR consists of three steps: heat denaturation, primer annealing, and DNA synthesis with DNA polymerase. Each step requires a different reaction temperature and reaction time, so the range is set to an appropriate range depending on the base sequence of the DNA region to be amplified and its length. Thermal cyclers for such operations are commercially available. The detection sensitivity can be further improved by examining suitable PCR conditions such as the concentration of TaqDNA polymerase and MgCl ₂ and the number of reaction cycles, or using nested PCR.
PCR reaction products can be identified by immunoreaction or any way, but they can be electrophoresed and, if necessary, a clear band in the electrophoretic image using positive and negative controls. Can be confirmed that the detection substance (wSS II gene mutant wheat) is present in the test substance (food).

  Examples of the method for detecting a mutation in the gene sequence (1) to (3) include the LAMP method, LCR method, SDA method, RCR method, TMA method and the like in addition to the PCR method. Any method can be used as long as it can detect mutations in this gene sequence. For example, the LAMP method can be used as a report by Notomi et al. (Nucleic Acids Research, 2000, vol. 28, No. 12, e63) The primer is designed with reference to the method. If the sequence to be detected at this time is the sequence of (1), the primer may be designed using the region sandwiching positions 106 to 113 of SEQ ID NO: 16 as the detection region. If the sequence to be detected is the sequence of (2), a primer may be designed using a part of the sequence from position 5900 to 6074 of SEQ ID NO: 17 as a detection region. If the sequence to be detected is the sequence of (3), a primer may be designed with the region sandwiching 2354 to 2355 of SEQ ID NO: 18 as the detection region. In addition to the designed primer, template DNA solution necessary for the reaction, dNTP, BstDNApolymerase, and other reagents necessary for the reaction are added, the reaction is performed at 65 degrees, and the product is detected by an appropriate method, so that the wSS II gene mutation Wheat detection or wheat wSS II gene type can be determined. In other methods, similar detection and determination can be performed by drawing up the detection system according to the manual.

Any primer can be used as the primer used in PCR as long as it can detect the mutation of the gene sequences (1) to (3).
As a primer which can detect the variation | mutation of the gene sequence of (1), for example,
(I) a primer whose 3 ′ end hybridizes upstream of position 506 of the wSS II-A1 gene region of SEQ ID NO: 1 and a 5 ′ end thereof from position 794 of the wSS II-A1 gene region of SEQ ID NO: 1 In combination with a primer that hybridizes downstream,
(Ii) a primer that hybridizes so that the 3 ′ end of the wSS II-A1 gene region of SEQ ID NO: 1 is downstream of the position 794 of the wSS II-A1 gene region and spans the deletions 506 to 794; In combination with a primer that hybridizes downstream from position 794 of the SS II-A1 gene region, and (iii) its 3 ′ end hybridizes upstream from position 506 of the wSS II-A1 gene region of SEQ ID NO: 1 And a primer that hybridizes so that its 5 ′ end straddles the deletions 506 to 794 upstream of position 506 of the wSS IIa-A1 gene region of SEQ ID NO: 1.
Furthermore, the primers (i), (ii), and (iii) are preferably designed so that the wSS II-A1 gene region can be specifically detected. Specifically, it is a primer designed in a region that does not completely match a wSS II gene region derived from another genome (wSS II-B1, wSS II-D1).

As a primer which can detect the variation | mutation of the gene sequence of (2), for example,
(I) a primer whose 3 ′ end hybridizes upstream from position 5900 of the wSS II-B1 gene region of SEQ ID NO: 2 and a 5 ′ end thereof from position 5901 of the wSS II-B1 gene region of SEQ ID NO: 2 In combination with a primer that hybridizes downstream,
(Ii) a primer which hybridizes to a base whose 3 ′ end is inserted between positions 5900 to 5901 of the wSS II-B1 gene region of SEQ ID NO: 2 and wSS II-B1 whose 5 ′ end is SEQ ID NO: 2 A combination with a primer that hybridizes downstream from the position 5901 of the gene region,
(Iii) a primer whose 3 'end is hybridized upstream of position 5900 of the wSS II-B1 gene region of SEQ ID NO: 2 and a position 5900 to 5' end of the wSS II-B1 gene region of SEQ ID NO: 2 A combination with a primer that hybridizes to the base inserted between 5901, and (iv) its 3 ′ end hybridizes to the base inserted between positions 5900-5901 of the wSS II-B1 gene region of SEQ ID NO: 2. A combination of a primer that sows and a primer that hybridizes to a base whose 5 ′ end is inserted between positions 5900 to 5901 of the wSS II-B1 gene region of SEQ ID NO: 2 can be mentioned.
Furthermore, the primers (i), (ii), (iii), and (iv) are preferably designed so that the SS II-B1 gene region can be specifically detected. Specifically, it is a primer designed in a region that does not perfectly match other SSII gene regions (wSS II-A1, wSS II-D1) derived from other genomes.

