KR101314168B1 - Method for developing gene specific molecular markers for discriminatinig soybean cultivars - Google Patents

Abstract

The present invention relates to a method for developing a gene specific molecular marker for soybean varieties, more specifically MYB of Arabidopsis Gene base sequences were used to identify soybean MYB- like genes, and from these genomes, Medicago truncatula genomes were found to identify genes similar to soybean MYB- like genes, and polymorphisms were observed at the promoter regions of these genes. The present invention relates to a method for developing a gene-specific molecular marker that can discriminate the relationship between soybean varieties.
The present invention provides a method for developing a gene-specific molecular marker that can confirm the specificity at the genome level of several soybean varieties using MYB transcription factor genes of Arabidopsis. According to the invention, the band pattern amplified by PCR can be used to discover the difference of polymorphism for each bean variety and use it as a molecular marker. Molecular markers discovered by the present invention can be used to determine soybean varieties in terms of molecular biology.

Description

METHOD FOR DEVELOPING GENE SPECIFIC MOLECULAR MARKERS FOR DISCRIMINATINIG SOYBEAN CULTIVARS}

The present invention relates to a method for developing a gene specific molecular marker for soybean varieties, more specifically MYB of Arabidopsis MYB of Soybean Using Gene Sequencing Identify similar genes and use them to Medicago A gene-specific molecular marker was developed to identify genes similar to the nucleotide sequences of soybean MYB- like genes in the truncatula genomic genes and to observe the related polymorphism at the promoter region of these genes to determine the relationship between soybean varieties. It is about a method.

The development of molecular markers between different genetic lines provides a very important way to select more effectively and accurately in the breeding process.

Conventional molecular markers used for breeding include restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), simple sequence repeats (SSR), and amplified fragment length polymorphism (AFLP). Most of these markers have the technical commonality that polymorphisms between DNA sequences on the genome can be easily identified through DNA fragments amplified by PCR. However, these polymorphisms are not related to genes involved in the expression of specific traits.

As mentioned above, marker assistant selection (MAS) (Yuejin et al. 2002), which can identify the presence or extent of specific traits in the breeding process, makes the process of breeding easier and more favorable to all breeders. In the methods mentioned above, such as RFLP, RAPD, SSR, and AFLP, the process of finding molecular markers related to specific traits is largely technically dependent on coincidence. There is a significant shortcoming that it is not known whether it is related to a particular trait.

The MYB transcription factor is a transcriptional regulatory protein involved in plant secondary metabolism, intrinsic tolerability, development, differentiation, and signal transduction, with single or multiple conserved mutations between MYB genes at the DNA binding site. A repeating nucleotide sequence of (R2R3) is observed. In Arabidopsis, approximately 130 MYB genes have multiple (R2R3) repeat sequences and are classified into 24 subgroups (Allan et al. 2008 Trends in Plant Science 13 (3)). : 99-102).

Therefore, the inventors of the Medicago gene having a similar sequence to the MYB genes of Arabidopsis using the technique of comparative genomics A gene-specific molecular marker was developed to identify varieties by identifying the genome-level specificity of various soybean varieties in Korea and foreign countries from the sequencing database of truncatula .

The present invention has been made by the necessity of the above, the technical problem to be achieved by the present invention is to discover the genetic polymorphism of the promoter region of the MYB- like gene of soybeans to compare the specificity at the genome level of various soybean varieties and soybean varieties It can be used as a gene-specific molecular marker that can be discriminated.

In order to achieve the above object, the present invention provides a method for developing a gene-specific molecular marker for the bean variety determination.

In the present invention, the method for developing a molecular marker comprises the steps of: (1) extracting genomic DNA (genomic DNA) for each variety of soybeans; (2) Gycine max ) extracting a similar MYB gene of soybean having a nucleotide sequence similar to that of the MYB R2R3 repeat sequence of Arabidopsis, SEQ ID NO: 41 from the DNA sequencing database; (3) finding a gene having a nucleotide sequence similar to the MYB gene of the soybean extracted in step (2) from the DNA sequence database of Medicago truncatula ; (4) discovering a promoter region of the gene found in step (3) from the DNA sequence database of the Medicago truncatula ; (5) preparing a PCR primer (primer) capable of amplifying a specific site of the promoter; (6) using the genomic DNA of step (1) as a template and amplifying a promoter region of a specific gene using the primer of step (5); And (7) observing and analyzing polymorphism at the promoter region of the amplified specific gene to identify a pattern that can be used as a molecular marker between strains.

