KR102077963B1 - Primer set for Discrimination of Aromatic Rice and Use Thereof - Google Patents

Abstract

The present invention relates to a primer set for determining flavor varieties and uses thereof, wherein the primer set for determining flavor varieties of the present invention comprises mutations due to 3bp deletion of the Badh2 gene 5 ′ UTR; Mutation due to a 7bp deletion of exon 2; Mutations due to 8bp deletion of exon 7; Mutations due to 3bp deletion of exon 12; Mutation due to 3bp insertion of exon 13; And mutations due to 1bp insertion of exon 14; in the group consisting of one or more mutations in the Badh2 gene including one or more mutations can be detected specifically, so that the effect can be more quickly and accurately determine the general rice varieties and flavor varieties There is.
In addition, the primer set capable of detecting the Badh2 gene mutation by 1bp insertion of exon 14 can accurately identify a variety having aroma characteristics in the F2 generation prepared by artificial breeding, and thus can be useful for improving flavor varieties.

Description

Primer set for Discrimination of Aromatic Rice and Use Thereof}

The present invention relates to a primer set for discriminating flavor (香米) varieties and uses thereof, and more particularly, to functional alleles of betaine aldehyde dehydrogenase gene (badh2) in flavor varieties. The present invention relates to a primer set that specifically detects mutations caused by substitution, deletion, and insertion of bases, a flavor variety discrimination method using the same, and a flavor variety discrimination kit.

Rice is the second largest food crop in the world after wheat. More than half of the world's population uses rice as a staple food crop. More than 90% of rice is produced in Asia and most of it is consumed in Asia. Korea, like other Asian countries, uses rice as a staple food, and 15 to 20 new varieties are cultivated and distributed every year, but most of the varieties distributed in Korea are introduced as specific varieties, and most of the domestic consumers are these varieties. It is impossible to confirm.

In addition, due to the opening of import and export, some distributors supply imported rice domestically or mixed with imported rice.To prevent such illegal distribution, the government has institutionalized the country of origin labeling. It is in a very difficult situation, such as having to go through complex procedures such as tracking and verifying the country of origin. Moreover, since more scientific investigation techniques are needed for complaints and accusations related to rice varieties, such as the theft of crops occurring in some rural areas recently, a method to quickly and easily identify rice varieties grown and cultivated in Korea. It will be said that the establishment of technology or technology is urgent.

Recently, rice genetic resources have been used as a very important material for the preservation of useful traits and creation of new varieties. Therefore, rapid identification of the collected genetic resources between species and between subspecies along with the collection of domestic and foreign resources accurately manages and protects genetic resources. It is very important for you.

Recently, many studies have been conducted to identify rice varieties using various DNA markers. Until now, random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), and microsatellite methods have been used to identify varieties. For example, Wang et al. identified varieties using a restricted fragment length polymorphism (RFLP) marker (Wang, ZY, Tanksley, SD 1989 L. Genome. 32, 1113-1118), and Ryu used a protein capillary electrophoresis method. The variety was identified using (Ryu, DJ 1998 J. food science and nutrition. 3, 43-347). RAPD, microsatellite, sequence tagged site (STS), and PCR (polymerase chain reaction) methods have been used to identify 40 domestic cultivars, and have been reported by comparing the advantages and disadvantages of each method (Jeong OY et al., Korean 1998 J). Breed. 30, 136-137). The identification of 31 rice cultivars using RAPD and microsatellite was shown (Kwon, SJ et al., 1999 Korean J Crop Science 44, 112-116), and a method of identifying rice cultivars using image analysis was reported ( Kwon, SJ et al., 1998 Korean J Crop Science 43, 33-34).

On the other hand, fragrant rice (hereinafter referred to as flavor; aromatic rice, fragrant rice) is rice that gives off a rich odor, such as when frying corn or boiling beans when cooking rice, and the fragrant smell of fragrant rice, also called flavor, is freshly crushed. It is also produced in the state of rice or brown rice, and is also strongly produced in the leaves, stems, and pollen of rice. Flavor has long been cultivated in many Asian countries such as Pakistan, India, Sri Lanka, Nepal, Thailand, the Philippines and China. In Islamic countries, flavors are sometimes used during religious ceremonies. Flavored rice is also found in Thai and Japanese traditional rice, but it has almost disappeared from conventional varieties. Recently, the breeding research team in Korea developed and distributed'Hyangnam Rice' and'Hyangmi Rice No. 2'starting with a variety called'Hyangmi Rice No. 1'. When sake is made with flavor, the aroma evaporates during the fermentation process, but in the case of sikhye, the taste of sikhye and savory soongniok are harmonized to create a unique taste. Also, among the flavors, there are glutinous rice varieties and colored rice varieties, so their application as a food is very diverse.

It is known that the main aromatic compound of fragrant rice is 2-acetyl-1-pyrroline (2AP), and the compound is present in chromosome 8 of rice. It was investigated that this was due to the loss of function due to mutations caused by substitution, deletion, and insertion of the base of the betaine aldehyde dehydrogenase gene (Badh2) (US Patent No. 7847083).

In US Patent No. 731918, which is one of the prior art, transgenic rice with reduced Os2AP gene expression was prepared in order to increase the expression of 2-acetyl-1-pyrroline, and Korean Patent No. 1054459 Primer sets for discriminating various rice varieties including flavor varieties such as No. 1, Hyangmi Rice No. 2, Hyangnam Rice, and Mihyang Rice are provided.

In addition, most documents such as Chinese Patent No. 101736018 and Chinese Patent Laid-Open No. 103725778 disclose primer sets that detect mutations in the Badh2 gene caused by deletion or insertion of a base. Most of the haplotypes proven as functional mutations have a problem that cannot be detected by general PCR-based markers.

Accordingly, the present inventors have made diligent efforts to effectively identify flavor varieties by selecting primers that effectively detect haplotypes that have been proven to be functional mutations in the Badh2 gene. As a result, the 3bp deletion portion of the Badh2 gene 5'UTR, exon A set of 5 primers capable of specifically detecting the base portion including the 7bp deletion portion of 2, the 8bp deletion portion of exon 7, the 3bp deletion portion of exon 12, the 3bp insertion portion of exon 13, and the 1bp insertion portion of exon 14 Was selected, and the primer set for detecting the 1bp insertion portion of exon 14 was confirmed to be able to accurately discriminate varieties having aroma characteristics in the F2 generation, and the present invention was completed.

The present invention has been conceived to solve the above problems, and the first problem to be solved of the present invention is to provide a primer set for determining flavor varieties and a method for determining flavor varieties using the same.

The second problem to be solved of the present invention is to provide a kit for determining flavor varieties comprising the primer set for determining flavor varieties.

In order to solve the first problem of the present invention described above, the present invention provides a primer set of SEQ ID NO: 1 and SEQ ID NO: 2; A primer set of SEQ ID NO: 3 and SEQ ID NO: 4; A primer set of SEQ ID NO: 5 and SEQ ID NO: 6; The primer set of SEQ ID NO: 7 and SEQ ID NO: 8; and a primer set for determining flavor varieties comprising at least one primer set selected from the group consisting of.

According to a preferred embodiment of the present invention, the primer set for determining flavor varieties is betaine aldehyde dehydrogenase gene ( Badh2 ) present on chromosome 8 of rice (genbank accession no. AB096083 or SEQ ID NO: 11) ), mutations caused by deletion or insertion of a base can be specifically detected.

According to another preferred embodiment of the present invention, the primer sets of SEQ ID NO: 1 and SEQ ID NO: 2 specifically amplify base 19 to base 181 of the Badh2 gene represented by SEQ ID NO: 11 (genbank accession no. AB096083). By doing so, it is possible to detect a mutation in which 3bp (base pair) of the 5'UTR portion of the Badh2 gene is deleted,

The length of the amplified nucleotide sequence may be 163 bp in the case of a general rice cultivar and 160 bp in the case of a flavor cultivar.