As a primer capable of detecting a mutation in the gene sequence of (3), for example,
(I) a primer whose 3 ′ end hybridizes upstream from position 2355 of the wSS II-D1 gene region of SEQ ID NO: 3 and a 5 ′ end thereof from position 2417 of the wSS IIa-D1 gene region of SEQ ID NO: 3 In combination with a primer that hybridizes downstream,
(Ii) a primer that hybridizes in such a manner that its 3 ′ end straddles the deleted portion 2355 to 2417 downstream of position 2417 of the wSS II-D1 gene region of SEQ ID NO: 3, and its 5 ′ end is SEQ ID NO: 3 A combination with a primer that hybridizes downstream from position 2417 of the wSS II-D1 gene region, and (iii) its 3 ′ end hybridizes upstream from position 2355 of the wSS II-D1 gene region of SEQ ID NO: 3 And a primer that hybridizes so that the 5 ′ end straddles the deletion portions 2355 to 2417 upstream of the position 2355 of the wSS II-D1 gene region of SEQ ID NO: 3.
Furthermore, the primers (i), (ii), and (iii) are preferably designed so that the wSS II-D1 gene region can be specifically detected. Specifically, it is a primer designed in a region that does not completely match a wSS II gene region (wSS II-A1, wSS II-B1) derived from another genome.

  Specifically, (d) a combination of a primer containing the sequence set forth in any one of SEQ ID NOs: 4 to 6 and a primer containing the sequence set forth in any one of SEQ ID NO: 7 or 8, (e A combination of a primer comprising the sequence described in any one of SEQ ID NO: 9 or 10 and a primer comprising a sequence described in any one of SEQ ID NO: 11 or 12, or (f) a SEQ ID NO: 13 or 14 And a combination of a primer containing the sequence described in any one of the above and a primer containing the sequence described in any one of SEQ ID NO: 15. As the primer used in the method of the present invention, a primer containing at least one combination of the above primers is preferable.

The wSS II gene mutant wheat detected by the detection method of the present invention is a wSS II-A1 gene mutant in which at least nucleotides at positions 506 to 794 are deleted and / or substituted in the wSS II-A1 gene of SEQ ID NO: 1. In the wSS II-B1 gene of SEQ ID NO: 2, at least a position in the wSS II-B1 gene variant in which one or more bases are inserted at least between positions 5900-5901 and the wSS II-D1 gene of SEQ ID NO: 3 It includes at least one of the wSS II-D1 gene variants from which 2355 to 2417 bases have been deleted.
Examples of the wSS II-A1 gene variant include a wSS II-A1 gene comprising a DNA sequence represented by the sequence described in SEQ ID NO: 16 or a DNA sequence having a homology of 90% or more with the sequence described in SEQ ID NO: 16 Mutants are mentioned.
Examples of the wSS II-B1 gene variant include wSS II-B1 gene variant represented by the sequence shown in SEQ ID NO: 17 or wSS II-B1 having a homology with the sequence shown in SEQ ID NO: 17 of 90% or more. A genetic variant is mentioned.
Examples of the wSS II-D1 gene variant include the wSS II-D1 gene variant represented by the sequence set forth in SEQ ID NO: 18 or the wSS II-D1 sequence having a homology of 90% or more with the sequence set forth in SEQ ID NO: 18. A genetic variant is mentioned.
In addition, the wSS II gene type of the wheat can be classified according to the combination of the above-described gene variants possessed by the wheat. This makes it possible to produce wheat with various starch properties.
Accordingly, the classification method of the wSS II genotype of the present invention comprises (1) deletion and / or substitution of bases at positions 506 to 794 of the DNA sequence of SEQ ID NO: 1, and (2) position 5900 of the DNA sequence of SEQ ID NO: 2. By detecting all mutations of insertion of one or more bases between ˜59001 and (3) deletion of bases at positions 2355-2417 of the DNA sequence of SEQ ID NO: 3.