The present invention provides a method for developing a gene-specific molecular marker that can confirm specificity at the genome level of various soybean varieties using MYB transcription factor genes of Arabidopsis. According to the invention, the band pattern amplified by PCR can be used to discover the difference of polymorphism for each bean variety and use it as a molecular marker. Molecular markers discovered by the present invention can be used to determine soybean varieties in terms of molecular biology.

1 is a table classifying soybean varieties and genetic resources in accordance with an embodiment of the present invention by species color.
Figure 2 is a table classifying soybean varieties and genetic resources system according to an embodiment of the present invention by granularity (seed size).
Figure 3 is a nucleotide sequence having a high degree of similarity found in the Medicago truncatula sequencing database using the soybean MYB- like gene sequence shown in SEQ ID NO: 1.
Figure 4 is a nucleotide sequence having a high degree of similarity found in the Medicago truncatula sequencing database using the soybean MYB- like gene sequence shown in SEQ ID NO: 2.
Figure 5 is a nucleotide sequence having a high degree of similarity discovered in the Medicago truncatula sequencing database using the soybean MYB- like gene sequence shown in SEQ ID NO: 3.
Figure 6 is a nucleotide sequence having a high degree of similarity found in the Medicago truncatula sequencing database using the soybean MYB- like gene sequence shown in SEQ ID NO: 4.
7 is a nucleotide sequence having a high degree of similarity found in the Medicago truncatula sequencing database using the soybean MYB- like gene sequence shown in SEQ ID NO: 5.
Figure 8 is a nucleotide sequence having a high degree of similarity found in the Medicago truncatula sequencing database using the soybean MYB- like gene sequence shown in SEQ ID NO: 6.
Figure 9 is a nucleotide sequence having a high degree of similarity discovered in the Medicago truncatula sequencing database using the soybean MYB- like gene sequence shown in SEQ ID NO: 7.
10 is a nucleotide sequence having a high degree of similarity found in the Medicago truncatula sequencing database using the soybean MYB- like gene sequence shown in SEQ ID NO: 8.
FIG. 11 is a nucleotide sequence including the 2Kb promoter region at the 5 'end of the nucleotide sequence shown in SEQ ID NO: 9;
12 is a nucleotide sequence including a 2Kb promoter region at the 5 'end of the nucleotide sequence shown in SEQ ID NO: 10.
FIG. 13 is a nucleotide sequence including a 2Kb promoter region at the 5 'end of the nucleotide sequence shown in SEQ ID NO: 11; FIG.
14 is a nucleotide sequence including the 2Kb promoter region at the 5 'end of the nucleotide sequence shown in SEQ ID NO: 12.
FIG. 15 is a nucleotide sequence including a 5K terminal higher 2Kb promoter region of the nucleotide sequence set forth in SEQ ID NO: 13;
FIG. 16 is a nucleotide sequence including a 2Kb promoter region at the 5 'end of the nucleotide sequence shown in SEQ ID NO: 14. FIG.
FIG. 17 is a nucleotide sequence including a 5K terminal high 2Kb promoter region of the nucleotide sequence set forth in SEQ ID NO: 15.
FIG. 18 is a nucleotide sequence including the 2Kb promoter region at the 5 'end of the nucleotide sequence shown in SEQ ID NO: 16.
