Selection method for soybean plants resistant to plant diseases

Abstract

To provide a method for selecting soybeans resistant to new plant diseases. [Solution] A method for selecting soybean plants resistant to plant diseases, comprising detecting the presence of a marker region containing 50 or more consecutive base sequences in the base sequence shown in Sequence ID No. 1, and comprising 50 or more consecutive base sequences in a marker selected from Sequence ID No. 3 to 12, from the genomic DNA of a soybean plant to be tested.

Description

Technical Field

[0001] The present invention relates to a method for selecting a plant disease-resistant soybean plant.

Background Art

[0002] The self-sufficiency rate of soybeans in Japan is as low as about 8% and is dependent on imports. However, due to the increasing global demand for soybeans, there are concerns about the soaring prices of imported soybeans and the instability of supply. Therefore, an expansion in the production volume of domestic soybeans is required. One effective measure to increase production is to reduce losses caused by diseases.

[0003] Soybean stem rot is one of the damping-off diseases that is a problem both in Japan and abroad. Soybeans infected with stem rot die and cause a reduction in yield. The causative agent of stem rot is known to be Phytophthora sojae, an oomycete. Current control methods include control with fungicides and breeding of varieties into which a true resistance gene has been introduced.

[0004] However, in the case of control with fungicides, the use of fungicides poses problems such as the emergence of fungicide-resistant bacteria and environmental load. In the case of control with a true resistance gene, although the stem rot pathogen has been reported to have various pathotype differentiations, the true resistance gene can effectively suppress the infection and growth of strains with a specific pathotype, but it cannot be applied to all stem rot pathogens. Also, there is a problem that there is a risk of the emergence of pathogenic bacteria that can invade the resistance. To date, no true resistance gene effective against all pathotypes has been reported. Therefore, when a strain with a different pathotype or a strain whose pathotype has changed due to gene mutation occurs, there is a risk of its infection and spread. Generally, when a true resistance gene is used alone, it is considered that strains capable of invading the resistance will appear in 8 to 20 years (Non-Patent Document 1). Resistance genes against soybean stem rot have been reported in Patent Documents 1 and 2, and are also mentioned in Non-Patent Document 2, which is a review of resistance genes and gene regions against soybean stem rot.

[0005] Fukuyutaka is known as a field-resistant soybean variety against soybean stem blight, but until now, there has been no knowledge about the causative fungal strain or the effective resistance gene. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] U.S. Patent Publication No. 2004034890 [Patent Document 2] International Public Gazette No. 2016196787 [Non-patent literature]

[0007] [Non-Patent Document 1] Grau et al. (2004) FungalDiseases. In Soybeans: Improvement, Production, and Uses (eds RM Shibles, JE Harper, RF Wilson and RC Shoemaker). https: / / doi.org / 10.2134 / agronmonogr16.3ed.c14 [Non-Patent Document 2] Lin et al. “Breeding for disease resistance in soybean: a global perspective.” Theoretical and Applied Genetics(2022) 135:3773-3872 DOI: 10.1007 / s00122-022-04101-3 [Non-Patent Document 3] Takuma Sugimoto et al., "Genetic analysis of field resistance to soybean stem blight using indoor inoculation tests," Journal of the Plant Pathology Society of Japan 79(3): 258 (2013) [Non-Patent Document 4] Song, Q., et al., “Abundance of SSR Motifs and Development of Candidate Polymorphic SSR Markers(BARCSOYSSR_1.0) in Soybean.” Crop Sci., 50: 1950-1960 (2010). https: / / doi.org / 10.2135 / cropsci2009.10.0607 [Non-Patent Document 5] Takashi Sayama, et al., “Development and Application of a Whole-Genome Simple Sequence Repeat Panel forHigh-Throughput Genotyping in Soybean”, DNA RESEARCH, 18, 107-115, (2011),20https: / / doi.org / 10.1093 / dnares / dsr003 [Overview of the project] [Problems that the invention aims to solve]

[0008] The present invention aims to provide a method for selecting new soybean plants resistant to plant diseases. [Means for solving the problem]

[0009] As a result of diligent research to solve the above problems, the inventors of the present invention have found that the soybean variety "Fukuyutaka" has field resistance to at least 10 different strains of stem blight fungus with different pathogenic prototypes, and is highly versatile. Furthermore, they have identified candidate resistance gene regions in "Fukuyutaka" and found that plant disease-resistant soybean plants can be selected based on these regions, leading to the completion of the present invention.