According to another preferred embodiment of the present invention, the primer sets of SEQ ID NO: 3 and SEQ ID NO: 4 specifically target bases 414 to 491 of the Badh2 gene represented by SEQ ID NO: 11 (genbank accession no. AB096083). By amplification, it is possible to detect a mutation in which 7bp of exon 2 portion of the Badh2 gene is deleted,

The length of the amplified nucleotide sequence may be 78 bp for general rice varieties and 71 bp for flavor varieties.

According to another preferred embodiment of the present invention, the primer sets of SEQ ID NO: 5 and SEQ ID NO: 6 specifically target bases 2933 to 3083 of the Badh2 gene represented by SEQ ID NO: 11 (genbank accession no. AB096083). By amplification, a mutation in which 8bp of the 7 portion of the Badh2 gene exon is deleted can be detected,

The length of the amplified nucleotide sequence may be 151 bp for general rice varieties and 143 bp for flavor varieties.

According to another preferred embodiment of the present invention, the primer sets of SEQ ID NO: 7 and SEQ ID NO: 8 specifically target bases 5206 to 5397 of the Badh2 gene represented by SEQ ID NO: 11 (genbank accession no. AB096083). By amplification, it is possible to detect a mutation in which 3bp of exon 12 portion of Badh2 gene is deleted or a mutation in which 3bp of exon 13 portion is inserted can be detected,

The length of the amplified nucleotide sequence may be a case that when the normal Rice variations in Badh2 gene exon 12, part of 192bp, flavor varieties, if any, variation in Badh2 gene exon 13, part of 189bp, flavor varieties exist 195bp.

According to another preferred embodiment of the present invention, the primer set of SEQ ID NO: 9 and SEQ ID NO: 10 is specific to bases 5715 to 5979 of the Badh2 gene represented by SEQ ID NO: 11 (or genbank accession no. AB096083). By amplification, it is possible to detect a mutation in which 1 bp of exon 14 portion of the Badh2 gene is inserted,

The length of the amplified nucleotide sequence may be 265 bp for general rice varieties and 266 bp for flavor varieties.

According to another preferred embodiment of the present invention, the primer set of SEQ ID NO: 9 and SEQ ID NO: 10 is a restricted fragment length polymorphism (RFLP) marker, and the amplification product amplified by the primer set is treated with restriction enzyme BslI to react. In this case, an amplification product of 60bp and 205bp in the case of general rice varieties and 266bp in the case of flavor varieties may be generated.

According to another preferred embodiment of the present invention, the primer sets of SEQ ID NO: 9 and SEQ ID NO: 10 differentiate genotypes of flavor varieties in the F2 generation artificially crossed using a restricted fragment length polymorphism (RFLP) method. Can do,

In the case of flavor cultivar, 266 bp, general rice cultivar 205 bp, and heterozygous general rice cultivar 205 bp and 266 bp long amplification products can be produced.

In addition, the present invention (a) is a betaine aldehyde dehydrogenase gene (Betaine aldehyde dehydrogenase gene; Badh2 ) located on chromosome 8 of rice by separating DNA from a sample of a general rice cultivar or flavor cultivar, and performing an amplification reaction. Amplifying;

(b) using the Badh2 gene amplified in step (a) as a template and performing an amplification reaction using the primer set for determining flavor varieties; And

(c) analyzing the length of the base sequence of the amplified product generated by the amplification reaction to detect a mutation due to a base deletion or insertion;

It includes a method for determining the variety of flavor comprising a.

According to a preferred embodiment of the present invention, the Badh2 gene in step (a) is a primer set of SEQ ID NO: 12 and SEQ ID NO: 13; A primer set of SEQ ID NO: 14 and SEQ ID NO: 15; A primer set of SEQ ID NO: 16 and SEQ ID NO: 17; A primer set of SEQ ID NO: 18 and SEQ ID NO: 19; A primer set of SEQ ID NO: 20 and SEQ ID NO: 21; A primer set of SEQ ID NO: 22 and SEQ ID NO: 23; A primer set of SEQ ID NO: 24 and SEQ ID NO: 25; A primer set of SEQ ID NO: 26 and SEQ ID NO: 27; A primer set of SEQ ID NO: 28 and SEQ ID NO: 29; The primer set of SEQ ID NO: 30 and SEQ ID NO: 31; can be amplified using one or more primer sets selected from the group consisting of.

According to another preferred embodiment of the present invention, in the case of performing the amplification reaction using the primer sets of SEQ ID NOs: 1 and 2 in step (b), the nucleotide sequence length of the amplification product is 163 bp for general rice varieties, and flavor varieties. Is 160bp,

When performing the amplification reaction using the primer sets of SEQ ID NO: 3 and SEQ ID NO: 4, the nucleotide sequence length of the amplification product is 78 bp for general rice varieties and 71 bp for flavor varieties,

When performing the amplification reaction using the primer sets of SEQ ID NO: 5 and SEQ ID NO: 6, the nucleotide sequence length of the amplification product is 151 bp for general rice varieties and 143 bp for flavor varieties,

When performing the amplification reaction using the primer sets of SEQ ID NO: 7 and SEQ ID NO: 8, the nucleotide sequence length of the amplification product is 192 bp for general rice varieties and 189 bp or 195 bp for flavor varieties,

When performing the amplification reaction using the primer sets of SEQ ID NO: 9 and SEQ ID NO: 10, the length of the base sequence of the amplified product may be 265 bp for general rice varieties and 266 bp for flavor varieties.

According to another preferred embodiment of the present invention, when performing an amplification reaction using a primer set of SEQ ID NO: 9 and SEQ ID NO: 10 in step (b), restriction enzyme BslI is added to the amplification product generated by the amplification reaction. It may further include a; step of reacting by processing.

According to another preferred embodiment of the present invention, an amplification product of 60bp and 205bp for general rice varieties and 266bp for flavor varieties may be generated by treatment with restriction enzymes.

In order to solve the second problem, the present invention is a primer set of SEQ ID NO: 1 and SEQ ID NO: 2; A primer set of SEQ ID NO: 3 and SEQ ID NO: 4; A primer set of SEQ ID NO: 5 and SEQ ID NO: 6; A primer set of SEQ ID NO: 7 and SEQ ID NO: 8; A primer set for determining flavor varieties comprising at least one primer set selected from the group consisting of: a primer set of SEQ ID NO: 9 and SEQ ID NO: 10; And a reagent for carrying out an amplification reaction. It includes a kit for determining flavor varieties comprising a.

According to a preferred embodiment of the present invention, the reagent for performing the amplification reaction is dNTP (dATP, dCTP, dGTP and dTTP); DNA polymerase; And a buffer solution; may include any one or more selected from the group consisting of.

According to another preferred embodiment of the present invention, a primer set for amplifying the Badh2 gene of flavor varieties; may further include,

The primer set includes a primer set of SEQ ID NO: 12 and SEQ ID NO: 13; A primer set of SEQ ID NO: 14 and SEQ ID NO: 15; A primer set of SEQ ID NO: 16 and SEQ ID NO: 17; A primer set of SEQ ID NO: 18 and SEQ ID NO: 19; A primer set of SEQ ID NO: 20 and SEQ ID NO: 21; A primer set of SEQ ID NO: 22 and SEQ ID NO: 23; A primer set of SEQ ID NO: 24 and SEQ ID NO: 25; A primer set of SEQ ID NO: 26 and SEQ ID NO: 27; A primer set of SEQ ID NO: 28 and SEQ ID NO: 29; It may be one or more primer sets selected from the group consisting of; a primer set of SEQ ID NO: 30 and SEQ ID NO: 31.

The primer set for determining flavor varieties of the present invention is a mutation due to the deletion of 3bp of the 5'UTR of the Badh2 gene; Mutation due to 7bp deletion of exon 2; Mutation due to the 8bp deletion of exon 7; Mutation due to the 3bp deletion of exon 12; Mutation due to 3bp insertion of exon 13; And mutations caused by 1bp insertion of exon 14; since mutations in the Badh2 gene containing one or more selected mutations can be specifically detected, the effect of being able to more quickly and accurately discriminate between ordinary rice varieties and flavor varieties. There is.

