KR20120056520A - Method for developing gene specific molecular markers - Google Patents

Abstract

PURPOSE: A method for developing a frogeye leaf spot resistant gene-specific molecular marker is provided to distinguish different species at a genomic level. CONSTITUTION: A method for developing a molecular marker comprises: a step of isolating genomic DNA with different reactivities to frogeye leaf spot; a step of searching for a gene which is similar to a sequence of the MYB transcription regulatory gene of Glycine m from the DNA base sequence database of Medicago truncatula; a step of searching for a promoter site from the database; a step of designing a PCR primer for amplifying a specific site of the promoter; a step of amplifying the promoter site using a primer and genomic DNA; and a step of confirming a pattern.

Description

METHOD FOR DEVELOPING GENE SPECIFIC MOLECULAR MARKERS}

The present invention relates to a method for developing a soybean spot disease resistant gene-specific molecular marker, more specifically Medicago The genome genes of truncatula inherent in the genome of the relevant soybean MYB transcriptional regulators, genes similar to those found, and polymorphisms observed in the promoter region of these genes by examining and analyzing the flowering characteristics of the soybean varieties characterized by The present invention relates to a method for developing a gene-specific molecular marker that can be discriminated.

Bean spots Cercospora It is a disease caused by sojina Hara, and its damage is spreading all over the country in the northern part of Gyeonggi-do recently. It is an important disease that occurs in pods and seeds as well as leaves and stems of beans. Varieties of soybean varieties to this pathogen vary, but only recently in Korea, the distribution of strains causing spot pattern disease has been investigated, and the pathogenicity test and the discrimination of varieties have been reported (Kim et al. 2009. Regional Specialized Technology Development). Report Publication No. 11-1390000-002219-01).

Selection for the development of resistant varieties of mucus disease is distinguished from susceptibility and resistance on the basis of 2 mm of lesion size at 14 days and 21 days after inoculation of pathogens in the growth stage between soybean growing period V3 ~ V5. Due to this experiment process, a lot of difficulties arise when conducting large-scale outdoor and indoor experiments. Cercospora Many technical difficulties are accompanied, such as mass cultivation of sojina Hara, and control of the growing season of the selected system. Therefore, the development of molecular markers related to spot disease resistance has been suggested as an effective alternative to reduce the time and cost required for the selection of strains resistant to spot disease.

Currently, gene-specific molecular markers are discovering polymorphisms from coding regions that are primarily involved in protein synthesis of genes, but it is very difficult to find enough polymorphisms to construct PCR primers. There was a problem, and most of the reported polymorphisms of coding regions were observed and attempted as markers were single nucleotide polymorphisms (SNPs) with only a few nucleotides.

In the present invention, soybean spot disease-related gene-specific molecular markers have been developed using promoters of MYB transcription factor genes, which are transcriptional regulatory proteins related to plant disease. Using primers showing polymorphism between varieties when amplifying the promoter region of each of the MYB transcription control genes, and in each variety phenotype the resistance degree for the bean jeommunuibyeong looks different compared with the dielectric properties of each of the MYB transcription control genes We have developed a MYB gene-specific molecular marker for soybean spot disease resistance.

The present invention has been made in view of the above problems and the need for the above, the technical problem to be achieved by the present invention is a genetic polymorphism of the gene promoter region of MYB transcription regulation factor gene that is a transcriptional regulatory protein associated with plant injuries By exploring this, it is to provide a method for developing a primer that represents this polymorphism as a molecular marker associated with soybean spot disease.

In order to achieve the above object, the present invention provides a method for the development of soybean spot disease resistant gene specific molecular markers.

In the present invention, the method of developing the molecular marker comprises the steps of: (1) extracting genomic DNA (genomic DNA) for each variety of soybeans with different reactivity to soybean spot pattern disease; (2) finding a gene similar to the nucleotide sequence of the MYB electron regulator gene of soybean ( Glycine max ) from the DNA sequence database of Medicago truncatula ; (3) discovering a promoter region of the gene found in step (2) from the DNA sequence database of the Medicago truncatula ; (4) preparing a PCR primer capable of amplifying a specific site of the promoter; (5) using the genomic DNA of step (1) as a template and amplifying a promoter region of a specific gene using the primer of step (4); And (6) observing and analyzing the 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 new molecular marker associated with soybean spot disease, according to the present invention it is possible to discover the difference in polymorphism for each of the soybean varieties in a band pattern amplified by PCR and use it as a molecular marker. The molecular markers discovered by the present invention can distinguish varieties having different degrees of response to soybean spot disease at the genome level, and can be used as soybean spot disease specific molecular markers.

1 is a bean ( Glycine max ) Medicago discovered through BLAST using the MYB transcriptional regulator gene (SEQ ID NO: 1) as a query It is a similar MYB transcriptional regulator of truncatula .
2 is a bean ( Glycine max ) Medicago discovered through BLAST using MYB transcriptional regulatory gene (SEQ ID NO: 2) as a query It is a similar MYB transcriptional regulator of truncatula .
3 is a bean ( Glycine max ) Medicago discovered through BLAST using MYB transcriptional regulator gene (SEQ ID NO: 3) as a query It is a similar MYB transcriptional regulator of truncatula .
4 is a bean ( Glycine max ) Medicago discovered through BLAST using MYB transcriptional regulator gene (SEQ ID NO: 4) as a query It is a similar MYB transcriptional regulator of truncatula .
5 is a bean ( Glycine max ) Medicago discovered through BLAST using MYB transcriptional regulator gene (SEQ ID NO: 5) as a query It is a similar MYB transcriptional regulator of truncatula .
6 is a bean ( Glycine max ) Medicago discovered through BLAST using MYB transcriptional regulatory gene (SEQ ID NO: 6) as a query It is a similar MYB transcriptional regulator of truncatula .
7 is Medicago DNA bands amplified using primers (SEQ ID NOs: 19-30) designed to amplify the promoters of similar MYB transcriptional regulators of truncatula and genomic DNA of soybean varieties that respond differently to soybean spots Photo shows the pattern.
FIG. 8 is a phylogenetic tree showing the relationship between soybean varieties obtained by NT sys analysis of the band pattern of FIG. 7.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for developing a soybean spot disease resistant gene specific molecular marker.

In the present invention, the method of developing the molecular marker comprises the steps of: (1) extracting genomic DNA (genomic DNA) for each variety of soybeans with different reactivity to soybean spot pattern disease; (2) finding a gene similar to the nucleotide sequence of the MYB transcriptional regulator gene of soybean ( Glycine max ) from the DNA sequence database of Medicago truncatula ; (3) discovering a promoter region of the gene found in step (2) from the DNA sequence database of the Medicago truncatula ; (4) preparing a PCR primer capable of amplifying a specific site of the promoter; (5) using the genomic DNA of step (1) as a template and amplifying a promoter region of a specific gene using the primer of step (4); And (6) 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 soybean varieties are characterized in that at least one selected from the group consisting of Bokwang beans, Jangwon beans, Geumgang beans, Cheongja bean No. 3, Anpyeong beans, Daeman beans, Dachae beans and Taekwang beans.