[0010] In other words, the present invention relates to, for example, the following inventions. [1] A method for selecting soybean plants resistant to plant diseases, comprising detecting the presence of a marker region containing 50 or more consecutive base sequences in the base sequence shown in Sequence ID No. 1, and comprising 50 or more consecutive base sequences in a marker selected from Sequence ID Nos. 3 to 12, from the genomic DNA of a soybean plant under test. [2] A method for selecting soybean plants resistant to plant diseases, comprising detecting the presence of a DNA region consisting of a base sequence encoding a protein that has 90% or more sequence identity with at least one amino acid sequence selected from Sequence ID No. 13 to 22, and that can confer plant disease resistance to the soybean plant, from the genomic DNA of a soybean plant under test. [3] The above-mentioned plant diseases include soybean stem blight, and the selection method described in [1] or [2]. [4] The above detection is performed by PCR using the genomic DNA of the soybean plant under test as a template and a primer capable of detecting the above marker region or the above DNA region, as described in any of [1] to [3]. [5] A method for producing plant disease-resistant soybean plants, comprising introducing DNA into soybean plants consisting of a base sequence that has 90% or more sequence identity with at least one amino acid sequence selected from Sequence IDs 13 to 22, and that encodes a protein capable of conferring plant disease resistance to soybean plants. [6] The above-mentioned plant diseases include soybean stem blight, and the method of production described in [5]. [Effects of the Invention]

[0011] According to the present invention, it is possible to efficiently select soybean plants resistant to plant diseases. Furthermore, according to the present invention, it is possible to create soybean plants resistant to plant diseases that have high versatility. [Brief explanation of the drawing]

[0012] [Figure 1]Figure 1 shows the results of verifying the field resistance of "Fukuyutaka", "Fukuminori" and "Himeshirazu" against 10 different pathogenic strains of soybean stem rot. The names described at the top of each graph are the types of soybean stem rot strains, and the vertical axis of the graph indicates the survival rate when the bacterial cells are mixed in the soil and cultivated. [Figure 2] Shows the QTL analysis of a recombinant inbred line ("Fukuyutaka" / "Himeshirazu" recombinant inbred line) of the resistant cultivar "Fukuyutaka" and the susceptible cultivar "Himeshirazu" in an indoor inoculation test of soybean stem rot pathogen. [Figure 3] Shows the QTL analysis of a recombinant inbred line ("Fukuyutaka" / "Himeshirazu" recombinant inbred line) of the resistant cultivar "Fukuyutaka" and the susceptible cultivar "Himeshirazu" in a field inoculation test of soybean stem rot pathogen. [Figure 4] Shows the QTL analysis of a recombinant inbred line ("Fukuyutaka" / "Enrei" recombinant inbred line) of the resistant cultivar "Fukuyutaka" and the susceptible cultivar "Enrei" in a field inoculation test of soybean stem rot pathogen. [Figure 5] Shows fine mapping using a recombinant inbred line ("Fukuyutaka" / "Himeshirazu" recombinant inbred line) of the resistant cultivar "Fukuminori" and the susceptible cultivar "Himeshirazu".

Mode for Carrying Out the Invention

[0013] <Method for Selecting Soybean Plants Resistant to Plant Diseases> A selection method for plant disease-resistant soybean plants according to one embodiment includes detecting the presence of a marker region containing 50 or more consecutive base sequences in the base sequence shown in Sequence ID No. 1, which is a marker region consisting of any of the base sequences shown in Sequence ID Nos. 3 to 12, from the genomic DNA of a soybean plant under test. Here, there may be one marker region or multiple marker regions. Any combination of marker regions containing 50 or more consecutive base sequences in each of the markers consisting of the base sequences shown in Sequence ID Nos. 3 to 12 may be, for example, a combination of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten.