In addition, a primer set capable of detecting a Badh2 gene mutation by 1bp insertion of exon 14 can accurately identify varieties having aroma characteristics in the F2 generation produced by artificial crossing, and thus can be usefully used for improving flavor varieties.

1 is a schematic diagram showing the origin of 45 rice varieties including flavor varieties analyzed in the present invention.
Figure 2 is a schematic diagram of 7 alleles of the functional badh2 gene consisting of 15 exons (green boxes) and 14 introns (gray boxes) (a: badh2-UTR-E7 / 8bp deletion in exon 7 And 5'UTR has a deletion of 3bp, b: badh2-E2/exon 2 has a 7bp deletion, c: badh2-E7/exon 7 has a deletion of 8bp, d: badh2-E12/exon 12 3bp deletion, e: badh2-E13/ exon 13 insertion of 3bp, f: badh2-E13.2/ exon 13 with one C/T SNP, g: badh2-E14/ exon 14 Has an insertion of 1 bp).
Figure 3 shows the alignment of the amino acid sequences of badh2 and badh2-E12.
FIG. 4 is a photograph of genotyping for 9 flavors (lanes 4-12) and 3 rice varieties (lanes 1-3) using five functional markers (A: FMU1-2 markers are used. 4-5 lane 5'UTR confirms 3bp deletion, B: uses FME2-7 marker to confirm 7bp deletion in 6-7 lane exon 2, C: FME7 marker uses 4-5 and 8-9 lanes Confirmation of 8bp deletion in exon 7, D: 3bp deletion of exon 12 in lane 10 and insertion of 3bp in exon 13 in lane 11-12 using FME12-3 marker, E: Using CAPs marker FME14Ⅰ and restriction enzyme BslⅠ Confirm the 1bp insertion of exon 14 in lanes 13-14).
Figure 5 shows the genotype of F2 obtained by crossing between non-fragrant general rice and Ar-30 varieties (having a badh2-E14 allele) and between Dongjin-wx (wild type) and Mongdon native (3bp deletion in exon 12). For each analysis, after PCR was performed using functional markers FME14I (SEQ ID NOs: 9 and 10) and FME12-3 (SEQ ID NOs: 7 and 8), each PCR product was electrophoresed. Genotyping photo of F2 crossing normal rice and Ar-30 with FME14Ⅰ primer, B: Genotype with FME12-3 primer for F2 crossing Dongjin-wx (wild type) and Mongdon native (3bp deletion in exon 12) Analyzed photo).

Hereinafter, the present invention will be described in more detail.

As mentioned above, the flavor of Badh2 In order to detect gene mutations, in the prior art, a primer set for detecting a Badh2 gene mutation caused by deletion or insertion of a base is disclosed, but the haplotype of the haplotype proved to be a large number of functional mutations present in the Badh2 gene. Most of them have a problem that cannot be detected by general PCR-based markers.

Accordingly, the present inventors have a primer set of SEQ ID NO: 1 and SEQ ID NO: 2; A primer set of SEQ ID NO: 3 and SEQ ID NO: 4; A primer set of SEQ ID NO: 5 and SEQ ID NO: 6; A primer set of SEQ ID NO: 7 and SEQ ID NO: 8; The primer set of SEQ ID NO: 9 and SEQ ID NO: 10; Solving the above-described problem was sought by providing a primer set for determining flavor varieties including at least one primer set selected from the group consisting of. Through this, it is possible to provide a new primer set capable of quickly and accurately discriminating ordinary rice varieties and flavor varieties and a discrimination method using the primer set.

In the present invention, the term'accession' is used as a term that encompasses all varieties, species, high-generation lines, and intermediate mothers.

Accordingly, the present invention is a primer set of SEQ ID NO: 1 and SEQ ID NO: 2; A primer set of SEQ ID NO: 3 and SEQ ID NO: 4; A primer set of SEQ ID NO: 5 and SEQ ID NO: 6; A primer set of SEQ ID NO: 7 and SEQ ID NO: 8; The primer set of SEQ ID NO: 9 and SEQ ID NO: 10; and a primer set for determining flavor varieties comprising at least one primer set selected from the group consisting of.

The "primer" of the present invention is a nucleic acid sequence having a short free 3'-hydroxyl group, which can form a complementary template and a base pair, and is a starting point for copying the template. It refers to a short nucleic acid sequence that functions as. Primers can initiate DNA synthesis in the presence of reagents for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at an appropriate buffer and temperature. PCR conditions, the length of the sense and antisense primers can be modified based on those known in the art. These oligonucleotide primers can be easily prepared and used by a person skilled in the art according to a method known in the art.

In addition, the "primer" of the present invention is a single-stranded oligonucleotide, in suitable conditions (the presence of four different nucleoside triphosphates and a polymerase such as DNA or RNA polymerase), a suitable temperature and suitable It is meant to serve as a starting point from which template-directed DNA synthesis can be initiated in a buffer.

The suitable length of the primer is determined by the characteristics of the primer to be used, but it can be generally used in a length of 80 to 500 bp. The primer does not need to be exactly complementary to the sequence of the template, but a primer that is complementary enough to form a hybrid complex with the template may be used.

In the "flavor", the main aromatic compound is known to be 2-acetyl-1-pyrroline (2AP), and the compound is present in chromosome 8 of rice. It was studied that this was due to the loss of function due to mutations caused by substitution, deletion, and insertion of the base of the betaine aldehyde dehydrogenase gene ( Badh2) (US Patent No. 7847083).

Mutations occurring in the functional alleles of the Badh2 gene are known to occur by substitution, deletion and insertion of bases, and there are about 23 haplotypes that are highly related to aroma formation. Was found. When one or more mutations in these functional alleles occur, the function of the Badh2 gene is lost, so it is important to effectively detect the haplotypes proven by various functional mutations present in the Badh2 gene.

The flavor varieties primer set for the determination of the present invention can effectively detect a single-phase-type (haplotype) demonstrated in a variety of functional mutations present in gene Badh2, Badh2 gene 5 'UTR of the variation due to the 3bp deletion; Mutation due to 7bp deletion of exon 2; Mutation due to the 8bp deletion of exon 7; Mutation due to the 3bp deletion of exon 12; Mutation due to 3bp insertion of exon 13; And mutations due to 1bp insertion of exon 14; mutations in the Badh2 gene including one or more selected mutations may be specifically detected.

The betaine aldehyde dehydrogenase gene ( Badh2 ) is basically genbank accession no. It is characterized in that it is represented by the nucleotide sequence of AB096083 or SEQ ID NO: 11, but a part of the nucleotide sequence is different depending on the rice varieties present in a myriad of numbers, so the primer for determining flavor varieties of the present invention is Badh2 of SEQ ID NO: 11 Not only the mutations present in the gene are limitedly detected, but various mutations present in the functional allele of the rice-derived Badh2 gene having 90 to 100% homology with the nucleotide sequence of SEQ ID NO: 11 can be detected.

The primer sets of SEQ ID NO: 1 and SEQ ID NO: 2 of the present invention specifically amplify the 19th to 181th bases of the Badh2 gene represented by SEQ ID NO: 11 (or genbank accession no. AB096083), and the Badh2 gene 5'UTR A mutation in which a partial 3bp (base pair) is deleted may be detected, and the length of the amplified nucleotide sequence may be 163bp for a general rice variety and 160bp for a flavor variety.

The primer sets of SEQ ID NO: 3 and SEQ ID NO: 4 of the present invention specifically amplify the 414th to 491th bases of the Badh2 gene represented by SEQ ID NO: 11 (or genbank accession no. AB096083), and the Badh2 gene exon 2 portion It is possible to detect a mutation in which 7bp of is deleted, and the length of the amplified nucleotide sequence may be 78bp for general rice varieties and 71bp for flavor varieties.