Although the measurement object is not limited, the method of the present invention can be detected with high reproducibility when used for the measurement of single-grain wheat, double-grain wheat, ordinary wheat, and timofebie wheat.
DNA is stable to heat and can be detected in trace amounts in processed foods. Moreover, since it can be used at any stage from the raw material stage to the processing stage of food, SSII gene mutant wheat can be detected by the method of the present invention not only at the raw material stage but also at each stage of the food production process.

1. Evaluation of markers for production of wSS II gene mutant wheat (A) Sample sample 1: Chinese Spring (wild type variety)
Sample 2: N7AT7D (Chinese Spring's 7A chromosome is replaced with 7D chromosome, and the genomic organization is B, D, D for chromosome 7.)
Sample 3: N7BT7D (Chinese Spring's 7B chromosome is replaced with 7D chromosome. Genome organization is A, D, and D for chromosome 7.)
Sample 4: N7DT7B (Chinese Spring's 7D chromosome is replaced with 7B chromosome. Genome composition is A, B, B for chromosome 7.)
Sample 5: wSS produced with reference to the method described in the literature (M. Yamamori, S. Fujita, K. Hayakawa, J. Matsuki, T. Yasui (2000) Theor. Appl. Genet 101: 21-29) II gene mutant wheat (wSS II gene is A, B, D all variants).
The seed of the above sample was germinated, genomic DNA was extracted from young leaves as follows, and subjected to PCR.

(B) Extraction method Genomic DNA was extracted from young leaves using a DNeasy plant mini kit manufactured by Qiagen. The operation followed the manual. 100 mg of germinated seed young leaves were ground in liquid nitrogen until powdered. The ground sample was transferred to a 1.5 ml tube, and then buffer AP1 and RNase solution attached to the kit were added and heated at 65 ° C. for 10 minutes. Next, buffer AP2 was added and allowed to stand on ice for 5 minutes, and then the deposited precipitate was removed by centrifugation. After adding buffer AP3 to the supernatant and mixing, the entire amount was applied to a mini spin column, and centrifuged to adsorb the DNA to the membrane. After washing with buffer AW twice, buffer AE was added and allowed to stand for 5 minutes, and the DNA solution was collected by centrifugation.

(C) Detection of wheat wSS II-A1 gene mutant A PCR reaction solution was prepared as follows. LA Taq (TaKaRa) was used for the reaction. A primer manufactured by FASMAC was used.

PCR増幅反応液は3％アガロースゲルによる電気泳動に供した後、エチジウムブロマイド染色を行い、各プライマーによる至適サイズのDNA増幅バンドを確認することによってサンプル中の野生型及び変異型遺伝子の有無を判定した。 GeneAmp PCR System 9700 (Applied Biosystems) was used as the PCR amplification apparatus, and the reaction conditions were set as follows.

After subjecting the PCR amplification reaction solution to electrophoresis on a 3% agarose gel, ethidium bromide staining is performed, and the presence or absence of wild-type and mutant-type genes in the sample is confirmed by confirming DNA amplification bands of optimal size with each primer. Judged.

The following three primer sets were used to determine whether the PCR fragment obtained by PCR of each sample under the above conditions was a wild type or a mutant type. The results are shown in FIGS.

  Since samples 1, 3 and 4 have the wild-type wSS II-A1 gene derived from the A genome, the band appeared at the wild-type position. However, in sample 2, the 7A chromosome was replaced with the 7D chromosome, and no wSS II gene derived from the A genome was present, so no band appeared. The band of sample 5 appeared at the position of the mutant type. Based on the above, the primer set used specifically amplified the wSS II-A1 gene in the A genome, and was able to distinguish between the wild type and the mutant type.

(D) Detection of wSS II-B1 gene mutant in wheat
The PCR reaction solution was prepared as follows. LA Taq (TaKaRa) was used for the reaction. A primer manufactured by FASMAC was used.