[0014] The plant disease may be any plant disease, but soybean stem blight is preferred. The causative agent of soybean stem blight (hereinafter sometimes simply referred to as "stem blight fungus") is Phytophthora sojae, but Phytophthora sojae may be of different pathogenic types (strains). For example, the strains held in Hyogo Prefecture (N1, HR1, Th1) and the strains obtained from the gene bank (Ps060619-3-2:MAFF 247625, F9:MAFF 247661, Ps080626-1-7:MAFF 247657, Ps070702-4-1:MAFF 247649, Ps74:MAFF 247663, Ps060828-1-1:MAFF 247646, Ps060626-6-1:MAFF 247630) may be the causative agent.

[0015] In this specification, soybeans refer to Glycine max(L.)Merr., a species of the Fabaceae family. This includes cultivated soybeans (Glycine max(L.)Merr.subsp.max) and wild soybeans (Glycine max(L.)Merr.subsp.soja(Siebold&Zucc.)H.Ohashi=Glycine soja Siebold&Zucc.). Cultivated soybeans include varieties such as 'Himeshirazu' and 'Enrei' that are susceptible to the causative agent of soybean stem blight, and species such as 'Fukuyutaka' and 'Fukuminori' that are resistant to the causative agent of soybean stem blight.

[0016] In this specification, unless otherwise specified, "plant" includes the whole plant, plant cells, plant protoplasts, plant callus, or parts of a plant, such as embryos, pollen, ovules, gametes, seeds, leaves, flowers, branches, fruits, stems, roots, anthers, etc.

[0017] The selected soybean plants resistant to plant diseases are preferably those that have resistance to soybean stem blight, and more preferably those that have resistance to a wide range of strains (races) of the soybean stem blight fungus. For example, soybean plants that have resistance to multiple (two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten) of the above ten types of fungal strains.

[0018] Here, resistance refers to exhibiting higher field resistance compared to the disease-prone varieties "Himeshirazu" or "Enrei". Resistance may also be evaluated by the survival rate of soybean plants inoculated with the soybean stem blight fungus after a certain period. For example, under similar inoculation conditions with the same fungal strain, the survival rate (percentage of surviving individuals) of "Himeshirazu" or "Enrei" is 0.3 or less, 0.2 or less, or 0.1, while the survival rate of resistant soybean plants is 0.5 or higher, 0.6 or higher, 0.7 or higher, 0.8 or higher, 0.9 or higher, 0.95 or higher, or 1. Inoculation methods include mixing the soybean stem blight fungus into the soil, spraying a fungal solution containing the soybean stem blight fungus, or directly applying it to the seeds, leaves, or stems of soybean plants. The inoculation amount is not particularly limited, but it should be an amount that can cause soybean stem blight in disease-prone varieties. The specified period may be, for example, 7 days, 14 days, or 21 days after inoculation. Resistance can also be evaluated by the condition of the plants, such as the disease rate and the degree of browning of the roots. Specifically, plants exhibiting symptoms characteristic of soybean stem blight (water-soaked lesions) were defined as diseased plants. The degree of browning was evaluated on a scale of 0 to 5 relative to the percentage of browning of the entire root. 0: No browning, 1: Browning of 0% or more but less than 20%, 2: Browning of 20% or more but less than 40%, 3: Browning of 40% or more but less than 60%, 4: Browning of 60% or more but less than 80%, 5: Browning of 80% or more but less than 100%.

[0019] The base sequence shown in Sequence ID No. 1 is a base sequence of approximately 3 Mb in length around 45 Mb in the genomic DNA of chromosome 15 of 'Fukuyutaka' (nucleotides 43640337 to 46577019 of the genomic DNA of chromosome 15). The full sequence of the genomic sequence of chromosome 15 of 'Fukuyutaka' was determined by the inventors through analysis. As demonstrated in Experimental Example 1 described later, 'Fukuyutaka' is resistant to at least 10 pathogenic types of stem blight fungi, and therefore may be resistant to a wider range of strains than conventionally known resistant varieties (for example, resistant varieties into which true resistance genes have been introduced). Furthermore, as demonstrated in Experimental Examples 2 and 3, the approximately 260kb base sequence around 45Mb of chromosome 15 (nucleotides 45401325 to 45662116 of the genomic DNA of chromosome 15; Sequence ID No. 2; total 260,792 bp), which has the highest contribution to resistance, is considered a candidate region for resistance genes.