The primer set of SEQ ID NO: 5 and SEQ ID NO: 6 of the present invention specifically amplifies base 2933 to 3083 of the Badh2 gene represented by SEQ ID NO: 11 (or genbank accession no. AB096083), and the 7 portion of the Badh2 gene exon It is possible to detect a mutation in which 8bp of is deleted, and the length of the amplified nucleotide sequence may be 151 bp for a general rice cultivar and 143 bp for a flavor cultivar.

The primer sets of SEQ ID NO: 7 and SEQ ID NO: 8 of the present invention specifically amplify the 5206th base to the 5397th base of the Badh2 gene represented by SEQ ID NO: 11 (or genbank accession no. AB096083), and the Badh2 gene exon 12 portion It is possible to detect a mutation in which 3bp of is deleted or a mutation in which 3bp of the exon 13 part is inserted can be detected, and the length of the amplified nucleotide sequence is 192bp in the case of a general rice cultivar, and a mutation exists in the exon 12 part of the Badh2 gene of the flavor cultivar. If there is a mutation in the exon 13 portion of the Badh2 gene of the flavor cultivar 189 bp, it may be 195 bp.

The primer set of SEQ ID NO: 9 and SEQ ID NO: 10 of the present invention specifically amplifies the 5715th base to 5979th base of the Badh2 gene represented by SEQ ID NO: 11 (or genbank accession no. AB096083), and the Badh2 gene exon 14 portion 1 bp of the inserted mutation can be detected, and the length of the amplified nucleotide sequence may be 265 bp for a general rice variety and 266 bp for a flavor variety.

The primer set of SEQ ID NO: 9 and SEQ ID NO: 10 can be used as a restricted fragment length polymorphism (RFLP) marker, and when reacting the amplified product amplified by the primer set by treatment with restriction enzyme Bsl I, an amplification product of a general rice cultivar By cleaving by the restriction enzyme BslI, an amplification product having a size of 60bp and 205bp can be produced, and in the case of a flavor variety, an amplification product of 266bp can be produced.

In addition, the primer set of SEQ ID NO: 9 and SEQ ID NO: 10 can distinguish the genotype of flavor varieties in the F2 generation artificially crossed using the RFLP (restricted fragment length polymorphism) method, and in the case of flavor varieties, 266 bp , In the case of general rice varieties, 205 bp, in the case of heterozygous general rice varieties, 205 bp and 266 bp long amplification products can be produced.

The primer set of SEQ ID NO: 1 and SEQ ID NO: 2 of the present invention; A primer set of SEQ ID NO: 3 and SEQ ID NO: 4; A primer set of SEQ ID NO: 5 and SEQ ID NO: 6; A primer set of SEQ ID NO: 7 and SEQ ID NO: 8; The primer set of SEQ ID NO: 9 and SEQ ID NO: 10; Using a set for determining flavor varieties including at least one primer set selected from the group consisting of, it is possible to discriminate between ordinary rice varieties and flavor varieties, and the specific method is as follows:

(a) separating DNA from a sample of a general rice cultivar or flavor cultivar, and performing an amplification reaction to amplify the betaine aldehyde dehydrogenase gene ( Badh2 ) located on chromosome 8 of the rice;

(b) using the Badh2 gene amplified in step (a) as a template and performing an amplification reaction using the primer set for determining flavor varieties; And

(c) analyzing the length of the base sequence of the amplified product generated by the amplification reaction to detect a mutation due to a base deletion or insertion;

It includes a method for determining the variety of flavor comprising a.

First, step (a) is to amplify the betaine aldehyde dehydrogenase gene (Badh2 ) located on chromosome 8 of rice by separating DNA from a sample of a general rice cultivar or flavor cultivar, and performing an amplification reaction. It is a step to do.

In the present invention, whole genome sequencing was performed for 25 flavor cultivars and 295 cultivars (accession) in order to discriminate common rice cultivars and flavor cultivars. Rice varieties were selected from varieties from various countries.

In one embodiment of the present invention, in order to separate genomic DNA from each rice plant, leaf tissues of seedlings of rice grown in the field were obtained, and then DNA was separated by the CTAB (Cetyltrimethylammonium bromide) method. Was used.

In the present invention, the Badh2 gene in step (a) is a primer set of SEQ ID NO: 12 and SEQ ID NO: 13; A primer set of SEQ ID NO: 14 and SEQ ID NO: 15; A primer set of SEQ ID NO: 16 and SEQ ID NO: 17; A primer set of SEQ ID NO: 18 and SEQ ID NO: 19; A primer set of SEQ ID NO: 20 and SEQ ID NO: 21; A primer set of SEQ ID NO: 22 and SEQ ID NO: 23; A primer set of SEQ ID NO: 24 and SEQ ID NO: 25; A primer set of SEQ ID NO: 26 and SEQ ID NO: 27; A primer set of SEQ ID NO: 28 and SEQ ID NO: 29; The primer set of SEQ ID NO: 30 and SEQ ID NO: 31; can be amplified using one or more primer sets selected from the group consisting of.

The primer set (Table 2) can specifically amplify the Badh2 gene of 25 flavor cultivars and 5 general rice cultivars, and uses previously known primers, which can completely amplify the entire Badh2 gene of rice. The primer can be used without limitation. The genotypes of 295 core sessions were prepared and used by a resequencing system.

The re-sequencing system basically uses standard genome data that has been sequenced and published in a database, and is a method in which sequence fragments to be sequenced are aligned and continued for a long time.

In the present invention, genotypes of a total of 295 cultivars (accession) were analyzed, and cultivars were selected based on exon mutations of the Badh2 gene of rice. FIG. 2 is a schematic diagram showing a haplotyping based on exon mutation of the Badh2 gene of rice, and a green block indicates an exon portion.

Next, step (b) is a step of performing an amplification reaction using the Badh2 gene amplified in step (a) as a template and using the primer set for determining flavor varieties.

In another embodiment of the present invention, a total of five primer sets were prepared to detect mutations contained in the functional allele of the Badh2 gene.

As shown in Table 3, the primer set of SEQ ID NO: 1 and SEQ ID NO: 2; A primer set of SEQ ID NO: 3 and SEQ ID NO: 4; A primer set of SEQ ID NO: 5 and SEQ ID NO: 6; A primer set of SEQ ID NO: 7 and SEQ ID NO: 8; Each of the primer sets of SEQ ID NO: 9 and SEQ ID NO: 10 is FMU1-2; FME2-7; FME7; FME12-3; And FME14I; and each of the primer sets includes a Badh2 gene mutation in which 3bp (base pair) of the 5'UTR portion is deleted; Badh2 gene mutation in which 7bp of exon 2 part was deleted; Badh2 gene mutation in which 8bp of exon 7 portion was deleted; Is inserted Badh2 mutation in exon 12, part of the 3bp deletion mutation or a Badh2 3bp of exon 13 parts; And a mutation of the Badh2 gene into which 1 bp of the 14 portion of the exon is inserted; can be detected.

Finally, step (c) is a step of analyzing the nucleotide sequence length of the amplified product generated by the amplification reaction to detect a mutation due to a base deletion or insertion.

In the present invention, in the case of performing the amplification reaction using the primer sets of SEQ ID NOs: 1 and 2 in step (b), the nucleotide sequence length of the amplified product is 163 bp for general rice varieties and 160 bp for flavor varieties,

As shown in Figure 4A, as a result of checking the size of the amplified product by electrophoresis, it was confirmed that the size of the amplification product of the general rice cultivar and the flavor cultivar were different, and flavoring using the primer sets of SEQ ID NO: 1 and SEQ ID NO: 2 It was confirmed that the variety can be discriminated.

When performing the amplification reaction using the primer sets of SEQ ID NO: 3 and SEQ ID NO: 4, the nucleotide sequence length of the amplification product is 78 bp for general rice varieties and 71 bp for flavor varieties,

As shown in Fig. 4B, as a result of checking the size of the amplified product by electrophoresis, it was confirmed that the size of the amplification product of the general rice cultivar and the flavor cultivar were different, and flavoring using the primer sets of SEQ ID NO: 3 and SEQ ID NO: 4 It was confirmed that the variety can be discriminated.