Step (3) of the present invention discovers a promoter at the top 1.5 ~ 2 Kb region of the 5 'direction of the gene sequence found in step (2), step (4) is the start codon including the promoter of a specific gene It characterized in that a plurality of primers are produced in 10 Kb in the 5 'direction from.

Soybean-related beans found in the present invention ( Glycine max ) MYB transcriptional regulator is characterized in that at least one selected from the nucleotide sequences set forth in SEQ ID NO: 1 to 6, Medicago Sequences similar to the specific genes found in the truncatula DNA sequencing database are set forth in SEQ ID NOs: 7-12.

In the present invention, soybean ( Glycine) described in SEQ ID NOs: 7 to 12 max ) Promoter sites discovered from MYB transcriptional regulator-like sequences are shown in SEQ ID NOs: 13-18, and primers capable of amplifying each promoter site are described in SEQ ID NOs: 19-30 .

The present invention also provides a soybean spot disease related gene specific molecular markers produced by the method of developing the molecular marker.

Hereinafter, the present invention will be described in more detail with reference to Examples.

However, the following examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not interpreted to be limited by these examples.

Example  1. Soybean Spotted Disease Identification DNA  extraction

The different varieties of soybeans that have different reactions such as resistance (R) or susceptibility (S) to soybean spot pattern disease are Kim et al. (2009, Regional Technology Development Report Publication No. 11-1390000-002219 -01). According to Kim et al., Bokwang soybeans are resistant to all types of races (Cs-2, Cs-10, Cs-14, Cs-16, Cs-19, and Cs-20) that cause soy spots in Korea. On the other hand, Taekwang showed sensitivity to all of the races.

In the present invention, Bogwang beans, Jangwon beans, Geumgang beans, Cheongja beans No. 3, Anpyeong beans, Daemong beans, Dachae beans, Taegong beans, which are known as varieties capable of discriminating the race of soybean spot pathogens in Korea, were used as samples.

5 g of leaves of each bean variety are placed in a mortar and finely ground in a 15 ml tube while supplying liquid nitrogen, DNA extraction buffe (100 ml of 5 M NaCl, 1 M Tris-HCl (pH 8.0) 100 ㎖, 200 ml of 0.25 M EDTA, 62.5 ml of 20% SDS, and 6 ml of 0.38 g / buffer 100 ml of Sodium bisulfite were added and placed in a 65 ° C. water bath for 20 minutes. An equal amount of chloroform isoamylalchohol (24: 1 (v / v)) was added thereto, shaken for about 20 minutes, and centrifuged at 3,000 rpm in a 4 ° C centrifuge.

The supernatant was then taken, transferred to a new tube, and DNA was precipitated by adding 2x ethanol or 2 / 3x isopropanol. The tangled DNA was washed in 70% ethanol, transferred to a 1.5 ml tube and dried in air. Next, the dried DNA was dissolved in 300 μl of TE buffer (TE buffer; 10 mM Tris-HCl, 1 mM EDTA, pH7.4), and placed in a thermostat at 50-60 ° C. to remove DNase activity and RNase (1 mg / ml). ) 4 µl of the solution was shaken well, stored at 37 ° C. for about 1 hour, RNA was removed, and DNA was extracted.

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

Soybean-related beans ( Glycine) used in the present invention max ) the nucleotide sequence of MYB transcriptional regulator genes are set forth in SEQ ID NOs: 1-6.

Medicago using the nucleotide sequence as a query BLAST results from the truncatula sequence database (sequencing database, GenBank, www.ncbi.nlm.nih.gov ) are summarized in Figures 1 to 6, the MYB transcriptional regulator of soybean ( Glycine max ) and the level of e-20 or more The base sequences of Medicago truncatula with similarities are set forth in SEQ ID NOs: 7-12. Each base sequence set forth in SEQ ID NOS: 7-12 is Medicago as shown in FIGS. It was discovered at AC137831, AC137831, AC153001, AC166313, CT025844 and AC122724 of the BAC sequencing library of the truncatula sequence database. In addition, gastric beans ( Glycine) as shown in Figures 1 to 6 max ) MYB transcriptional regulator genes of SEQ ID NO: 1, 2, 5 is one nucleotide sequence, SEQ ID NO: 3, 4, 6 was shown that one or more nucleotide sequences have a level of e-20 or more similarity.

Example  3. Medicago truncatula of MYB  Identification of Promoter Sequences from Transcription Gene-like Sequences

Based on the similarity of the gene base sequence found in Example 2 , the base sequence in the 5 'direction of the base sequence matched as above can be estimated to include a site serving as a promoter, and the base of the part serving as the promoter. In order to extract the sequence, the program get_seq2 programmed in C language was developed and used.

Medicago beans excavated from truncatula (Glycine max ) using the base sequence (SEQ ID NOS: 7-12) having a high degree of significance with MYB transcriptional regulator gene of about 2 Kb from the 5 'end to the start of the corresponding base (5' direction) Sequence of the same Medicago Extracted from truncatula genomic sequence database. Medicago discovered in this way The base sequences comprising the promoter region of each MYB- like gene of truncatula are set forth in SEQ ID NOs: 13-18.

Example  4. Related to soybean spot disease resistance gene Molecular Marker  Development

4-1) Amplify Specific Promoter Sites primer  making

Each primer was prepared to amplify a specific site of the nucleotide sequence (SEQ ID NOS: 13-18) including the respective promoter sites, and is represented by SEQ ID NOs: 19-30. The primers set forth in SEQ ID NOs: 19 and 20 represent specific regions of the nucleotide sequence of SEQ ID NO: 13, SEQ ID NOs 21 and 22, SEQ ID NO: 14, SEQ ID NOs: 23 and 24, SEQ ID NO: 15, SEQ ID NOs: 25 and 26, SEQ ID NO: 16, SEQ ID NOs: 27 and 28 were designed to amplify a specific site of SEQ ID NO: 17, SEQ ID NOs: 29 and 30 of SEQ ID NO: 18.

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

PCR mixture contained 20 ng genomic DNA, 10 mM Tris-HCl (pH 9.0), 40 mM KCl, 1.5 mM MgCl ₂ , 250 μM dNTP, 10 pmole each primer, 1 unit Taq DNA polymerase The reaction solution was 20 µl.