[0020] To efficiently select soybean plants resistant to plant diseases, it is advisable to detect the presence of the candidate resistance gene region shown in Sequence ID No. 2. Specifically, soybean plants resistant to plant diseases can be selected based on the presence or absence of the full-length sequence of Sequence ID No. 2. If the sequence of Sequence ID No. 2 is present, the plant can be selected as a soybean plant resistant to plant diseases.

[0021] On the other hand, plant disease-resistant soybean plants can be selected not only by detecting the nucleotide sequence of Sequence ID No. 2, but also by detecting the presence of the surrounding region. For example, the presence of a nucleotide sequence upstream or downstream of the nucleotide sequence shown in Sequence ID No. 2 in the genomic DNA of chromosome 15 of 'Fukuyutaka' is considered useful as it suggests the presence of the candidate region for the resistance gene. The nucleotide sequence shown in Sequence ID No. 2 and the range including its upstream and downstream regions may be, for example, the nucleotide sequence shown in Sequence ID No. 1, and the presence of the full-length or partial nucleotide sequence of the nucleotide sequence shown in Sequence ID No. 1 is considered to suggest the presence of a resistance gene, and plant disease-resistant soybean plants can be selected.

[0022] Some of the sequences in sequence number 1 include, for example, consecutive sequences of 50 or more, 60 or more, 80 or more, 100 or more, 120 or more, 150 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, or 500 or more in sequence number 1, and are 3M (3,000,000) or less, 2M (2,000,000) or less, 1M (1,000,000) or less, 500k (500,000) or less, 100k (10 The base sequence may be 0,000 or less, 50,000 or less, 10,000 or less, 5,000 or less, 4,500 or less, 4,000 or less, 3,000 or less, 2,500 or less, 2,000 or less, 1,500 or less, 1,000 or less, 500 or less, 400 or less, 300 or less, 250 or less, 200 or less, 150 or less, or 100 or less, and soybean plants resistant to plant diseases can be selected based on the detection of base sequences within this range. The range of the partial sequence in Sequence ID No. 1 may be any range at any position in the base sequence shown in Sequence ID No. 1, and may include, for example, a range of a partial base sequence of Sequence ID No. 2 (for example, a range of base sequences of 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, and with lengths of 5,000 or less, 4,500 or less, 4,000 or less, 3,000 or less, 2,500 or less, 2,000 or less, 1,500 or less, 1,000 or less, 500 or less, 400 or less, 300 or less, 250 or less, 200 or less, 150 or less, or 100 or less), or may be a range of base sequences that do not include the base sequence of Sequence ID No. 2.