19 shows DNAs amplified by using primers (SEQ ID NOs: 25, 26) prepared for amplifying a specific site of the promoter represented by SEQ ID NO: 17 (FIG. 11) and each soybean variety genomic DNA as a template. Band pattern. A1 indicates the position of the band used for NTSYS analysis. 19 to 26, M: Standard marker, lane 1: Namhae soybean, lane 2: golden soybean, lane 3: Bokwang soybean, lane 4: black soybean No. 1, lane 5: Gwangdu, lane 6: Northeast Asia, lane 7 : Real bean 2, lane 8: Haman bean, lane 9: Bugwang bean, lane 10: Baiyun bean, lane 11: galaxy bean, lane 12: radiant bean, lane 13: Hannam bean, lane 14: Advent bean, lane 15 : Green bean, lane 16: New Paldal No. 2, lane 17: big ol bean, lane 18: Dongdong bean 69, lane 19: Dongdong bean 127, lane 20: Manly bean, lane 21: Shinnong black bean, lane 22: Gwangan bean , lane 23: Dawon bean, lane 24: pioneer bean No. 1, lane 25: myeong bean, lane 26: daol bean, lane 27: new ol bean, lane 28: sobaek bean, lane 29: celadon bean, lane 30: hwaham foot bean, lane 31: black bean 4, lane 32: black bean, lane 33: daewon bean, lane 34: dapung bean, lane 35: solok bean, lane 36: taekwang bean, lane 37: rhubarb bean, lane 38: samnam bean, lane 39 : Chengdu bean, lane 40: Cheongseoritae, lane 41: black and blue bean, lane 42: black bean, lane 43: black bean 3, lane 44: rose bean, lane 45: manor bean, lane 46: real bean, lane 47: Pungsan bean, lane 48: black frosted) .
FIG. 20 shows DNAs amplified by using primers (SEQ ID NOs 27 and 28) prepared for amplifying a specific site of the promoter represented by SEQ ID NO: 18 (FIG. 12) and respective soybean varieties genomic DNA. Band pattern. B1 and B2 indicate the position of the band used for NTSYS analysis.
21 shows DNAs amplified by using primers (SEQ ID NOs: 29 and 30) prepared for amplifying a specific site of the promoter described in SEQ ID NO: 19 (FIG. 13) and respective soybean varieties genomic DNAs. Band pattern. C1 and C2 indicate the position of the band used for NTSYS analysis.
FIG. 22 shows DNAs amplified by using primers (SEQ ID NOs: 31 and 32) prepared for amplifying a specific site of the promoter represented by SEQ ID NO: 20 (FIG. 14) and respective soybean varieties genomic DNA. Band pattern. D1 and D2 indicate the position of the band used for NTSYS analysis.
Fig. 23 shows DNAs amplified by using primers (SEQ ID NOs: 33, 34) prepared for amplifying a specific site of the promoter represented by SEQ ID NO: 21 (Fig. 15) and respective soybean varieties genomic DNAs as templates. Band pattern. F1 and F2 indicate the position of the band used for NTSYS analysis.
FIG. 24 shows DNAs amplified by using primers (SEQ ID NOs: 35 and 36) prepared for amplifying a specific site of the promoter represented by SEQ ID NO: 22 (FIG. 16) and respective soybean varieties genomic DNA. Band pattern. E1 and E2 indicate the position of the band used for NTSYS analysis.
FIG. 25 shows DNAs amplified by using primers (SEQ ID NOs: 37 and 38) prepared for amplifying a specific site of the promoter represented by SEQ ID NO: 23 (FIG. 17) and respective soybean varieties genomic DNA. Band pattern. G1 and G2 indicate the position of the band used for NTSYS analysis.
FIG. 26 shows DNAs amplified by using primers (SEQ ID NOs: 39 and 40) prepared for amplifying a specific site of the promoter set forth in SEQ ID NO: 24 (FIG. 18) and respective soybean varieties genomic DNA. Band pattern. H1 and H2 indicate the position of the band used for NTSYS analysis.
FIG. 27 is a phylogenetic tree showing a relationship between soybean varieties obtained by NTSYS analysis of the band patterns of FIGS. 19 to 26.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for developing a gene-specific molecular marker for discriminating bean varieties.