When performing the amplification reaction using the primer sets of SEQ ID NO: 5 and SEQ ID NO: 6, the nucleotide sequence length of the amplification product is 151 bp for general rice varieties and 143 bp for flavor varieties,

As shown in Fig. 4C, as a result of checking the size of the amplified product by electrophoresis, it was confirmed that the size of the amplification product of the general rice cultivar and the flavor cultivar were different, and flavoring using the primer sets of SEQ ID NO: 5 and SEQ ID NO: 6 It was confirmed that the variety can be discriminated.

When performing the amplification reaction using the primer sets of SEQ ID NO: 7 and SEQ ID NO: 8, the nucleotide sequence length of the amplification product is 192 bp for general rice varieties and 189 bp or 195 bp for flavor varieties,

As shown in Fig. 4D, as a result of confirming the size of the amplified product by electrophoresis, it was confirmed that the size of the amplification product of the general rice cultivar and the flavor cultivar were different, and the flavor cultivar and Badh2 gene in which the Badh2 gene exon 12 was mutated. It was confirmed that it was also possible to discriminate the flavor variety in which the mutation occurred in the 13 part of the exon.

In the case of performing the amplification reaction using the primer sets of SEQ ID NO: 9 and SEQ ID NO: 10 in step (b), the amplification product generated by the amplification reaction is treated with restriction enzyme BslI to react; may further include In addition, an amplification product of 60 bp and 205 bp in the case of general rice varieties and 266 bp in the case of flavor varieties can be produced by treatment with restriction enzymes.

As shown in Fig. 4E, as a result of confirming the size of the amplified product by electrophoresis, it can be seen that the size of the amplified product of the general rice cultivar and the flavor cultivar are different.

In an embodiment of the present invention, it was confirmed whether the genotype of the flavor variety of the F2 generation can be determined using the primer sets of SEQ ID NOs: 7 and 8 or SEQ ID NOs: 9 and 10. In the F2 generation, 14 species were selected to extract genomic DNA from rice and amplify the Badh2 gene in the same manner as in Example 2, and the amplified products amplified using each primer set were treated with restriction enzyme BslI. After the reaction, the size of the amplified product was measured by electrophoresis.

That is, it was confirmed that varieties having aroma characteristics can be accurately identified in the F2 generation produced by artificial crossing using the primer sets of SEQ ID NO: 9 and SEQ ID NO: 10 of the present invention, which is SEQ ID NO: 9 and sequence of the present invention. The number 10 primer set means that it can be usefully used in improving flavor varieties.

Therefore, the primer set for determining flavor varieties of the present invention is a mutation due to the deletion of 3bp of the Badh2 gene 5'UTR; Mutation due to 7bp deletion of exon 2; Mutation due to the 8bp deletion of exon 7; Mutation due to the 3bp deletion of exon 12; Mutation due to 3bp insertion of exon 13; And mutations caused by 1bp insertion of exon 14; since mutations in the Badh2 gene containing one or more selected mutations can be specifically detected, the effect of being able to more quickly and accurately discriminate between ordinary rice varieties and flavor varieties. It was confirmed that there was.

The present invention also includes a primer set of SEQ ID NO: 1 and SEQ ID NO: 2; A primer set of SEQ ID NO: 3 and SEQ ID NO: 4; A primer set of SEQ ID NO: 5 and SEQ ID NO: 6; A primer set of SEQ ID NO: 7 and SEQ ID NO: 8; A primer set for determining flavor varieties comprising at least one primer set selected from the group consisting of: a primer set of SEQ ID NO: 9 and SEQ ID NO: 10; And a reagent for carrying out an amplification reaction. It includes a kit for determining flavor varieties comprising a.

In the kit of the present invention, the reagent for performing the amplification reaction is not particularly limited as long as it is usually included in the kit for breed identification, but preferably dNTP (dATP, dCTP, dGTP and dTTP), DNA polymerase and buffer ( buffer solution) and the like.

In addition, the kit of the present invention may further include a user's guide describing the optimum reaction performance conditions. The guide is a printout explaining how to use the kit, e.g., how to prepare PCR buffer, suggested reaction conditions, and so on. The guide may include a brochure in the form of a pamphlet or leaflet, a label affixed to the kit, and a description on the surface of the package containing the kit. In addition, the guide may include information disclosed or provided through an electronic medium such as the Internet.

The primer set included in the kit is a primer set for identifying flavor varieties capable of specifically detecting mutations occurring in the functional allele of the Badh2 gene of general rice varieties and flavor varieties, and the primer set included in the kit is A person skilled in the art can easily design using the primer set for determining flavor varieties of the present invention, and the designed primer set is included within the scope of the present invention. Preferably, a mutation due to a 3bp deletion of the 5'UTR of the Badh2 gene; Mutation due to 7bp deletion of exon 2; Mutation due to the 8bp deletion of exon 7; Mutation due to the 3bp deletion of exon 12; Mutation due to 3bp insertion of exon 13; And mutations due to 1bp insertion of exon 14; a primer set may be designed based on mutations in the Badh2 gene including at least one selected mutation.

In addition, the kit of the present invention may further include a primer set for amplifying the Badh2 gene of the flavor variety,

The primer set includes a primer set of SEQ ID NO: 12 and SEQ ID NO: 13; A primer set of SEQ ID NO: 14 and SEQ ID NO: 15; A primer set of SEQ ID NO: 16 and SEQ ID NO: 17; A primer set of SEQ ID NO: 18 and SEQ ID NO: 19; A primer set of SEQ ID NO: 20 and SEQ ID NO: 21; A primer set of SEQ ID NO: 22 and SEQ ID NO: 23; A primer set of SEQ ID NO: 24 and SEQ ID NO: 25; A primer set of SEQ ID NO: 26 and SEQ ID NO: 27; A primer set of SEQ ID NO: 28 and SEQ ID NO: 29; It may be one or more primer sets selected from the group consisting of; a primer set of SEQ ID NO: 30 and SEQ ID NO: 31.

Hereinafter, the present invention will be described in detail with reference to preferred embodiments so that those of ordinary skill in the art can easily implement the present invention. However, the present invention may be implemented in various different forms and is not limited to the embodiments described herein.

Example 1. Selection of rice varieties

In the present invention, 25 kinds of flavor cultivars and 5 kinds of ordinary rice cultivars were selected for discrimination of general rice cultivars and flavor cultivars. The rice varieties were grown at Kongju National University during the rice cultivation period in 2012. During 2013, F2 was obtained through crossing of'Comrade-wx' and'Mongdon native (3bp deletion in exon 12)', and the F2 seeds obtained at this time were planted and cultivated in 2014. Another F2 was obtained through crossing between non-fragrant normal rice and A30 (inserted 1 bp in exon 14).

Example 2. Sensory evaluation of flavor

The evaluation of young leaf scent was conducted by the experimenter's sensory evaluation according to the method of Sood and Siddiq (Sood B, Siddiq E et al., Indian Journal of Genetics and Plant Breeding, 38(2):268-275, 1978). Became. About 2 g of young leaves were put in a 15 ml tube, and 10 ml of 1.7% KOH aqueous solution was mixed. Next, the tube was put in a 30 °C water bath for 10 minutes and incubated, and then the fragrance was evaluated.

According to the scent sensory evaluation, 25 species out of 30 species (accession) were observed as flavors, and the remaining 5 species were general tastes without scent. In addition, 13 species of 295 rice varieties were observed as flavor (see Table 1).

Presence or absence of alleles and fragrances for the selected 30 species (accession) rice Alleles Sample ID Fragrant Badh2* A01~A05 - badh2-UTR-E7 A09, A10 + badh2-E2 A06, A07, A08 + badh2-E7 A11~A23, RWG-042, RWG-061, RWG-064, RWG-069, RWG-179, RWG-201, RWG-236, RWG-272, RWG-295 + badh2-E12 RWG-031 + badh2-E13 A24, A25, RWG-038 + badh2-E13.2 A26, A27, A28, RWG-088, RWG-191, RWG-197 + badh2-E14 A29, A30 +

*: Wide type +/-: Yes/No

Example 3. Analysis of exon base polymorphism of badh2 gene in rice

In the present invention, genotypes of a total of 295 cultivars (accession) were analyzed, and 30 cultivars based on exon mutations of the Badh2 gene of rice were selected.