[0023] Furthermore, the detection of specific markers (variety identification markers) for "Fukuyutaka" in the base sequence shown in Sequence ID No. 2 is also useful for selecting soybean plants resistant to plant diseases. The inventors selected BARCSOYSSR_15_1228, BARCSOYSSR_15_1233, BARCSOYSSR_15_1235, indel-1, indel-4, indel-5, indel-7, indel-8, BARCSOYSSR_15_1254, and BARCSOYSSR_15_1262 as specific markers (variety identification markers) for "Fukuyutaka" around 45 Mb of the genomic DNA of chromosome 15 of "Fukuyutaka" (Experimental Example 3). If these markers are detected, it is highly likely that the candidate resistance gene region (SEQ ID NO: 2) located around 45 Mb on chromosome 15 of 'Fukuyutaka' is present, and if this candidate resistance gene region is present, the variety may have broad resistance to soybean stem blight. (1) BARCSOYSSR_15_1228 (nucleotides 44875099 to 44875370 of SEQ ID NO: 1; SEQ ID NO: 3; total length 272 bp) (2) BARCSOYSSR_15_1233 (nucleotides 45043203 to 45043404 of SEQ ID NO: 1; SEQ ID NO: 4; total length 202 bp) (3) BARCSOYSSR_15_1235 (nucleotides 45096185 to 45096444 of SEQ ID NO: 5; total length 260 bp) (4) indel-1 (nucleotides 45206691 to 45206873 of SEQ ID NO: 1; SEQ ID NO: 6; total length 183 bp) (5) indel-4 (nucleotides 45401325 to 45401626 of SEQ ID NO: 1; SEQ ID NO: 7; total length 302 bp) (6) indel-5 (nucleotides 45479601 to 45479862 of SEQ ID NO: 1; SEQ ID NO: 8; total length 262 bp) (7) indel-7 (nucleotides 45620743 to 45621013 of SEQ ID NO: 1; SEQ ID NO: 9; total length 271 bp) (8) indel-8 (nucleotides 45662116 to 45662387 of SEQ ID NO: 10; total length 271 bp) (9) BARCSOYSSR_15_1254 (nucleotides 45670807 to 45671126 of SEQ ID NO: 11; total length 320 bp) (10) BARCSOYSSR_15_1262 (nucleotides 45794555 to 45794709 of SEQ ID NO: 12; total length 155 bp)

[0024] For the ten markers listed in (1) to (10) above, the presence of the entire marker or a portion thereof can also be used to select soybean plants resistant to plant diseases. For example, it is thought that soybean plants resistant to plant diseases can be selected by detecting the presence of a marker region containing 50 or more consecutive base sequences in the base sequence shown in Sequence ID No. 1 from the genomic DNA of a soybean plant under test, which is one of the ten markers selected from above (a marker consisting of a base sequence shown in any of Sequence ID Nos. 3 to 12).

[0025] The above-mentioned "marker region" may be a sequence of 50 or more base pairs and may include the entire length or a portion thereof of each marker (a marker consisting of a base pair selected from sequence numbers 3 to 12). If a portion of the marker is included, for example, it may include portions of 60 or more, 80 or more, 100 or more, 120 or more, 150 or more, 200 or more, 250 or more, or 300 or more of the marker, or it may include portions of 300 or less, 250 or less, 200 or less, 150 or less, or 100 or less.

[0026] "A sequence of 50 or more consecutive bases in the marker" may be a portion of the sequence of 50 or more bases of each marker (a marker consisting of a sequence of bases selected from sequence numbers 3 to 12), or it may be the entire length of each marker. "A sequence of 50 or more consecutive bases in the marker" may be, for example, 60 or more, 80 or more, 100 or more, 120 or more, 150 or more, 200 or more, 250 or more, 300 or more, or 350 or more, and less than or equal to the entire length of each marker.

[0027] Furthermore, the length of the "marker region" may be the total length of each marker, or it may be longer or shorter than the total length of each marker. The upper limit of the "marker region" may be, for example, 5000 or less, 4500 or less, 4000 or less, 3000 or less, 2500 or less, 2000 or less, 1500 or less, 1000 or less, 500 or less, 400 or less, 300 or less, 250 or less, 200 or less, 150 or less, or 100 or less.

[0028] In another embodiment of the method for selecting plant disease-resistant soybean plants, it is preferable to detect the presence of a DNA region consisting of a base sequence encoding a protein that has 90% or more sequence identity with at least one amino acid sequence selected from SEQ ID NOs: 13-22, and that can confer plant disease resistance to the soybean plant, from the genomic DNA of the soybean plant under test. As mentioned above, the base sequence of approximately 260kb around 45Mb of chromosome 15 (nucleotides 45401325-45662116 of the genomic DNA of chromosome 15; SEQ ID NO: 2), which has the highest contribution rate to resistance, is a candidate region for resistance genes. Therefore, the protein expressed by the gene in this approximately 260kb region is highly likely to be a protein that confers plant disease resistance, and if these proteins are present in the genomic DNA of a soybean plant, it is highly likely to be a plant disease-resistant soybean plant.