In the present invention, the development method of the molecular marker comprises the steps of: (1) extracting genomic DNA (genomic DNA) for each variety of soybeans; (2) Gycine max ) extracting a similar MYB gene of soybean having a nucleotide sequence similar to that of the MYB R2R3 repeat sequence of Arabidopsis, SEQ ID NO: 41 from the DNA sequencing database; (3) finding a gene having a nucleotide sequence similar to the MYB gene of the soybean extracted in step (2) from the DNA sequence database of Medicago truncatula ; (4) discovering a promoter region of the gene found in step (3) from the DNA sequence database of the Medicago truncatula ; (5) preparing a PCR primer (primer) capable of amplifying a specific site of the promoter; (6) using the genomic DNA of step (1) as a template and amplifying a promoter region of a specific gene using the primer of step (5); And (7) observing and analyzing polymorphism at the promoter region of the specific amplified gene to identify a pattern that can be used as a molecular marker between lines.

In the present invention, the step of discovering the promoter is characterized in that the excavation in the upper 1.5 ~ 3 Kb region of the 5 'direction of the gene sequence found in the step (3), the step of preparing a primer is a promoter of a specific gene It is characterized by producing a plurality of primers within 10 Kb in the 5 'direction from the start codon included.

Soybean ( Glycine) discovered in the present invention max ) MYB- like gene is characterized in that at least one selected from the nucleotide sequence of SEQ ID NO: 1 to 8, the nucleotide sequence similar to the MYB- like gene of the soybean found from the DNA sequence database of Medicago truncatula is SEQ ID NO: 9 To 16 (FIGS. 3 to 10).

In the present invention, the promoter region discovered from the soybean MYB transcriptional regulator analogous nucleotide sequence shown in SEQ ID NOs: 9 to 16 (FIGS. 3 to 10) is described as SEQ ID NOs: 17 to 24 (FIGS. 11 to 18), Primers capable of amplifying each promoter site are the nucleotide sequences set forth in SEQ ID NOs: 9-24.

The present invention also provides a gene-specific molecular marker for discriminating soybean varieties produced by the method of developing the molecular marker.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

Example  1. Varieties of Beans DNA  extraction

For the test materials, Korean soybean varieties and 48 genetic resources were used. Specifically, Namhae, Golden, Bokwang, Black No. 1, Gwangdu, Northeast Tae, Gen. No. 2, Haman, Bugwang, Baiyun, Galaxy, Radiation, Hannam, Advent , Blue, Sinpaldal No.2, Bigol, Dongsan No.69, Dongsan No.127, Manly, Sinnong Black, Gwangan, Dawon, Pioneer No.1, Soybean, Daol, Saol, Sobaek, Celadon, Hwaeomfoot, Black No.4, Blackol , Daewon, Dafeng, Sorok, Taekwang, Rhubarb, Samnam, Chengdu, Cheongseoritae, Black Blue, Galmi, Black No.3, Rose, Changwon Bean, Genuine, Poongsan, Black Surrey Seed size) and the like are summarized in FIGS. 1 and 2. Among the test materials, bean varieties and gene source systems of which the seed color was not yellow were 9 having black seedlings, such as No. 1, and 6 having other seedlings, such as green and brown (Fig. 1). In addition, the classification of soybean varieties and genetic resources by the seed size is shown in FIG.

In order to extract DNA, soybeans were germinated under sterile conditions for 7 days and then samples were taken. For seed sterilization, each seed was well shaken with 95% alcohol for 1 minute, then rinsed three times with sterile water, then shaken well with 50% sodium hypochlorite for 5 minutes, and then rinsed three times with sterilized water. . The sterilized seeds were placed in a petri dish containing 1% Bacto agar medium in a clean bench, sealed, and germinated at 30 ° C. in a growth chamber with light blocking.

DNA extraction of germinated soybean seeds was performed using Plant SV mini DNA extraction kit of GENEALL (Korea). Seed germinated soybean seed with liquefied nitrogen, take 100 mg into a micro tube, add 400 μL PL buffer and 4 μL RNase in the kit and vortex. After incubation for 15 minutes in a heating block adjusted to 65 ℃ (140 μl PD buffer) and put on ice for 5 minutes. Next, after centrifuging the mixture at 13,000 × g for 5 minutes, the supernatant was separated into an Easy sep filtert column, and again centrifuged at 13,000 × g for 2 minutes, and filtered through a column. Transfer the liquid to a new tube. Put the BD buffer corresponding to 1.5 times the filtered liquid, stir and vortex and transfer to the SV column and centrifuge for 30 seconds at 13,000 × g. Discard the liquid below, add 700 μl CW buffer to the SV column and centrifuge for 30 seconds at 13,000 × g. Then, add 300 μl CW buffer and centrifuge for 2 minutes at 13,000 × g. do. Subsequently, remove the filter part of the SV column, place it in a new tube, add 50 μl AE buffer, react for 5 minutes at room temperature, and centrifuge for 1 minute at 13,000 × g. genomic DNA) was extracted.