PCR conditions were GeneAmp PCR System 2700 (Applied Biosystems USA), the first PCR, the initial DNA denaturation 94 ℃ 5 minutes, DNA denaturation 94 ℃ 30 seconds, annealing 49.3 ℃ 30 seconds, DNA extension (DNA extension) 25 cycles were performed at 72 ° C. for 30 seconds, and the final DNA extension was performed at 72 ° C. for 7 minutes.

The second PCR contained 5 μl of the first PCR product, 10 mM Tris-HCl (pH 9.0), 40 mM KCl, 1.5 mM MgCl ₂ , 250 μM dNTP, 10 pmole each primer, 1 unit Taq DNA polymerase, total reaction The solution was 20 μl and 25 cycles were performed under the same conditions as the first PCR. The second PCR product was electrophoresed with a 2% agarose gel (agarose gel) and then confirmed the band (band) with a UV eluminator (UV eluminator) (Fig. 7).

4-3) Medicago truncatula of MYB  Development of Polymorphism Between Soybean Varieties Using Pseudogene Promoter Sequences

Medicago , a model plant of legume crops The primers are prepared using the nucleotide sequence of the genomic DNA sequencing database of truncatula , and the reason for discovering polymorphism among soybean varieties using the genomic DNA of soybean as a template is 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). Genomic DNA sequence of Williams82 (PI518671). 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, according to the experiment in Williams82's genomic DNA sequence database, the promoter sequence was identified based on the nucleotide sequence of a specific gene, a primer was prepared to amplify the promoter region, and the polymorphism was compared. If you want to dig, 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 Excavating the nucleotide sequence of the analogous gene from truncatula, and extracts the promoter nucleotide sequence based on it, and then create the primer, using the techniques of comparative genomics Medicago We tried to compare the diversity between soybean varieties based on the truncatula nucleotide sequence as a polymorphism of the promoter region of a specific gene.

Medicago for the above reasons A primer was prepared to amplify the promoter region of MYB- like gene extracted from truncatula 's genomic DNA database and observed in band pattern amplified when genomic DNA of various soybean varieties responding to soybean spot disease was used as a template. NT SYS analysis was performed to investigate the relationship between soybean varieties. The bands used for this NT SYS analysis are shown in FIG. 7 and Table 2.

This polymorphism among soybean varieties can be seen as a characteristic at the genome level of genomic DNA of different soybean varieties. As a result of analyzing the polymorphism, the relationship between the varieties which show various responses to the race of the bacteria which cause soybean spot disease is shown in FIG. 8.

Table 1 compares the differences between the soybean varieties that respond to the race of soybean spotted germs. Bogwang soybeans are resistant to race Cs-2, 10, 14, 16, 19, and 20. Sensitive to race, Cheongja soybean 3 was susceptible to Cs-10, 14, 19, while Daemyung, Dachae, and Taekwang were generally susceptible to soy spot disease.

Based on the inherent characteristics of the genome level, the difference in the response between the varieties to the soybean spot disease is represented by the phenotype of each soybean variety, and the polymorphism of the band pattern mentioned above is inherent in the genome level of each soybean variety. Comparing the relationship between latex and phenotypes, we can observe that Bogwang beans, which are resistant to all six soybean spot germ races, and Taek, Soybean, and Daemang beans, which are generally susceptible, are divided into genetically distant groups. In addition, it can be observed that Kumgang beans and Cheongja 3 Ho beans, which are sensitive and resistant depending on race, are separated from these resistant and sensitive varieties. Therefore, by analyzing the intrinsic properties of the genome level shown by the primers described in SEQ ID NOs: 19 to 30, it can be utilized as a molecular marker for predicting the response of a specific bean variety to soybean spot bacteria.

The specific parts of the present invention have been described in detail above, and it should be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus 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.