[0029] The protein consisting of the amino acid sequence shown in Sequence ID No. 13 is expressed by a gene encoded at nucleotides 45405479-45406373 of the genomic DNA on chromosome 15, and is presumed to be an Argonaute family protein.

[0030] The protein consisting of the amino acid sequence shown in Sequence ID No. 14 is expressed by a gene encoded at nucleotides 45450555 to 45453275 of the genomic DNA on chromosome 15, and is presumed to belong to the LYR family of Fe / S cluster biogenesis proteins.

[0031] The protein consisting of the amino acid sequence shown in Sequence ID No. 15 is expressed by a gene encoded at nucleotides 45480220 to 45488369 of the genomic DNA on chromosome 15, and is presumed to be TGACG motif-binding factor 6.

[0032] The protein consisting of the amino acid sequence shown in Sequence ID No. 16 is expressed by a gene encoded at nucleotides 45552507 to 45554401 of the genomic DNA on chromosome 15, and is presumed to be a disease resistance protein (TIR-NBS-LRR class).

[0033] The protein consisting of the amino acid sequence shown in Sequence ID No. 17 is expressed by a gene encoded at nucleotides 45571662 to 45573915 of the genomic DNA on chromosome 15, and is presumed to belong to the Disease resistance protein (TIR-NBS-LRR class) family.

[0034] The protein consisting of the amino acid sequence shown in Sequence ID No. 18 is expressed by a gene encoded at nucleotides 45577674 to 45579607 of the genomic DNA on chromosome 15, and is presumed to be a disease resistance protein (TIR-NBS-LRR class).

[0035] The protein consisting of the amino acid sequence shown in Sequence ID No. 19 is expressed by a gene encoded at nucleotides 45583112 to 45583288 of the genomic DNA on chromosome 15, and its function is unknown.

[0036] The protein consisting of the amino acid sequence shown in Sequence ID No. 20 is expressed by a gene encoded at nucleotides 45596606 to 45600352 of the genomic DNA on chromosome 15, and is presumed to be an NB-ARC domain-containing disease resistance protein.

[0037] The protein consisting of the amino acid sequence shown in Sequence ID No. 21 is expressed by a gene encoded at nucleotides 45625254 to 45627056 of the genomic DNA on chromosome 15, and is presumed to be a protein kinase / ubiquitin-protein ligase.

[0038] The protein consisting of the amino acid sequence shown in Sequence ID No. 22 is expressed by a gene encoded at nucleotides 45628762 to 45632588 of the genomic DNA on chromosome 15, and is presumed to be nudix hydrolase homolog 16.

[0039] A protein that has 90% or more sequence identity with at least one amino acid sequence selected from Sequence IDs 13 to 22 and can confer plant disease resistance to soybean plants (hereinafter sometimes referred to as "plant disease resistance protein") may be the above 10 proteins of "Fukuyutaka" or their mutant proteins, and preferably has 95% or more, 98% or more, or 99% or more sequence identity with any of the above 10 proteins. To determine whether a protein can confer plant disease resistance to soybean plants, for example, if the DNA encoding the mutant protein is introduced into a soybean plant variety susceptible to stem blight fungus and the survival rate after inoculation with stem blight fungus is higher than that of the parent plant, it can be determined that the protein can confer plant disease resistance.

[0040] The soybean plants used as test subjects can be any soybean plant, and may be of known or unknown varieties, or even hybrid varieties of multiple varieties. In particular, hybrid varieties are preferred in breeding.

[0041] The detection method is not particularly limited as long as it is a method that can be used for detecting base sequences in the art, but for example, it may be detected by PCR using the genomic DNA of the soybean plant under test as a template and primers that can detect the marker region or the DNA region. Genomic DNA is extracted using a commercially available DNA extraction kit and purified using a DNA purification kit if necessary. As primers that can detect the marker region, a primer set can be designed so that it can amplify the entire length of the marker region and includes a region in the primer that contains at least a portion of each marker and its complementary sequence in Sequence ID No. 1, for example, 15 bp, 20 bp, or 25 pb. As primers that can detect the DNA region, any primer set that can amplify the DNA region is sufficient, and a primer set can be designed based on the base sequence of the DNA region. PCR reaction conditions can be appropriately determined by those skilled in the art, taking into account the length of the DNA to be amplified and the length of the primers. If the PCR product is analyzed by electrophoresis and a band of the desired length is confirmed, the soybean plant under test is likely to have resistance to plant diseases. The marker region or the DNA region may also be detected by a sequencer.