Example  2. Beans Glycine max )of MYB  Having a nucleotide sequence similar to a transcriptional regulator Medicago truncatula  of  Finding similar genes

Arabidopsis thaliana ) R2, R3 repetitive sequence (SEQ ID NO: 41, cttcg atgga ccaat tatct aagac cggac atcaa gagag gaaga ttctc gttcg) which is commonly found in the coding region of the MYB transcription factor genes of thaliana aggaa gaaga aacta tcat tcagc tacac agtgt tatgg gaaac aa: Marin C and Paz-Ares J. 1998 MYB transcription factors in plant, Trends Genet 13 (2): 67-73) Soybean ( Glycine) max ) nucleotide sequence was extracted from GenBank ( www.ncbi.nlm.nih.gov ), soybean MYB It was used as a similar gene (SEQ ID NOS: 1-8).

Medicago by querying the base sequence Glycine by BLAST in truncatula sequencing database (GenBankk, www.ncbi.nlm.nih.gov) max ) in MYB Medicago with a level of e-20 or greater similarity with the transcriptional regulator gene The base sequence of truncatula is set forth in SEQ ID NOs: 9-16 (FIGS. 3-10). The base sequences described in SEQ ID NOs: 9 to 16 (FIGS. 3 to 10) were discovered at AC137831, AC153128, AC124961, AC166313, AC140544, AC140524, AC122724 and AC148762 of the BAC sequencing library of the Medicago truncatula sequence database, respectively.

Example  3. Beans MYB  Similar to genes Medicago truncatula  Discovery of Promoter Sequences from Genes

Using the base sequence SEQ ID Nos. 9 to 16 (Figs. 3 to 10) of Medicago truncatula having high significance with the MYB transcriptional regulator gene of soybean extracted in Example 2, the 5 'terminal at which the corresponding base starts from the top (5 'direction) the same Medicago the base sequence of the approximately 2 Kb Extracted from the truncatula genomic sequence database, the program for sequencing was developed by self-program get_seq2 programmed in C language.

The base sequences set forth in SEQ ID NOs: 17 to 24 (FIGS. 11 to 18) are each Medicago discovered by this method. Nucleotide sequence containing the promoter region of the MYB- like gene of truncatula . For example, an underlined portion at the 5 'end of the base sequences of FIGS. 3 to 10 indicates a portion corresponding to the underlined base sequence at the 3' end of FIGS. 11 to 18, which is each promoter site sequence.

Example  4. Varieties of Beans Of a molecular marker  Development

4-1) Medicago truncatula Similar to MYB  Can amplify the gene promoter region primer  making

Medicago , a model plant of legume crops The primers are generated using the nucleotide sequence of the genomic DNA sequencing database of truncatula , and the polymorphisms between the soybean varieties based on the genomic DNA of the soybean are as follows. Currently, the genomic DNA sequence of soybeans presented in NCBI GenBank and Phytozome (www.phytozome.net) is the result of a genome sequencing project conducted at the Joint Genome Institute of the US Department of Energy (US DOE). (PI518671) genomic DNA sequence. Thus, the nucleotide sequence of the genomic DNA sequence database in this document is the nucleotide sequence of one particular soybean cultivar, based on which it is possible to identify primers and to identify polymorphisms at the genome level related to various quantitative and qualitative traits compared to other soybean varieties. There is a limit to using it.

If Williams' genomic DNA sequence database was used in this experiment, we could identify a polymorphism by discovering a promoter sequence based on the nucleotide sequence of a particular gene, and creating a primer to amplify the promoter region and comparing the amplified patterns. If you want to do so, you should compare several different soybean varieties based on the specific bean variety Williams82. This approach has many limitations and limitations in comparing the specific (phenotype) and polymorphism (genotype) at the genome level between soybean varieties.

Therefore, in the present invention, the same glycine (Glycine) genus and soybean ( Glycine) max ) is known to be genetically very similar to Medicago Based on the nucleotide sequence of Medicago truncatula based on the nucleotide sequence of Medicago truncatula, the gene sequence polymorphism of a specific gene is identified by extracting the nucleotide sequence of a similar gene from truncatula , extracting the promoter sequence based on it, and preparing a primer. To compare with.

For this reason, Medicago has a highly similar sequence with the MYB transcriptional regulator of soybean, which was discovered using the R2R3 sequence of Arabidopsis. After excavating from truncatula 's genomic DNA database and preparing primers for amplifying the promoter regions of these genes, the polymorphism observed in band patterns amplified when genomic DNA of various soybean varieties is used as a template is NT SYS. The relationship between soybean varieties was investigated.