<110> Konkuk University Industrial Cooperation Corp. <120> METHOD FOR DEVELOPING GENE SPECIFIC MOLECULAR MARKERS <130> P10-E414 <160> 30 <170> KopatentIn 1.71 <210> 1 <211> 684 <212> DNA <213> Glycine max <400> 1 gggtctttgg tcaccagaag aagatgaaaa acttttgagg catattacta agtacggtca 60 tggatgttgg agctcagttc ctaagcaagc aggtactcca acactgtata gtctatacca 120 ccccatttag cataaaaatc aagggaagaa aactgtagtt cattttcccg tcaatgtcct 180 ttattggcag gtttgcagag gtgcggcaaa agctgcaggc ttaggtggat caattattta 240 aggcctgatt tgaagagagg tacattttca caagaggagg aaaatctcat cattgaactt 300 catgcagtgc tagggaacag gtaaatctaa caatccttct ttacatgatt ttattagtct 360 ctgactctcg gccgatggat acctgttttc ttgtgattgg gtcatcctag atatcttgac 420 atggatatcc ttttgttttc ttcttctcag atggtctcaa attgcagcac aattgccagg 480 gaggactgac aatgaaataa agaatctgtg gaattcttgc ttaaagaaga aactcaggca 540 aagaggaata gaccctgtga cacataagcc actgtcagag gttgagaatg gagaggaggg 600 caaaacgagg agccaagaat tatccaatga actgaacctc ctgaattcag agagcttcaa 660 gtcagatgaa gggtcctatg agac 684 <210> 2 <211> 1001 <212> DNA <213> Glycine max <400> 2 gtttttgaat aatgaggact caagttagta agataacttc ttaggtaact aactttgtca 60 gcatatagta aaataattct gccatcagtt tttctaagta tcaatttttt ttatcttcag 120 tcacaagcat tttttttccc ttataatgtt ttcacttgat tatgatatca gcaaaggtgt 180 cccaccattc ctgcaagagg catcaatgct cacaccttcg aaaagacatg gtacactgtt 240 tcaagtttca accccatttt tttcaccttg ccaaaataaa taaaccttcc ctcctttcaa 300 ctccgaccta tatacaacaa gaagtcctat ttcacattgt tgaatctcta tcagatagat 360 caaccatata attgtaattc gacccttaaa tacatttttt ttaaaataaa gaaaatacaa 420 ggaaccttgt tgactatgac ccttctcatg gggccttgct cttatattag ttcaacttct 480 tttctcagct gccagttcat tttccttctt catatcatct ctctcgctgt cacattctct 540 ctatctatct cagccactcc aatagctttg gttcactgcc acccctgctg acatttttct 600 cattggaata aacaaacttt gtgttaactg aaaaaggttt ttaagggaaa aagaaaaaat 660 tacaaaatgg gaaggcactc ttgttgttac aagcagaagc ttaggaaggg tctttggtct 720 cctgaggagg atgaaaaact tctgaggcat ataacaaagt atggtcatgg atgttggagt 780 tctgtgccta agcaagcagg taattaatca aattatccaa ctaagcctcc tttaatctcc 840 tttgtaacaa aaagagaaat ttttttattc atctctctgt gaatgatatg aattggcagg 900 tttgcaaagg tgtggtaaga gctgcaggct taggtggatc aattacttaa ggcctgattt 960 aaagagagga acattctcac aagaagaaga aaaccttatc a 1001 <210> 3 <211> 723 <212> DNA <213> Glycine max <400> 3 tggatgttaa gaaaggtggg tctgtagtac aagcacaagt gaagttgcag aagcataacg 60 aaaaggagat gggcatgaga aaaggtccat gggcggttga ggaggacacc attctggtca 120 attacatcgc cacacacggt gaaggccact ggaattccgt ggcacgatgt gcaggcatgc 180 atgcatctta attataattt aatttctgca tgtgccaagt aataatggta tatatgttaa 240 ttatgagatt tatcaattac aggtctaagg aggagtggga agagttgcag attaaggtgg 300 ctaaactact tgcgcccaga cgtgcggcgt ggaaatatca cactccaaga acaaatatta 360 attctcgacc ttcactctcg ctggggcaac aggtatggta taaaaatatt ccactctctc 420 tctctctcta tatatatata ttaatttgtt ttataaggtt ggatgtacac atatataaca 480 tagtatgaat gaatgaatgt tttttgatag gtggtcaaag attgctcaac agctgccagg 540 aagaacagac aacgaaataa agaactattg gaggaccaga gtgataaaac aagcgaagca 600 gctaaagtgc gatgtgaata gcaaacagtt cagagacacg ttgcgttacg tttggatgcc 660 gcgcttgctg gagcggcttc agcccacatc acaagcactg gagccaaacc aaagtggact 720 tgt 723 <210> 4 <211> 506 <212> DNA <213> Glycine max <400> 4 agacctgaag gggctaagga aaattagaag gacacacaag tataaaggcg gtgaaataaa 60 agagaaagac aagaagaaga catgggaaga ccaccttgtt gtgacaaaga aggggtcaag 120 aaagggcctt ggactcctga agaagacatc atattggtgt cttatattca ggaacatggt 180 cctggaaatt ggagggcagt tcctgccaaa acaggttaat taattagccc tacactcaat 240 ttgatgatta ttttgcataa tattggttct tgttctttgt tgtgtttatt agatcattat 300 ttaaagaagt ttctgattga aatattggcc atttgggttt cattgtttta tctttggatc 360 tgaattgatt tttccatttt ggggttattt tcagggttgt caagatgcag caagagttgc 420 agacttagat ggactaatta cctgaggcca ggaatcaaac ggggcaactt cacagaacaa 480 gaggagaaga tgataatcca tcttca 506 <210> 5 <211> 544 <212> DNA <213> Glycine max <400> 5 aaaaagntgg ngctccaccg cggtggcggc cgctctagaa ctagtggatc ccccgggctg 60 caggaattcg gcacgaggat acaaggtcac ttagggaaca agaatataaa agaaggaaaa 120 agaagacgac atagcaaagc ctagaaacat gggtcaccat tcttgctgca accaacagaa 180 ggttaaaaga ggactttggt ctcctgagga agacgaaaaa ctcatcagat acattactac 240 tcatggatat ggctgttggg gtgaagttcc agaaaaagct gggcttctaa ggtgtggcaa 300 agagttgtcg cttgagatgg attaattatt taagacccga tattaaaaga ggcaggttta 360 cacctgagga ggagaagtta attataactc ttcacggggt cgtgggaaac agatgggctc 420 acattgcaag ccacttgcct gggtccgaac tgacaatnga ataaagaact attgggactc 480 atgggttana aangnangat aagaaaagac ntccgtgtct tcaaccaccn antgcacaaa 540 gtnt 544 <210> 6 <211> 434 <212> DNA <213> Glycine max <400> 6 tctaactcct tttgtgctat ataaagaagt agaaaagtca ccagtgttta tcactgttcc 60 cactttctcc aaaaatggga agatcaccat gctgtgacaa ggtgggtttg aagaagggac 120 catggacgcc agaggaagat cagaagctct tggcttatat tgaagaacat ggccatggaa 180 gctggcgtgc tttgccagca aaagctggta aataatctta tatacatgtt gatatataaa 240 tatcttgtaa ttaaagtaaa acttgccgtg taatttgtga agtgaattag ttgctggcta 300 ctgtgttcag gacttcagag gtgtggcaaa agctgcagat tgagatggac taattatcta 360 aggcctgata ttaagagggg gaaattcagt atgcaagaag aacaaaccat cattcaactt 420 catgctctct tagg 434 <210> 7 <211> 107 <212> DNA <213> Medicago truncatula <400> 7 agctgtaggc ttaggtggat taattactta aggcctgatt taaaaagagg tacattttca 60 caagaagaag aaaatctcat cattgaactt cattcagtac tagggaa 107 <210> 8 <211> 128 <212> DNA <213> Medicago truncatula <400> 8 tggtcacaaa ttgctgcaca attgccaggg aggactgata atgaaataaa gaatctttgg 60 aattcttgct taaagaagaa actgagacaa aagggtatag accctgtcac tcataaacca 120 ctttctga 128 <210> 9 <211> 127 <212> DNA <213> Medicago truncatula <400> 9 aggagaagtg gtaaaagttg tagattaagg tggttaaact acttgcgtcc cgatttacgt 60 cgtggaaata tcacacttca agaacagata ttgattcttg acctccactc tcgctggggc 120 aataggt 127 <210> 10 <211> 124 <212> DNA <213> Medicago truncatula <400> 10 acatgggaag accaccttgt tgtgacaaag aaggtgttaa