In order to isolate genomic DNA from each cultivar, leaf tissues of rice seedlings grown in a field (Suwon, Korea) were obtained, and then DNA was separated and used by the CTAB (Cetyltrimethylammonium bromide) method. .

Primers capable of specifically amplifying the Badh2 gene of rice varieties were prepared by referring to previously known papers (shi et al., Mol. Breed, 22:185, 2008) (see Table 2). For PCR amplification, 20 µl of a PCR reaction solution consisting of 2 µl of 10X buffer, 4 µl of dNTP, 0.2 µl of taq polymerase, 0.5 µl of 10ppm primer, and 50 ng genomic DNA was prepared, and the Badh2 gene portion of each rice cultivar was amplified. (Thermal Cycler S1000, bio-red).

Primer sequence for amplifying Badh2 gene Primer name Primer sequence(5'-3') annealing temperature (℃) Products size (bp) P1 F: GTCTCTCCTCCAACATGCTC SEQ ID NO: 12 61 995 R: GTGGTGTCAGGTGTGGAGTG SEQ ID NO: 13 P2 F: CCGAAGTCCGTACCAACTGC SEQ ID NO: 14 58 972 R: CAATCAGCCATGCTTCCAAC SEQ ID NO: 15 P3 F: GTGCGTATCCTCTGTTCTGG SEQ ID NO: 16 50 855 R: CAAGCGTCTCTAGTCTAGCC SEQ ID NO: 17 P4 F: CTGAGCTGGCTAGACTAGAG SEQ ID NO: 18 58 1025 R: CCATCAGGAGAGGATAGTTC SEQ ID NO: 19 P5 F: CCTCCGTGTTAATGCAGCTC SEQ ID NO: 20 61 1026 R: CATAGCAAGTGGCATGTACC SEQ ID NO: 21 P6 F: GGTTGGTCTTCCTTCAGGTG SEQ ID NO: 22 50 926 R: GTCCTTCCTAACTGCCTTCC SEQ ID NO: 23 P7 F: CCAGACGAGCAGGATGCAAG SEQ ID NO: 24 61 984 R: CATGGTCAGGAGCAAGAAGC SEQ ID NO: 25 P8 F: GTGGCAAGGAAGGCAGTTAG SEQ ID NO: 26 61 912 R: GCAATAAGGCTTGGAAGGAC SEQ ID NO: 27 P9 F: TTTCAGCTTCTTGCTCCTGA SEQ ID NO: 28 50 1023 R: CAGTTAATCTCTGGCATCGC SEQ ID NO: 29 P10 F: GGCCAACGATACTCAGTGAG SEQ ID NO: 30 50 1083 R: CCGGTCATCAGCTAACTTCC SEQ ID NO: 31

The badh2 nucleotide sequence of 295 varieties could be obtained by re-sequencing 295 whole genomes by performing genome mapping based on IRGSP-1.0 (http://rapdb.dna.affrc.go.jp/download/archive/irgsp1/IRGSP). -1.0_genome.fasta.gz).

Thereafter, nucleotide sequence alignment was performed, and through this, the present inventors discovered five alleles (see Table 3).

Badh2 exon base polymorphism analysis Alleles Location Sequence divergences Marker development Segregation test badh2-p-5'UTR 5'UTR 3-bp deletion N - badh2-E1.1 Exon 1 2-bp deletion N - badh2 -E1.2 Exon1 - intron1 junction G/A snp Y Y badh2 -E2.1 Exon 2 7-bp deletion Y Y badh2-E2.2 Exon 2 75-bp deletion N - badh2-E4-5.1 Exon4 to exon5 806-bp deletion N - badh2-E4-5.2 Exon4 to exon5 803-bp deletion Y N badh2 -E7 Exon 7 8-bp deletion Y Y badh2-E8 Exon 8 7-bp insertion N - badh2-E10.1 Exon 10 1-bp insertion N - badh2-E10.2 Exon 10 1-bp deletion N - badh2-E10.3 Exon 10 G/T snp N - badh2-E10.4 Exon 10 G/A snp N - badh2 -E12* Exon 12 3-bp deletion Y Y badh2 -E13.1 Exon 13 3-bp insertion Y Y badh2-E13.2 Exon 13 C/T snp N - badh2-E14.1 Exon 14 1-bp insertion Y N badh2-E14.2 Exon 14 G/T snp N -

*: Discovered in this study

Of the 25 flavors, 13 have an 8bp deletion in exon 7, 3 have a 7bp deletion in exon 2, 3 have one C/T SNP in exon 13, 2 have 3bp insertion in exon 13, and 2 have exon It has an insertion of 1 bp at 14. These results have been reported previously. However, two of these were the deletion of 3bp of 5'UTR and 8bp of exon 7, which is the first to be reported.

We found a novel badh2 allele from data resequencing the entire genome of 295 varieties. 8bp deletion in exon 7 appeared in 9 species, and C/T SNP in exon 13 appeared in 3 species. A new allele containing a 3bp deletion in exon 12 was found in the Korean native species'Mongdon Native'. One amino acid was damaged by the 3bp deletion, which gave a fragrance to the rice (see FIG. 3).

Example 4. Preparation of a primer for detecting flavor varieties

We designed a functional marker to detect 3 bp deletion in 5'UTR, 7 bp deletion in exon 2, 8 bp deletion in exon 7 and 3 bp insertion in exon 13, and a CAP marker to detect 1 bp insertion in action 14. For the newly discovered allele badh2-E12, the inventors designed various markers according to the lateral sequence. After that, an optimal primer to generate a PCR product of a clear and suitable size was selected.

A truncated amplified polymorphic sequence (CAPS) marker that marks a functional insertion into exon 14 of the Badh2 gene is Primer Premier v. 5.0 software (Lalitha 2000) and NEBcutter v. It was developed using 2.0. The remaining four primers were developed using Primer Premier v.5.0 software.

As a result, a total of 5 primer sets were prepared (see Table 4). The four primers FMU1-2, FME2-7, FME7-1 and FME14 were selected from well-known alleles. In addition, FME12-3 was selected as an optimal primer for detecting 3bp insertion into exon 12, a new trait. The FME12-3 marker can also detect a 3bp deletion in exon 13. FME12-3 can amplify a 142 bp region in a 3 bp deletion in exon 12 and a 3 bp insertion in exon 13.

Badh2 gene mutation detection primer set Primer Sequencing(5'-3') Product size Start End Target region FMU1-2 F: TCCCACCACCACTCCACA SEQ ID NO: 1 163/160 19 181 3bp deletion in 5'UTR R: ACGAAGAGCTGCCGCTGC SEQ ID NO: 2 163/160 19 181 3bp deletion in 5'UTR FME2-7 F: ACGAAGAGCTGCCGCTGC SEQ ID NO: 3 78/71 414 491 7bp deletion in exon 2 R: GCGATTGCGCGGAGGTACT SEQ ID NO: 4 78/71 414 491 7bp deletion in exon 2 FME7 F: TCCTGTAATCATGTATACCC SEQ ID NO: 5 151/143 2933 3083 8bp deletion in exon 7 R: AATTTGGAAACAAACCTT SEQ ID NO: 6 151/143 2933 3083 8bp deletion in exon 7 FME12-3 F: TTGGTCCAGTGCTCTGTGTG SEQ ID NO: 7 192/189 5206 5397 3bp deletion in exon 12
R: AGCACCAGCCAGACCATAAC SEQ ID NO: 8 192/195 5206 5397 3bp insertion in exon 13 FME14I F:TCGATGCCGGAATTATCTGGGTGA SEQ ID NO: 9 265/266 (or 60, 205/266) 5715 5979 1bp insertion in exon 14 R:TCCCCACGGCTCATCGGAG SEQ ID NO: 10 60, 205/266 5715 5979 1bp insertion in exon 14

FMU1-2 primers generate 163/160 bp PCR products in normal/flavor, respectively, and FME2-7 primers and FME7 primers generate 78/71 and 151/143 bp PCR products, respectively. FME14I generates a PCR product of 265 bp in normal rice and 266 bp in flavor, and when Bsl I restriction enzyme is treated therein, the non-fragrant normal rice is separated into 205 bp and 60 bp fragments, and a 266 bp fragment of flavor is detected. Finally, the FME12-3 primer generates a PCR product of 192/189 bp for the allele of exon 12 (badh2-E12) and a PCR product of 192/195 bp for the allele of exon 13 (badh2-E13). .