プライマーセットVIについて

PCR増幅反応液は3％アガロースゲルによる電気泳動に供した後、エチジウムブロマイド染色を行い、各プライマーによる至適サイズのDNA増幅バンドを確認することによってサンプル中の野生型及び変異型遺伝子の有無を判定した。 GeneAmp PCR System 9700 (Applied Biosystems) was used as the PCR amplification apparatus, and the reaction conditions were set as follows.
About primer sets IV and V

About Primer Set VI

After subjecting the PCR amplification reaction solution to electrophoresis on a 3% agarose gel, ethidium bromide staining is performed, and the presence or absence of wild-type and mutant-type genes in the sample is confirmed by confirming DNA amplification bands of optimal size with each primer. Judged.

Using the following three primer sets, it was determined by electrophoresis whether the PCR fragment obtained by PCR of each sample under the above conditions was a wild type or a mutant type. The results are shown in FIGS.

  Since samples 1, 2 and 4 have the wild-type wSS II-B1 gene derived from the B genome, the band appeared at the wild-type position. However, in sample 3, the 7B chromosome was replaced with the 7D chromosome, and no band appeared because it did not have the wSS II-B1 gene derived from the B genome. The band of sample 5 appeared at the position of the mutant type. From the above, it can be seen that the primer set used specifically amplifies the B genome wSS II-B1 gene.

(E) Detection of wSS II-D1 gene mutant in wheat
The PCR reaction solution was prepared as follows. LA Taq (TaKaRa) was used for the reaction. A primer manufactured by FASMAC was used.

PCR増幅反応液は3％アガロースゲルによる電気泳動に供した後、エチジウムブロマイド染色を行い、各プライマーによる至適サイズのDNA増幅バンドを確認することによってサンプル中の野生型及び変異型遺伝子の有無を判定した。 GeneAmp PCR System 9700 (Applied Biosystems) was used as the PCR amplification apparatus, and the reaction conditions were set as follows.

After subjecting the PCR amplification reaction solution to electrophoresis on a 3% agarose gel, ethidium bromide staining is performed, and the presence or absence of wild-type and mutant-type genes in the sample is confirmed by confirming DNA amplification bands of optimal size with each primer. Judged.

Using the following three primer sets, it was determined by electrophoresis whether the PCR fragment obtained by PCR of each sample under the above conditions was a wild type or a mutant type. The results are shown in FIGS.

  Since samples 1 to 3 have the SS IIa gene derived from the wild-type D genome, the band appeared at the wild-type position. However, in sample 4, since the 7D chromosome was replaced with the 7B chromosome, and no wSS II-D1 gene derived from the D genome was present, no band appeared. The band of sample 5 appeared at the position of the mutant type. From the above, it can be seen that the primer set used specifically amplifies the wSS II-D1 gene of the D genome.

It is an electrophoretic diagram of the amplification product derived from A genome by primer set I obtained by electrophoresis. It is an electrophoretic diagram of the amplification product derived from A genome by primer set II obtained by electrophoresis. It is an electrophoretic diagram of the amplification product derived from A genome by primer set III obtained by electrophoresis. It is an electrophoretic diagram of the amplification product derived from B genome by primer set IV obtained by electrophoresis. It is an electrophoretic diagram of the amplification product derived from B genome by primer set V obtained by electrophoresis. It is an electrophoretic diagram of the amplification product derived from B genome by primer set VI obtained by electrophoresis. It is the electrophoretic diagram of the amplification product derived from D genome by the primer set VII obtained by electrophoresis. It is the electrophoretic diagram of the amplification product derived from D genome by primer set VIII obtained by electrophoresis.