aaaaggacca tggactcctg 60 aagaagatat catacttgtt acttatatac aagaacatgg tcctggaaat tggagggctg 120 ttcc 124 <210> 11 <211> 140 <212> DNA <213> Medicago truncatula <400> 11 agaaagatgg gtcaccattc atgctgtaat caacagaagg ttaagagagg cctttggtca 60 cctgaagaag atgagaaact cattagatac attactactc atggttatgg ttgctggagt 120 gaagttcctg agaaagctgg 140 <210> 12 <211> 137 <212> DNA <213> Medicago truncatula <400> 12 aaaatgggac gttcaccatg ttgtgacaaa gttggtttaa agaaaggtcc atggactcca 60 gaagaagatc aaaaactctt agcttatatt gatgaacatg gtcatggaag ttggcgtgct 120 ttgcccgcta aagctgg 137 <210> 13 <211> 2059 <212> DNA <213> Medicago truncatula <400> 13 gaaaatgtac ctctttttaa atcaggcctt aagtaattaa tccacctaag cctacagctc 60 ttaccacatc tttgaagacc tatgccatgc catgccatgc caataaaata cattatatat 120 caacaacagt ttcttctcta atctcataat tatatgctaa aaggagattg aaatattaaa 180 atttggatta tatataaata tatatacctg cttgcttagg aactgaactc caacatccat 240 gaccatattt agtaatatga ttcaaaagtt tttcatcctc ctcaggtgac catagacctt 300 tcctaagctt ctgcttgtag caacaagagt gtcttcccat ttgtagtttg tttgtggttt 360 ggtccctcaa acttaaaaat acaatgaagt gcgaaagttt ttgataaaca gaaagattgt 420 tggatttgtg cagcaataaa caaagaccgt gacaggtaaa atatgagtga gagagataga 480 gagagagaga ttatattatg aagtgtgaaa gatggaaaat gaagtggcag ctgagaaaat 540 gagttgaggc cccatgaatt atgtcatata gtcattaagg taccttctat ttatcttatt 600 ttttcctttt ttgagaatat acgtgtcctt atgtttcttt atttattttt tctatctttt 660 tatttaaaat gtgtataatg acaagtgaaa tagaatacat catgcattaa aatcaaatta 720 tttgttcatt agattagata tcttgtaatg taataaaaaa gtgaaatgtt tatagaatgg 780 aaggtcattt cgtcatgctt tttccatttt tttttttata aacgttattt tattttttaa 840 agggaaagac ttaatggtac tacgatgcat gagatcaggg tcgtcttaaa caatttgttg 900 gccccgtttt aatcttataa attggccccc ggataaaaat agaaagaaaa aaaacaattt 960 tttaaagtaa attatgataa catcccaata atgtaaataa catcaaaaag tgattttatg 1020 ttcaaatcac taacatcaaa ataaacgagc cactcttcca acattttttg aatagtaacc 1080 acattttcaa gaactccaac acctagcggg agttgaatcc ctgtccatgc ggtcttgtac 1140 gtaacgctct accaccgaag tattcttaac aatttgtcaa gttctctctt tcacctctat 1200 ataacactta ctataggacc ttacatataa aaaaaaaaat atgggcccat agcctgtcag 1260 agcttgatta acgcattaat gaaaaaaaat aatcaaatta caaacttgcc ctttagcttg 1320 actttgattt caaaaagaaa agttggagtt ttttatccaa accaaaaggt gttcaatgct 1380 aaaacacaca ttctattttc ttgatgggag tttcataaca aaactatata tatatatata 1440 tatatatata tatatatata tataagtttt taatagcctt tttaaattgt agaacaagtt 1500 tttatggatg acagttaggg aatacaacca cttttgtaat tacaacaaac caattaaaaa 1560 actaaaattt ctagatgatt gaaatatttt attttttgaa aaataaagtt tatgtgagtt 1620 aggtttcaat aattcataag gatcagagta tacaatattt gtctgaacaa agtcttagcg 1680 ttgataatga aagttgaaca tatgtccata ctatgtatac aaatttctta taagaatgta 1740 tataaatttt tcattaacaa agtcttaacg ctgataatga aaattgaata cgtgtctata 1800 ctatttatga atcaacttct taacaattga atgacaattt tatgcatttt tattttattt 1860 ttctcgagaa tatattcttt cacttttaaa caaagcttta atagtaaccg aaaaaaacaa 1920 agctttaatt atggacaact aattaactat ttggctcata tgttgtttca ctgtctaata 1980 ctaacgacaa tgagtttatt aaatctcatt aacgtcacaa ctattccacc ggcactgtat 2040 ggtatggtta tggtataat 2059 <210> 14 <211> 2059 <212> DNA <213> Medicago truncatula <400> 14 caaagattct ttatttcatt atcagtcctc cctggcaatt gtgcagcaat ttgtgaccat 60 ctaagaaaac acatcatatg catgtcaata aggtttgaaa aataaaaaga atgatccaac 120 aaccacataa caattaaaaa atacgccaga gtctaacgca agtggttggt gtgtcgtgtg 180 tgagtgttgt aaactctggt aaatcatact taaaaaagaa ggattgaatt taattaccta 240 ttccctagta ctgaatgaag ttcaatgatg agattttctt cttcttgtga aaatgtacct 300 ctttttaaat caggccttaa gtaattaatc cacctaagcc tacagctctt accacatctt 360 tgaagaccta tgccatgcca tgccatgcca ataaaataca ttatatatca acaacagttt 420 cttctctaat ctcataatta tatgctaaaa ggagattgaa atattaaaat ttggattata 480 tataaatata tatacctgct tgcttaggaa ctgaactcca acatccatga ccatatttag 540 taatatgatt caaaagtttt tcatcctcct caggtgacca tagacctttc ctaagcttct 600 gcttgtagca acaagagtgt cttcccattt gtagtttgtt tgtggtttgg tccctcaaac 660 ttaaaaatac aatgaagtgc gaaagttttt gataaacaga aagattgttg gatttgtgca 720 gcaataaaca aagaccgtga caggtaaaat atgagtgaga gagatagaga gagagagatt 780 atattatgaa gtgtgaaaga tggaaaatga agtggcagct gagaaaatga gttgaggccc 840 catgaattat gtcatatagt cattaaggta ccttctattt atcttatttt ttcctttttt 900 gagaatatac gtgtccttat gtttctttat ttattttttc tatcttttta tttaaaatgt 960 gtataatgac aagtgaaata gaatacatca tgcattaaaa tcaaattatt tgttcattag 1020 attagatatc ttgtaatgta ataaaaaagt gaaatgttta tagaatggaa ggtcatttcg 1080 tcatgctttt tccatttttt tttttataaa cgttatttta ttttttaaag ggaaagactt 1140 aatggtacta cgatgcatga gatcagggtc gtcttaaaca atttgttggc cccgttttaa 1200 tcttataaat tggcccccgg ataaaaatag aaagaaaaaa aacaattttt taaagtaaat 1260 tatgataaca tcccaataat gtaaataaca tcaaaaagtg attttatgtt caaatcacta 1320 acatcaaaat aaacgagcca ctcttccaac attttttgaa tagtaaccac attttcaaga 1380 actccaacac ctagcgggag ttgaatccct gtccatgcgg tcttgtacgt aacgctctac 1440 caccgaagta ttcttaacaa tttgtcaagt tctctctttc acctctatat aacacttact 1500 ataggacctt acatataaaa aaaaaaatat gggcccatag cctgtcagag cttgattaac 1560 gcattaatga aaaaaaataa tcaaattaca aacttgccct ttagcttgac tttgatttca 1620 aaaagaaaag ttggagtttt ttatccaaac caaaaggtgt tcaatgctaa aacacacatt 1680 ctattttctt gatgggagtt tcataacaaa actatatata tatatatata tatatatata 1740 tatatatata taagttttta atagcctttt taaattgtag aacaagtttt tatggatgac 1800 agttagggaa tacaaccact tttgtaatta caacaaacca attaaaaaac taaaatttct 1860 agatgattga aatattttat tttttgaaaa ataaagttta tgtgagttag gtttcaataa 1920 ttcataagga tcagagtata caatatttgt ctgaacaaag tcttagcgtt gataatgaaa 1980 gttgaacata tgtccatact atgtatacaa atttcttata agaatgtata taaatttttc 2040 attaacaaag tcttaacgc 2059 <210> 15 <211> 2059 <212> DNA <213> Medicago truncatula <400> 15 cgtaaatcgg gacgcaagta gtttaaccac cttaatctac aacttttacc acttctcctc 60 aaacctgtat aattaattag ttttgtttag aatagattat gagaatcaat aagctattca 120 tgcagctatg tcataagcta