Example 5. Rice Badh2 Genetic variation detection

DNA was extracted from rice leaves according to the CTAB procedure (doyle 1991). A total of 20 μl volume of PCR reactions included 10 mM Tris-HCl, 50 mM KCl, 0.1% Triton X-100, 1.8 mM MgCl2, 0.1 mM dNTPs, 0.2 mM primer, 1 U Taq DNA polymerase, and 50 ng genomic DNA. Since exon 2 exhibits a high GC content, 1 µl of DMSO per 10 µl was added to the PCR reaction.

The PCR reaction was carried out with Thermal Cycler S1000, followed by initial denaturation at 94°C for 5 minutes, followed by 30 seconds at 94°C, 30 seconds at 50-61°C, and 1 minute at 72°C, for a total of 30 cycles. Was carried out. Then, the final stretching process was performed at 72°C for 10 minutes.

When PCR was performed using the E14I primer set of SEQ ID NO: 9 and SEQ ID NO: 10, the resulting amplification product was treated with BslI, a restriction enzyme, according to the manual provided by BioLabs. 1U enzyme was added to 1µg DNA so that the total reaction volume became 50µl, and then reacted at 55°C for 1 hour.

Among the PCR amplified products amplified by the five primer sets, the PCR products of FME2-7, FME7 and FME14I were isolated on a 3% agarose gel, and the PCR products of FMU1-2 and FME12-3 were 6% denatured polyacrylamide. Separated from the gel.

When performing the amplification reaction using the primer sets of SEQ ID NO: 1 and SEQ ID NO: 2, the nucleotide sequence length of the amplification product is 163 bp for general rice varieties and 160 bp for flavor varieties,

As shown in Figure 4A, as a result of checking the size of the amplified product by electrophoresis, it was confirmed that the size of the amplification product of the general rice cultivar and the flavor cultivar were different, and flavoring using the primer sets of SEQ ID NO: 1 and SEQ ID NO: 2 It was confirmed that the variety can be discriminated.

When performing the amplification reaction using the primer sets of SEQ ID NO: 3 and SEQ ID NO: 4, the nucleotide sequence length of the amplification product is 78 bp for general rice varieties and 71 bp for flavor varieties,

As shown in Fig. 4B, as a result of checking the size of the amplified product by electrophoresis, it was confirmed that the size of the amplification product of the general rice cultivar and the flavor cultivar were different, and flavoring using the primer sets of SEQ ID NO: 3 and SEQ ID NO: 4 It was confirmed that the variety can be discriminated.

When performing the amplification reaction using the primer sets of SEQ ID NO: 5 and SEQ ID NO: 6, the nucleotide sequence length of the amplification product is 151 bp for general rice varieties and 143 bp for flavor varieties,

As shown in Fig. 4C, as a result of checking the size of the amplified product by electrophoresis, it was confirmed that the size of the amplification product of the general rice cultivar and the flavor cultivar were different, and flavoring using the primer sets of SEQ ID NO: 5 and SEQ ID NO: 6 It was confirmed that the variety can be discriminated.

When performing the amplification reaction using the primer sets of SEQ ID NO: 7 and SEQ ID NO: 8, the nucleotide sequence length of the amplification product is 192 bp for general rice varieties and 189 bp or 195 bp for flavor varieties,

As shown in Fig. 4D, as a result of confirming the size of the amplified product by electrophoresis, it was confirmed that the size of the amplification product of the general rice cultivar and the flavor cultivar were different, and the flavor cultivar and Badh2 gene in which the Badh2 gene exon 12 was mutated. It was confirmed that it was also possible to discriminate the flavor variety in which the mutation occurred in the 13 part of the exon.

As shown in Figure 4E, it was confirmed that the amplification product of the general rice cultivar was cut by the restriction enzyme BslI to produce 60bp and 205bp amplification products, and since the flavor cultivar was not cut by the restriction enzyme, 266bp of It was confirmed that the amplification product was generated.

Example 6. Identification of flavor varieties of the F2 generation

Analyzing genotypes of F2 obtained by crossing between non-fragrant normal rice and Ar-30 varieties (having the badh2-E14 allele), and between Dongjin-wx (wild type) and Mongdon native (3bp deletion in exon 12). For this purpose, functional markers FME14I (SEQ ID NOs: 7 and 8) and FME12-3 (SEQ ID NOs: 9 and 10) were used.

In the F2 generation, 14 species were randomly selected to extract genomic DNA from rice and amplify the Badh2 gene in the same manner as in Example 2, and amplification amplified using a primer set of SEQ ID NO: 9 and SEQ ID NO: 10. The product was reacted with restriction enzyme BslI, and then subjected to electrophoresis to measure the size of the amplified product.

The present inventors selected 13 individuals from the F2 generation obtained by crossing an Ar-30 variety with a non-fragrant general rice. 1 bp insertion was found in only one of 13 subjects, heterozygous in 8 subjects, and wild type in 4 subjects. The 1bp-inserted individual had a scent, whereas the heterozygous and wild-type had no scent (FIG. 5A).

According to the band pattern in the F2 generation (badh2/badh2-E12) obtained through crossing of Dongjin-wx and Mongdon native varieties, 2 individuals had a 3bp deletion, 7 individuals with two bands were heterozygous, and 4 individuals. It was wild type (FIG. 5B). Here, the individuals with 3bp deletion smelled, and the rest of the individuals did not. These results imply that badh2 is a recessive factor for rice aroma.

As described above, specific parts of the present invention have been described in detail, and for those of ordinary skill in the art, these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereby. It will be obvious. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.