Claims (18)

A method for detecting a wSS II gene mutant wheat by detecting one or more of the mutations generated on the DNA sequence of the wheat wheat starch synthase II (wSS II) gene of the following (1) to (3):
(1) deletion and / or substitution of bases at positions 506 to 794 of the DNA sequence of SEQ ID NO: 1,
(2) insertion of one or more bases between positions 5900-5901 of the DNA sequence of SEQ ID NO: 2, or (3) deletion of bases at positions 2355-2417 of the DNA sequence of SEQ ID NO: 3.   The method according to claim 1, wherein the method for detecting one or more mutations generated on the DNA sequence of the wheat wSS II gene is PCR. The method of claim 2, wherein the reagent used in the PCR comprises at least one of the following primers:
(A) a primer designed to detect mutation (1),
(B) Primer designed to detect mutation (2), or (c) Primer designed to detect mutation (3). The method according to claim 3, wherein the reagent used in PCR comprises at least one of the following primer combinations:
(D) a combination of a primer comprising the sequence set forth in any one of SEQ ID NOs: 4 to 6 and a primer comprising the sequence set forth in any one of SEQ ID NO: 7 or 8,
(E) A combination of a primer comprising the sequence described in any one of SEQ ID NO: 9 or 10 and a primer comprising the sequence described in any one of SEQ ID NO: 11 or 12, or (f) SEQ ID NO: 13 Or a combination of a primer comprising the sequence according to any one of 14 and a primer comprising the sequence according to any one of SEQ ID NO: 15. PCR reagents for detection of wSS II gene mutant wheat containing at least one of the following primer combinations:
(D) a combination of a primer comprising the sequence set forth in any one of SEQ ID NOs: 4 to 6 and a primer comprising the sequence set forth in any one of SEQ ID NO: 7 or 8,
(E) A combination of a primer comprising the sequence described in any one of SEQ ID NO: 9 or 10 and a primer comprising the sequence described in any one of SEQ ID NO: 11 or 12, or (f) SEQ ID NO: 13 Or a combination of a primer comprising the sequence according to any one of 14 and a primer comprising the sequence according to any one of SEQ ID NO: 15. The method for classifying wheat wSS II gene types by the method that can detect all of the mutations generated on the DNA sequence of the wheat wSS II gene of the following (1) to (3):
(1) deletion and / or substitution of bases at positions 506 to 794 of the DNA sequence of SEQ ID NO: 1,
(2) insertion of one or more bases between positions 5900-5901 of the DNA sequence of SEQ ID NO: 2, and (3) deletion of bases at positions 2355-2417 of the DNA sequence of SEQ ID NO: 3.   7. The method of claim 6, wherein the method of detecting one or more mutations that occur in the wheat genomic DNA sequence is PCR. 8. The method of claim 7, wherein the reagent used in PCR comprises at least one of the following primers:
(A) a primer designed to detect mutation (1),
(B) Primer designed to detect mutation (2), or (c) Primer designed to detect mutation (3). 9. The method of claim 8, wherein the reagent used in PCR comprises at least one of the following primer combinations:
(D) a combination of a primer comprising the sequence set forth in any one of SEQ ID NOs: 4 to 6 and a primer comprising the sequence set forth in any one of SEQ ID NO: 7 or 8,
(E) A combination of a primer comprising the sequence described in any one of SEQ ID NO: 9 or 10 and a primer comprising the sequence described in any one of SEQ ID NO: 11 or 12, or (f) SEQ ID NO: 13 Or a combination of a primer comprising the sequence according to any one of 14 and a primer comprising the sequence according to any one of SEQ ID NO: 15.   A wSS II-A1 gene variant in which at least nucleotides at positions 506 to 794 are deleted and / or substituted in the wSS II-A1 gene of SEQ ID NO: 1.   The wSS II-A1 gene variant according to claim 10, comprising a DNA sequence represented by the sequence of SEQ ID NO: 16.   The wSS II-A1 gene variant according to claim 10, comprising a DNA sequence having a homology of 90% or more with the sequence of SEQ ID NO: 16.   A wSS II-B1 gene variant in which one or more bases at least between positions 5900-5901 are inserted in the wSS II-B1 gene of SEQ ID NO: 2.   The wSS II-B1 gene variant according to claim 13, represented by the sequence set forth in SEQ ID NO: 17.   The wSS II-B1 gene variant according to claim 13, which has a homology with the sequence shown in SEQ ID NO: 17 of 90% or more.   A wSS II-D1 gene variant in which at least nucleotides at positions 2355 to 2417 are deleted from the wSS II-D1 gene of SEQ ID NO: 3.   The wSS II-D1 gene variant according to claim 16, which is represented by the sequence shown in SEQ ID NO: 18.   The wSS II-D1 gene variant according to claim 16, which has a homology of 90% or more with the sequence shown in SEQ ID NO: 18.

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