ataaataaaa gagctagaag aaaatatata aatatataca 180 tgcctgcaga agatgcaagg gtattccagt gaccttcacc gtgtattgta atgtaatcaa 240 ccagaatggt gtcctcttct aacgtccaag gaccttttct tacccctgtc tcattttcct 300 ttgcagaccc acctttctga accttaatat ccatacctct catgtatgtg tagctctcaa 360 taattgttac tgaaattaaa agtgatttag ttttgtgaaa ggaagaacaa acagtatgaa 420 atatatatga gaaacgaaga aacgtgtaag attttgattt aattgatggt ttcacttatt 480 gggggcatgt ggagttattg tgtatatata ataatagagt cggataagga taggtatact 540 tcaaggtttg cactagtcta caaaatataa attaaaaggg tacggaatag tgacgacttg 600 gtacacgaat aatgcaccag tttgagtaaa tttaattaat attactactt taggattaag 660 atggaaccaa gcttatagtt tggggtttga ctggttgact cttttgttta tatttttggt 720 aagtcatgcg catggaaata ttgatgtcag tgaatactct tttcacaatc taaactgatt 780 tggctttagc tctatcagct cactgttaag aaagacaaat tttactaaaa aataataata 840 gaataaattg ttattgtaaa tttgtaatct catattgtta ttccctaact aataattaaa 900 ctcatgtgat taataaattt taattgagaa ggattcttta gaagatttga attcaaattc 960 atattcgggc agatggagac attaatgaaa gtaaaaaaaa aataaaaatt agagagaaca 1020 atcaatttag ttattatact atcattttct catcaatttg gtcttaaaat taataaattt 1080 aagtaaaaaa tccataattt aaaaaaaaaa aaaaaaaaaa gggacaagcc aatctaaatt 1140 gaggagagtc aaacttggac cttaaaagta atagacgttc aaaccaaaac caactaaagt 1200 gagtttaaaa gaaattccat aaattttact ctcatccatc aatttgatga tctctcaatt 1260 aattagttta tgtatcaaat tgatgatgaa attatagtac aagacatttc acttgatttt 1320 aaaagtttag gaatcaaatt gacaattgat agtttatgaa ctaaatttac ttattattta 1380 tattatgtgt gagattaccg tgtaatattt gttttttttc aaagcaacta tagattgttg 1440 gttaataaag gagatttttt ccttcttttt cctactaatc cagaatggca ttgacggtag 1500 aatgagaagc aacactaaca cagcagccac caccttttca cacaagtgca taaatttata 1560 tcattcacga ttcttaagtg tttgtttgtt tcatgtaatg ttgcatcctg agaattacat 1620 ttgggcacta aaaagcttag tagtgaatag aaagacgaag aaatagactt gttgtgatta 1680 actcattaat taacgcagga ctagaaggaa gaaaattagt tgcgataatc attgcacgtt 1740 ttcactttga ccaaaacact aagaattgga aacaatccca tcgcagtcgc acgatcatgt 1800 ggatttggtt aaatacttcg tattgtctca ataataaaca aaaaattggt caaaatgccc 1860 ataagacacg ttcaagctcc aagttttcaa ggacatcata ccatcgacga caatgtacgg 1920 tgaattgttc cctccatacc caagttatat aagtaaaaaa aatatcaaag aaagttcatg 1980 tatttgaatc aaattttaaa tcttctatac ctcaactcaa tgtctgtcat tcaattggct 2040 aactattaaa cttggtaat 2059 <210> 16 <211> 2050 <212> DNA <213> Medicago truncatula <400> 16 aggagtccat ggtccttttt taacaccttc tttgtcacaa caaggtggtc ttcccatgtt 60 tctttgtttc tcttttactt ttagtaacac ttgtatatgt tgccactaat ttctattcta 120 tagacctttt tcttcctctt cctctctcgg tgtatgatca cttatcttta atttttagtt 180 ctctctaaaa cctcttgatt tgataggttt caaaaggcaa gtgtccaagc ctaacctttc 240 catggtgtaa atatatagct aagaaaatag ggtgtattta gcataagcta ggatttttta 300 tttttttatt tttttttaaa gataagctag gaaactgact taaaaaagta gagaaaaaat 360 aaagagtcaa aatttgacag caaagagcat taactgaagg aaggaaaaag ctaaattatt 420 tactatagtt atgttgtgta ccactcaaaa ccgcgtagag cattgaatgt gtagctctat 480 tacactggga aagctcaatt ttatttcagc ttttaaagtt tctataaatt aacacaaaca 540 tgtcattaat tgtaaatttg tgatatagct gacttttgca agaaagctag taattaagct 600 atcaccatta tgtctttcta tttgcctacc tggtgatcac caatgaacaa gccctttcta 660 gatactttaa cttgtctata aaatcaatta attgcattta tattttgtta attagacagt 720 gactaaaact gacacccttg cttcattcta gaaagggata gctgatctat ttgcattatt 780 gtagtagaat tctaatatcc catattttgt taaagaaaaa tatactagta taatttaaga 840 atggtatttt tctttaagag aaaatatact agtaatatcg cattgttact gatttttgtg 900 aaataaaata ttagtcgttt aatgtttgtg cacgattatt aaggaaatga ttaattgtgt 960 tgatttcaac ggtaaaatta gtcatttact acaacaccct tattaattgt agttagtgga 1020 gtagttaagt aggatgaaat tcaataaata agggtataat agtgaaaaaa aataataaat 1080 gctgcattgg tattgtaaag tgtcaattat tttgaaaaaa agaaaaaatg ctaaagagat 1140 aataagtggg aaacggatgg agtatttttt atttaagaga aaaaaaatag gattatcaaa 1200 taaaataaac gtgtttaaat tttgggtttt gatgccctga gggcactttg ttaaggatac 1260 aaaaggggat gcttaacaag aagtgatctt tgttaagtaa aaaaccttta aggaattcca 1320 taagagatgc tctaagagca ctcgttagca cttcccttaa ataaaattat cactttataa 1380 caaaaaattt cagagattac aacttgtaaa taaaattaaa tatgtttcaa atgagtagtt 1440 gttccgagta gcttttccat aaaactaatt tttcataact ggttctaaaa aaaatatttt 1500 cattttgata atgaaatata gaataacttc tgctttaaaa aaaaaaaatc tccatcaatt 1560 tggtcttaga gttatcaccc tcttttttaa ttaatcttcg gactataaaa atatatacta 1620 atgagtttgt ctgacatgta caatattaca catgatatgg tagctaaaaa taacaacttt 1680 ttatttaaaa attatatact ccaatactaa ttttgaactt cttatttgag cttccaaaat 1740 ggaccgacaa tatactctta cgaatctaaa agacctccag aaggactttt agactctttt 1800 agaggcctca tcgtcctcca taagggctat gaggcccaag gcgcgccctc cccagggcgt 1860 cgactcaatc tgcttctagg aacctctaga aagccgccca aatacttaga tacccctgtt 1920 atttactgcc taagtaacca caaatcagac ccatagagag ctataaatac caacccttga 1980 gagccttctc aaggcatgac caatacaata caaaccctaa tacgattatt gtacttttag 2040 caagtacaca 2050 <210> 17 <211> 2767 <212> DNA <213> Medicago truncatula <400> 17 ctgatttttc cgtccttctc acactgtcac tcatattatg ttttagagga ttctactcga 60 agttattata aaataaaaat ttaattaaaa tgttatatcc atttttttag accacactat 120 caatgttaaa ttaaaagttc ttatttagta ctccctccgt ctctaaataa tagtcgttta 180 aggttgatgc acagttatta aggaaatgat taattgtgtt gattttgatg gtaaaattag 240 tactaaaaca cccttattaa ttgtagttag tgtgcagtag ttaaattgga taaagttcaa 300 taaataaggg tattataatg aaaaaataaa ataaatgctg cattgatatt ctaaagtgac 360 aattattttg agaaaaagaa aaataggtaa agagacaatt attgtgagac ggagggagta 420 gatcgacgtt ttttctaaat attttagttc ttttttggaa aaactaaaag aataatatta 480 agttatggat catgctaact tatgttctta ggacacatgt taagaatttc attagtggta 540 aatatttact atgaaaaatt aattcttaac ttcttgaaaa atacaagagt ttcttaacat 600 gtgtctattt tagatatctt aacatgtgtt ttaaaaggac aagtttgtat aagttattaa 660 caaaagcttg cacaaaaagt ggctagggga gaaatacaag agtttccaca atccatgaca 720 aatgagtgaa cacatgaaaa acaaattaca caaatgagcc acaaacaaaa acccaactct 780 taatgagtgt catgtcggtt ccttattaat aaataaaaaa