<110> KONGJU NATIONAL UNIVERSITY INDUSTRY-UNIVERSITY COOPERATION FOUNDATION <120> Primer set for Discrimination of Aromatic Rice and Use Thereof <130> 1066008 <160> 31 <170> KoPatentIn 3.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> FMU1-2 F primer <400> 1 tcccaccacc actccaca 18 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> FMU1-2 R primer <400> 2 acgaagagct gccgctgc 18 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> FME2-7 F primer <400> 3 acgaagagct gccgctgc 18 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> FME2-7 R primer <400> 4 gcgattgcgc ggaggtact 19 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FME7 F primer <400> 5 tcctgtaatc atgtataccc 20 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> FME7 R primer <400> 6 aatttggaaa caaacctt 18 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FME12-3 F primer <400> 7 ttggtccagt gctctgtgtg 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FME12-3 R primer <400> 8 agcaccagcc agaccataac 20 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> FME14I F primer <400> 9 tcgatgccgg aattatctgg gtga 24 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> FME14I R primer <400> 10 tccccacggc tcatcggag 19 <210> 11 <211> 1512 <212> DNA <213> Artificial Sequence <220> <223> badh2 gene sequence <400> 11 atggccacgg cgatcccgca gcggcagctc ttcgtcgccg gcgagtggcg cgcccccgcg 60 ctcggccgcc gcctccccgt cgtcaacccc gccaccgagt cccccatcgg cgagatcccg 120 gcgggcacgg cggaggacgt ggacgcggcg gtggcggcgg cgcgggaggc gctgaagagg 180 aaccggggcc gcgactgggc gcgcgcgccg ggcgccgtcc gggccaagta cctccgcgca 240 atcgcggcca agataatcga gaggaaatct gagctggcta gactagagac gcttgattgt 300 gggaagcctc ttgatgaagc agcatgggac atggacgatg ttgctggatg ctttgagtac 360 tttgcagatc ttgcagaatc cttggacaaa aggcaaaatg cacctgtctc tcttccaatg 420 gaaaacttta aatgctatct tcggaaagag cctatcggtg tagttgggtt gatcacacct 480 tggaactatc ctctcctgat ggcaacatgg aaggtagctc ctgccctggc tgctggctgt 540 acagctgtac taaaaccatc tgaattggct tccgtgactt gtttggagct tgctgatgtg 600 tgtaaagagg ttggtcttcc ttcaggtgtg ctaaacatag tgactggatt aggttctgaa 660 gccggtgctc ctttgtcatc acaccctggt gtagacaagg ttgcatttac tgggagttat 720 gaaactggta aaaagattat ggcttcagct gctcctatgg ttaagcctgt ttcactggaa 780 cttggtggaa aaagtcctat agtggtgttt gatgatgttg atgttgaaaa agctgttgag 840 tggactctct ttggttgctt ttggaccaat ggccagattt gcagtgcaac atcgcgtctt 900 attcttcata aaaaaatcgc taaagaattt caagaaagga tggttgcatg ggccaaaaat 960 attaaggtgt cagatccact tgaagagggt tgcaggcttg ggcccgttgt tagtgaagga 1020 cagtatgaga agattaagca atttgtatct accgccaaaa gccaaggtgc taccattctg 1080 actggtgggg ttagacccaa gcatctggag aaaggtttct atattgaacc cacaatcatt 1140 actgatgtcg atacatcaat gcaaatttgg agggaagaag tttttggtcc agtgctctgt 1200 gtgaaagaat ttagcactga agaagaagcc attgaattgg ccaacgatac tcattatggt 1260 ctggctggtg ctgtgctttc cggtgaccgc gagcgatgcc agagattaac tgaggagatc 1320 gatgccggaa ttatctgggt gaactgctcg caaccctgct tctgccaagc tccatggggc 1380 gggaacaagc gcagcggctt tggacgcgag ctcggagaag ggggcattga caactaccta 1440 agcgtcaagc aagtgacgga gtacgcctcc gatgagccgt ggggatggta caaatcccct 1500 tccaagctgt aa 1512 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> P1 forward primer <400> 12 gtctctcctc caacatgctc 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> P1 reverse primer <400> 13 gtggtgtcag gtgtggagtg 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> P2 forward primer <400> 14 ccgaagtccg taccaactgc 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> P2 reverse primer <400> 15 caatcagcca tgcttccaac 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> P3 forward primer <400> 16 gtgcgtatcc tctgttctgg 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> P3 reverse primer <400> 17 caagcgtctc tagtctagcc 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> P4 forward primer <400> 18 ctgagctggc tagactagag 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> P4 reverse primer <400> 19 ccatcaggag aggatagttc 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> P5 forward primer <400> 20 cctccgtgtt aatgcagctc 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> P5 reverse primer <400> 21 catagcaagt ggcatgtacc 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> P6 forward primer <400> 22 ggttggtctt ccttcaggtg 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> P6 reverse primer <400> 23 gtccttccta actgccttcc 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> P7 forward primer <400> 24 ccagacgagc aggatgcaag 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> P7 reverse primer <400> 25 catggtcagg agcaagaagc 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> P8 forward primer <400> 26 gtggcaagga aggcagttag 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> P8 reverse primer <400> 27 gcaataaggc ttggaaggac 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> P9 forward primer <400> 28 tttcagcttc ttgctcctga 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> P9 reverse primer <400> 29 cagttaatct ctggcatcgc 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> P10 forward primer <400> 30 ggccaacgat actcagtgag 20 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 10 reverse primer <400> 31 ccggtcatca gctaacttcc 20

Claims (9)

A primer set for identifying a domestic native flavor variety comprising a primer sequence represented by SEQ ID NO: 3 and SEQ ID NO: 4, characterized by specifically amplifying the 414th to 491th bases of the Badh2 gene represented by SEQ ID NO: 11. .
The method of claim 1,
The flavor variety primer set is caused by the base deletion or insertion of the aldehyde dehydrogenase gene ( Badh2 ) represented by betaine SEQ ID NO: 11 present on chromosome 8 of rice. A primer set for discriminating domestic native flavor varieties, characterized in that it detects mutations specifically.
The method of claim 1,
The primer set can detect mutations in which 7bp of the Badh2 gene exon 2 portion is deleted,
The length of the amplified nucleotide sequence is 78bp in the case of normal rice varieties, 71bp in the case of flavor varieties, the domestic conventional flavor varieties set for discriminating.
(a) amplifying a betaine aldehyde dehydrogenase gene ( Badh2 ) located on chromosome 8 of rice by separating DNA from a sample of a general rice variety or a flavor variety and performing an amplification reaction;
(b) using the Badh2 gene amplified in step (a) as a template and performing an amplification reaction using a primer set for discriminating flavor varieties according to claim 1; And
(c) analyzing the sequence length of the amplification product generated by the amplification reaction to detect a mutation due to base deletion or insertion;
Varieties determination method of domestic traditional flavor comprising a.
The method of claim 4, wherein
Badh2 gene in step (a) is a primer set of SEQ ID NO: 12 and SEQ ID NO: 13; Primer sets of SEQ ID NO: 14 and SEQ ID NO: 15; Primer sets of SEQ ID NO: 16 and SEQ ID NO: 17; Primer sets of SEQ ID NO: 18 and SEQ ID NO: 19; Primer sets of SEQ ID NO: 20 and SEQ ID NO: 21; Primer sets of SEQ ID NO: 22 and SEQ ID NO: 23; Primer sets of SEQ ID NO: 24 and SEQ ID NO: 25; Primer sets of SEQ ID NO: 26 and SEQ ID NO: 27; Primer sets of SEQ ID NO: 28 and SEQ ID NO: 29; And a set of primers of SEQ ID NO: 30 and SEQ ID NO: 31; varieties of domestic flavors, characterized in that amplification using one or more primer sets selected from the group consisting of.
The method of claim 4, wherein
When performing the amplification reaction using the primer set in the step (b), the length of the nucleotide sequence of the amplification product is a varietal determination method of the domestic traditional flavor, characterized in that 78bp for rice, 71bp for flavor varieties.
A primer set comprising a primer sequence represented by SEQ ID NO: 3 and SEQ ID NO: 4, characterized by specifically amplifying the 414th to 491th bases of the Badh2 gene represented by SEQ ID NO: 11; And reagents for carrying out amplification reactions; Domestic traditional flavor varieties kit comprising a.
The method of claim 7, wherein
Reagents for performing the amplification reaction are dNTP (dATP, dCTP, dGTP and dTTP); DNA polymerase; And a buffer (buffer solution); domestic traditional flavor discrimination kit comprising at least one selected from the group consisting of.
The method of claim 7, wherein
The kit may further include a primer set for amplifying the Badh2 gene represented by SEQ ID NO: 11 of the flavor variety.
The primer set comprises a primer set of SEQ ID NO: 12 and SEQ ID NO: 13; Primer sets of SEQ ID NO: 14 and SEQ ID NO: 15; Primer sets of SEQ ID NO: 16 and SEQ ID NO: 17; Primer sets of SEQ ID NO: 18 and SEQ ID NO: 19; Primer sets of SEQ ID NO: 20 and SEQ ID NO: 21; Primer sets of SEQ ID NO: 22 and SEQ ID NO: 23; Primer sets of SEQ ID NO: 24 and SEQ ID NO: 25; Primer sets of SEQ ID NO: 26 and SEQ ID NO: 27; Primer sets of SEQ ID NO: 28 and SEQ ID NO: 29; A kit for discriminating domestic native flavor varieties, comprising one or more primers selected from the group consisting of SEQ ID NO: 30 and SEQ ID NO: 31 primer set.

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