Medicago by Example 3 PCR primers capable of amplifying a specific site of the imaginary promoter sequences SEQ ID NOs 17 to 24 (Figs. 11 to 18) found in the truncatula 's genomic sequence database were prepared and represented by SEQ ID NOs: 25 to 40. Primer pairs prepared for amplifying a specific region of SEQ ID NO: 17 (FIG. 11) are SEQ ID NOs: 25 and 26, below SEQ ID NOs: 27 and 28 are SEQ ID NO: 18 (FIG. 12), and SEQ ID NOs: 29 and 30 are SEQ ID NO: 19 (Fig. 13), SEQ ID NOs: 31 and 32 are SEQ ID NO: 20 (Fig. 14), SEQ ID NOs: 33 and 34 are SEQ ID NO: 21 (Fig. 15), SEQ ID NOs: 35 and 36 are SEQ ID NO: 22 (Fig. 16), SEQ ID NO: 37 And 38 are SEQ ID NO: 23 (FIG. 17), SEQ ID NO: 39 and 40 are primer pairs prepared for amplifying a specific site of SEQ ID NO: 24 (FIG. 18).

4-2) PCR  And polymorphism ( polymorphism or polymorphic pattern Excavation

Polymerase chain reaction (PCR) was performed using a PCR premix of Bioneer (Seoul, Korea). To each 20 μl of reaction solution, 5 ng of premixture and genomic DNA, 16 μl of dH2O, 10 pmole (1 μl) of forward primer and reverse primer were added, and the premixture was Taq DNA polymerase 1 U (unit). , dNTP 0.25 mM, Tris-HCL (pH 9.0) 10 mM, KCl 30 mM, MgCl ₂ 1.5 mM, Stabilizer and tracking dye.

DNA amplification was performed using a Veriti 96-well thermal cycler (Applied Biosystesm, USA). PCR conditions were initially denatured at 94 ° C. for 5 minutes, denaturing at 94 ° C. for 30 seconds, annealing for 30 seconds at the annealing temperature of each primer, and 1 minute at 72 ° C. After 35 cycles of amplifying (cycle), and finally DNA extension (DNA extension) was performed for 5 minutes at 72 ℃. The product amplified by PCR was subjected to 2 hours at 70 V using 1.0% agarose gel containing 0.5 × TBE buffer (45 mM tris-borate, 1 mM EDTA) and ethidium bromide. During electrophoresis. Agarose gels were photographed under UV light and used for band analysis. The electrophoretic test results are shown in FIGS. 19 to 26.

4-3) Discovery of genotype polymorphisms between different soybean lines

Medicago having high similarity with MYB gene of soybean having R2R3 nucleotide sequence found in Example 1 above Based on the nucleotide sequence of truncatula (Figs. 3 to 10), a pair of primers (SEQ ID NOs: 25 to 40) capable of amplifying a portion of a portion of about 2 Kb in the upstream direction from the 5 'end of the nucleotide sequence Using this, genomic DNA of each soybean variety was amplified by PCR and separated by electrophoresis on agarose gel to examine polymorphism according to band size and amplification. . Varietal polymorphism was observed in the promoters of the amplified genes, and the experimental results are shown in FIGS. 19 to 26. In addition, the phylogenetic tree analyzing the genetic relationship between varieties using the NT SYS program is shown in FIG. 27.

4-4) Beans Lineage  Determination of Soybean Varieties by Genomic Level Polymorphism

Similar MYB in the present invention By using the polymorphism of the promoter region nucleotide sequence involved in the transcription and expression of genes, we tried to find the unique specificity at the genome level of various soybean varieties and use it as a marker. The coding region of the MYB transcriptional regulator is divided into DNA binding sites located in the 5 'terminal direction (N-terminal) and sites having various functions located in the 3' terminal direction (C-terminal). The base sequence of the 5 'direction DNA binding site is very similar between MYB genes, and the MYB gene family is determined by the presence of three repeated sequences R1, R2, and R3 (repeated sequence) located at this site. Many genes belonging to are classified.