tagaatattt gtgtgtcatg 840 tatgaagatg gtttccaggg tggatttgga cttaaaatat tagtgaggca caattttttt 900 tgttgaataa ttaaataaaa ctaaatttaa taagaataaa aagtgtttgt ttatgtttgt 960 gttgtgcgtt tggtgagaga aagaacaatg aaaaaggctt actgaagtga gaagtgaggg 1020 gaaaggtttt actgtgtact tgtgttttgg agtgttcgtg gcgcagtttg gttcgatttt 1080 gagactaaaa gtcatttgaa ccgtaagata aaaaaacatg cggtttggtt tggtttggtt 1140 gacttttaaa ataaattcag aaccaaatcg atgcggtttg ggttggatcg gttggtgcgg 1200 ttttttttac tagacaatac aattttttgt ttccaatttt aaaatcaagt taaaaagtta 1260 aaacacaaca tacttctggc aatgtttccg acaatggttt ttaatttcat acaacattaa 1320 aatttcattt tcactaaaca acatatacca aattaaaaat gtggacaaaa aagtaattct 1380 attatgtaag aaatgtaaaa gattaagatt ttatcatttt tttttaagga agattttatc 1440 attctttaaa agttcaaatt gcacataaat acatttttga aataaaaagc aacttaacaa 1500 aagaaaatat atgtcattta taaaagtttc catttagttt ctatgagtat tggcgtagat 1560 gacatatatt taattagtgt ttttcttatg ttgcggtttg attcggttca gttggtaaaa 1620 taaaaactgg aaaccgaacc aaaccatgcg gtagagtaaa aaagtgattt gaaattcaaa 1680 ccaaatgcag ttttttgcag tttcggttga ttcagtttgc ggtttttcta tagaattagt 1740 ttggctttga tcaccctatt gttttggact gggctgagct tgtggaaaga aataaaaaat 1800 cagcttgggg gtggtgtggc gcttgccaca ccaggacctg ttcaggtccg cccctgaggg 1860 ttccataaga tttgacaaga gtatcatatt tgagagtgtc atatgtgctt gtcacatgat 1920 tctttattaa ttatccatga tttccttaaa tccatagtct tccgaaagtt tgttataaat 1980 aaatgtattt ttagttaaga agcaaacgaa aaacaaataa aatgcatata aattttgacc 2040 ttttaaaaat acacacaaaa actaaatgat atcatattgg acatgtcaag gtatagttac 2100 ttctctgtta gaagcaccat gaagataaat acacacaaga aaactaaatg atatcatttt 2160 gatggattat ttcacgaaaa gtagatgaaa gtgtagtaat aatctacttt ttgcataaat 2220 tcaaaattaa tacttgcttc ctaattccta tctctttcac gtagctatag taaataaata 2280 ttatccactt attaataagt actgatgatg tataatgacg ttgttgaata tatagttggc 2340 agacaggaaa caaccagcaa caatactcct tctttctttt ttcagcttcc tctcgttatg 2400 tatatatttt ggagagactt gttatatatg gtaaaatgtg aagaattttg ttatgtaata 2460 ctacacatgt acgtagaaaa ttgctagaat aaagttgatg gaacgtcact ttagggttag 2520 atggagatat gggtcctaca tgcaggttgt aaatgtcgac ttcagttccc tcagttttaa 2580 aagaaggaaa gatagaaaga gaaagagata actattgcat tgcacttgca cttgcacttg 2640 cacacataca ctgatatatg aatgaaggta caatttacta aacaaaaaga gacaggcata 2700 gaacaagaga aagatgggtc accattcatg ctgtaatcaa cagaaggtta agagaggcct 2760 ttggtca 2767 <210> 18 <211> 2054 <212> DNA <213> Medicago truncatula <400> 18 cagatgtcca tggccctttc tttaaaccaa ccttttcaca acatggtgat ctccccattg 60 gaaatgttct aatgaaaaac tatatagtaa attgtgtata tggttgagaa gttggaggag 120 aaacttggaa gagaaagaga aagaaaggag aatatataag ttgatcaagt agctatgtac 180 aagaaattaa tgagatgggc agagaagaat ttgtcagcgg cataaaaggg ataagaaatt 240 aaattgatac tcatcaatat aaatcattat acgaggagaa tagtaaaact ttaatttaac 300 tgcagtatag tacagcacat tcactacact ttgcgcattt tttgctgcat catttatttg 360 cagtacattc tacatttata tgcatataaa ttgggtggtt tttatgatag acaaacttac 420 ataaacattt tttttggaaa gatatctact ttttttttaa ggaaaggaaa gatatctact 480 taacggcgag taaatatatt tttttaaaca ggtaaaatga taatattatc acaaaaaaaa 540 acacaatatc accaaatcga tttagcttaa aaaaacacaa tatctacttt agtacaagat 600 atgcgaggag gaaaatagtt ttatatcacc aaatcgaaag gataaaacat aacaattcag 660 tcaagaccca aacaagcaaa gggttgtttc aaccacatca aatgaccaaa aggaaaatta 720 acattttgag ctttcagcca ccaataggta gaaactttca ctttatccat cagcttcaga 780 atagatttag ctttatggct caggctacgg tacatgacct caatgggtct cctaccgtag 840 gtgacttgac tttttttttt tttacaaaat tgatgtcaac cattggatgt gcttgaaaac 900 ttttgaagac catagtatta ttatactctt taaacattag attattttca cactatgttc 960 catcatcttt ctctttccct ctccatatct cttactcgcc caacaacaac aacaacgcga 1020 tgcaccgcca ccgaaaaatc acaacgacaa tgcgccggaa aatcacaaca acaacgacga 1080 atttttggaa atcttaatcc aatcctttca acccaaaatt gcagaaaaca acattcatta 1140 aactcaaatt tcatttaatc taattcacct gaaaacaaaa ttgcagaaaa tattgatttc 1200 ttaattattc aattcattca acccaaagtt cacatattca tcactaaact cagatgaaag 1260 tgattaaatc cgtcgccaga ccaaatgtta cacaacaaaa caatcaaaat cggaatggaa 1320 aatctcacag aactcagatc tggagaggga tggtgtttaa gttcagatgt gaacacaatc 1380 tggtcaatgg aaaattacga atgaaaatag aataaaaaga acaaaaaaag atgtgttggt 1440 gttaaaaaaa aaaacgacga tttgtgttgg aggtataaca atcaacagaa agtgcagcaa 1500 caacaatgga gttgctacag tacacgtctc tcgcttagat ctaccaaatg aaaaaaatat 1560 atatagtgta gaaggattat ggtaagatca gtgtttttcg tcaatctaac cgcaacacat 1620 gtcatttttg ttataaaata aataaataaa acagtctttt ttaatgtctc ctcccgtggg 1680 agacctattc aagtctccta ccataaacgg cgccgagatt atgtctctaa gtctaaccat 1740 gatgatgtta tggacgacga tgataatgat ggagacatga ctgttttttt ttcttagaat 1800 aaaagagaag agtaatttag aagtaaagag aagagtaatt tagaataaaa gaaagtaatt 1860 cggtgtggga ggtatatagt aagcattttg atccaaagtt aatttacttt tatccaatgg 1920 ttcatattta ttttgccaaa aaaaaaaaag ccatgtctcc tacaggaaac agagccttag 1980 ctttattgtt aaagagtctg tttctctttt ctgtccacaa cattgaaaaa caacaaagcc 2040 atattagctg caaa 2054 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 19 tccacctaag cctacagctc 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 20 ccggtggaat agttgtgacg 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 21 atttcattat cagtcctccc 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 22 aacgctaaga ctttgttcag 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 23 gtaaatcggg acgcaagtag 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 24 atggagggaa caattcaccg 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 25 cacaacaagg tggtcttccc 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 26 tttgggcggc tttctagagg 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 27 tgcacaaaaa gtggctaggg 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 28 cagtgtatgt gtgcaagtgc 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 29 tcacaacatg gtgatctccc 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> {rimer <400> 30 actatatacc tcccacaccg 20