[0042] <Method for creating soybean plants resistant to plant diseases> One embodiment of a method for producing plant disease-resistant soybean plants may include introducing a protein (plant disease-resistant protein) into a plant disease-susceptible soybean plant that has 90% or more sequence identity with at least one amino acid sequence selected from Sequence IDs 13 to 22 and that can confer plant disease resistance to the soybean plant. That is, it includes introducing DNA consisting of a base sequence encoding a protein that has 90% or more sequence identity with at least one amino acid sequence selected from Sequence IDs 13 to 22 and that can confer plant disease resistance to the soybean plant into the soybean plant.

[0043] Another embodiment of the method for producing plant disease-resistant soybean plants involves introducing DNA into a soybean plant that has a DNA sequence containing the full length or a portion thereof of the continuous DNA sequence shown in Sequence ID No. 2 (nucleotides 45401325 to 45662116 of the genomic DNA on chromosome 15 of "Fukuyutaka"). The portion of the DNA sequence shown in Sequence ID No. 2 may include, for example, DNA encoding a protein consisting of at least one amino acid sequence selected from Sequence ID Nos. 13 to 22 (hereinafter sometimes referred to as the "plant disease resistance gene").

[0044] A further embodiment of a method for producing plant disease-resistant soybean plants may include introducing DNA into soybean plants that comprises a continuous region in SEQ ID NO: 1, which includes the nucleotide sequence shown in SEQ ID NO: 2 and a region of 500 to 2000 (e.g., about 500, about 800, about 1,000, about 1,500) on its 5' or 3' side. The 500 to 2000 region on the 5' or 3' side of the nucleotide sequence shown in SEQ ID NO: 2 typically contains a sequence that regulates the expression of the gene, such as a promoter.

[0045] The DNA to be introduced can be obtained by artificial synthesis or amplification by PCR. There are no particular limitations on the method of introduction into soybean plants, but for example, the DNA to be introduced is incorporated into an appropriate expression vector (e.g., an Agrobacterium-mediated plant transfer vector), and this vector is introduced into Agrobacterium tumefaciens. Methods of introduction include freeze-thaw cycles and electroporation. Subsequently, Agrobacterium is introduced into sections of soybean plant leaves or stems. Specifically, the plant sections are immersed in an Agrobacterium suspension and co-cultured. After gene introduction, the plant sections are transferred to an appropriate regeneration medium, the plants are regenerated, and individuals containing the introduced gene are selected from the regenerated plants using antibiotic resistance markers or PCR.

[0046] The plant disease resistance of the created candidate soybean plants may be evaluated using the resistance evaluation method described above. Here, plant diseases include soybean stem blight. [Examples]

[0047] The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

[0048] <Experimental Example 1: Field resistance of "Fukuyutaka"> Using 10 strains of soybean stem blight fungus with different pathogenic types, the field resistance of two varieties, 'Fukuyutaka' and 'Fukuminori' (registration number 22017, breeder: National Agriculture and Food Research Organization), was investigated. 'Fukuminori' is a variety developed by conferring beet armyworm resistance to 'Fukuyutaka', and therefore possesses field resistance derived from 'Fukuyutaka'. For comparison, 'Himeshirazu', a variety susceptible to soybean stem blight fungus, was also used.

[0049] Field resistance to 10 types of soybean stem blight fungi was confirmed as follows: Soybean stem blight fungi were cultured for about two weeks in V8 medium with 5 mm square fungal colonies, and the medium was crushed to serve as the inoculum. 0.1-1% (w / v) of the inoculum was mixed with soil (JA Nippi Horticultural Culture Soil No. 1: Sakata Super Mix A = 1:1) and filled into 4x4 cell trays. Moisturized soybean seeds were placed with the hilum facing downwards and covered with soil to the extent that the seeds were hidden. Watering was performed from the bottom, and resistance was evaluated by measuring the survival rate (percentage of surviving soybean plants) after 2-3 weeks. The survival rate of one tray (5 individuals) per experimental plot was investigated, and the average of four replicates was calculated to evaluate resistance.