The soybean-like MYB genes used in this experiment are the R2R3 MYB genes that are known to be present in many plants. Only a few functions have been reported until recently regarding the specific functions of proteins produced by these MYB transcriptional regulators. However, among these, Medicago had a high degree of similarity with the eight soybean-like MYB transcriptional regulators used in this experiment. Twenty- four soybean varieties and genetic relationships were independently isolated on the phylogenetic tree by primer pairs capable of amplifying the promoter region of the pseudo MYB transcriptional regulator of truncatula (FIG. 27). This is a result of demonstrating that statistical analysis of the PCR band pattern amplified using a primer set that amplifies the promoter region of the eight genes can determine the specificity at the genome level of 48 Korean soybean varieties. .

Although these results do not explain the specific functions of the genes in which the polymorphisms of the above promoter regions are found, the polymorphisms found in the promoters of these genes are determined at the genomic level of each soybean variety and gene source. Specificity means you can prove it.

Having described specific portions of the present invention in detail, those skilled in the art will appreciate that these specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

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Claims (15)

(1) extracting genomic DNA (genomic DNA) for each variety of soybeans;
(2) at least one selected from the nucleotide sequences set forth in SEQ ID NOs: 1 to 8, which are similar to the MYB R2R3 repeat sequences of Arabidopsis as depicted in SEQ ID NO: 41 from the DNA base sequence database of soybean ( Gycine max ); Discovering the MYB gene of the soybean having the nucleotide sequence;
(3) finding a gene having a nucleotide sequence similar to the MYB gene of the soybean extracted in step (2) from the DNA sequence database of Medicago truncatula ;
(4) discovering a promoter region of the gene found in step (3) from the DNA sequence database of the Medicago truncatula ;
(5) preparing a PCR primer (primer) capable of amplifying a specific site of the promoter;
(6) using the genomic DNA of step (1) as a template and amplifying a promoter region of a specific gene using the primer of step (5); And
(7) observing and analyzing the polymorphism at the promoter region of the specific amplified gene to identify a pattern that can be used as a molecular marker between lines;
Including, but the gene having a nucleotide sequence similar to the MYB gene of the soybean in step (3) is a molecular marker development method, characterized in that at least one selected from the nucleotide sequence shown in SEQ ID NO: 9 to 16.
The method of claim 1,
Step (4) is a molecular marker development method, characterized in that the excavation of the promoter in the 1.5 ~ 3 Kb region of the 5 'direction of the gene sequence found in step (3).
The method of claim 1,
Step (5) is a method for developing a molecular marker, characterized in that for producing a plurality of primers within 10 Kb in the 5 'direction from the start codon including the promoter of a specific gene.
delete delete The method of claim 1,
Promoter site discovered in step (4) is a molecular marker development method, characterized in that at least one selected from the base sequences described in SEQ ID NO: 17 to 24.
The method according to claim 6,
Primer capable of amplifying the promoter region described in SEQ ID NO: 17 is a molecular marker development method, characterized in that the primer pair described in SEQ ID NO: 25 and 26.
The method according to claim 6,
Primer capable of amplifying the promoter region described in SEQ ID NO: 18 is a molecular marker development method, characterized in that the primer pair described in SEQ ID NO: 27 and 28.
The method according to claim 6,
Primer capable of amplifying the promoter region described in SEQ ID NO: 19 is a molecular marker development method, characterized in that the primer pair described in SEQ ID NO: 29 and 30.
The method according to claim 6,
Primer capable of amplifying the promoter region described in SEQ ID NO: 20 is a molecular marker development method, characterized in that the primer pair described in SEQ ID NO: 31 and 32.
The method according to claim 6,
Primer capable of amplifying the promoter region described in SEQ ID NO: 21 is a molecular marker development method, characterized in that the primer pair described in SEQ ID NO: 33 and 34.
The method according to claim 6,
Primer capable of amplifying the promoter region described in SEQ ID NO: 22 is a molecular marker development method, characterized in that the primer pair described in SEQ ID NO: 35 and 36.
The method according to claim 6,
Primer capable of amplifying the promoter region described in SEQ ID NO: 23 is a molecular marker development method, characterized in that the primer pair described in SEQ ID NO: 37 and 38.
The method according to claim 6,
Primer capable of amplifying the promoter region described in SEQ ID NO: 24 is a molecular marker development method, characterized in that the primer pair described in SEQ ID NO: 39 and 40.
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