Claims (14)

(1) extracting genomic DNA (genomic DNA) according to varieties of beans having different reactivity to soybean spot disease;
(2) Medicago finding a gene similar to the nucleotide sequence of the MYB transcriptional regulator of soybean ( Glycine max ) from the DNA sequence database of truncatula ;
(3) the Medicago digging out the promoter region of the gene found in step (2) from the DNA sequence database of truncatula ;
(4) preparing a PCR primer capable of amplifying a specific site of the promoter;
(5) using the genomic DNA of step (1) as a template and amplifying a promoter region of a specific gene using the primer of step (4); And
(6) 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 lines;
Molecular marker development method comprising a.
The method of claim 1,
The bean varieties are Bogwang bean, Jangwon bean, Geumgang bean, Cheongja bean No. 3, Anpyeong bean, Daemangong bean, Dachae bean and Taekwang bean development method of molecular marker, characterized in that at least one selected from the group consisting of.
The method of claim 1,
MYB electron control gene of the soybean ( Glycine max ) is a molecular marker development method, characterized in that at least one selected from the base sequences described in SEQ ID NO: 1 to 6.
The method of claim 1,
Glycine max ) genes similar to the nucleotide sequence of MYB electron control gene of the molecular marker development method, characterized in that at least one selected from the nucleotide sequence shown in SEQ ID NO: 7 to 12.
The method of claim 1,
Step (3) is a method for developing a molecular marker, characterized in that the excavation of the promoter in the 1.5 ~ 2 Kb region of the 5 'direction of the gene sequence found in step (2).
The method of claim 1,
Step (4) 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.
The method of claim 1,
Promoter site discovered in step (3) is a molecular marker development method, characterized in that at least one selected from the base sequences described in SEQ ID NO: 13 to 18.
The method of claim 7, wherein
Primer capable of amplifying the promoter region described in SEQ ID NO: 13 is a molecular marker development method, characterized in that the primer pair described in SEQ ID NO: 19 and 20.
The method of claim 7, wherein
Primer capable of amplifying the promoter region described in SEQ ID NO: 14 is a molecular marker development method, characterized in that the primer pair described in SEQ ID NO: 21 and 22.
The method of claim 7, wherein
Primer capable of amplifying the promoter region described in SEQ ID NO: 15 is a molecular marker development method, characterized in that the primer pair described in SEQ ID NO: 23 and 24.
The method of claim 7, wherein
Primer capable of amplifying the promoter region described in SEQ ID NO: 16 is a molecular marker development method, characterized in that the primer pair described in SEQ ID NO: 25 and 26.
The method of claim 7, wherein
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: 27 and 28.
The method of claim 7, wherein
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: 29 and 30.
Claims 1 to 13 of the soybean disease produced by the method of any one of claims related gene-specific molecular markers.

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