[0050] The following strains of soybean stem blight fungus (Phytophthora sojae) were used: strains held in Hyogo Prefecture (N1, HR1, Th1) and strains obtained from the gene bank (Ps060619-3-2:MAFF 247625, F9:MAFF 247661, Ps080626-1-7:MAFF 247657, Ps070702-4-1:MAFF 247649, Ps74:MAFF 247663, Ps060828-1-1:MAFF 247646, Ps060626-6-1:MAFF 247630).

[0051] The survival rate results are shown in Figure 1. From Figure 1, it was found that 'Fukuyutaka' and 'Fukuminori' exhibited field resistance to all 10 strains tested. This indicates that 'Fukuyutaka' and 'Fukuminori' are resistant to a wide range of pathogenic types of soybean stem blight.

[0052] <Experimental Example 2: QTL Analysis in the Resistance Region> Regarding the genomic regions that contribute to field resistance to soybean stem blight, several QTLs have been reported from resistance evaluation of recombinant self-pollinating populations of the resistant variety "Fukuyutaka" and the susceptible variety "Himeshirazu," and from genotyping analysis using SSR markers. Among these, chromosome 15, with a contribution rate of 48%, is included (Non-Patent Literature 3). The present inventors decided to perform QTL analysis on chromosome 15 to identify the resistance region.

[0053] (Indoor inoculation test of soybean stem blight fungus) Quantitative trait locus mapping (QTL) analysis in the indoor inoculation test was performed as follows: A 5mm square colony of soybean stem blight fungus was placed on V8 medium (10cm diameter petri dish) and cultured for approximately two weeks to serve as the inoculum. A 3cm layer of vermiculite was placed at the bottom of a No. 4 pot (12cm diameter x 10cm height), the inoculum was placed on top, and another 7cm layer of vermiculite was placed on top to create the inoculation medium. Thirteen test soybean seeds were sown on the medium with the hilum facing downwards, and approximately 1cm of vermiculite was used to cover the seeds. Water was immediately applied to induce germination, and then watering was performed from the bottom. Resistance was evaluated 21 days after inoculation based on the disease rate and the degree of root browning. Specifically, plants exhibiting symptoms characteristic of soybean stem blight (water-soaked lesions) were considered diseased plants. The degree of browning was evaluated on a scale of 0 to 5 relative to the overall browning percentage of the roots. 0: No browning, 1: Browning of 0% to less than 20%, 2: Browning of 20% to less than 40%, 3: Browning of 40% to less than 60%, 4: Browning of 60% to less than 80%, 5: Browning of 80% to less than 100%. For the "Fukuyutaka" / "Himeshirazu" recombinant inbred lines and the "Fukuyutaka" / "Enrei" recombinant inbred lines, 13 plants per line and 2 to 4 replicates were used.

[0054] QTL analysis of chromosome 15 was performed using Windows QTL Cartographer 2.5, utilizing linkage map data from the 'Fukuyutaka' / 'Himeshirazu' recombinant inbred line and the 'Fukuyutaka' / 'Enrei' recombinant inbred line, as well as resistance evaluation values ​​and genotypes from field inoculation trials of the recombinant inbred lines. Specifically, the analysis format in QTL Cartographer was set to MAPMAKER / QTL, and the linkage map data, resistance evaluation values, and genotype data were loaded into the software. The analysis parameters were set as follows: Cross Type: RIL, Map Function: Kosambi, Analysis: Copmosite Interval Mapping, Threshold Value Setting: Manual Input, LR value: 11.5, Control method: Forward Backward Method.

[0055] The results of the QTL analysis are shown in Figure 2. From Figure 2, in the indoor inoculation test of soybean stem blight fungus, a resistance QTL with a high contribution rate to chromosome 15 was detected in the 'Fukuyutaka' / 'Himeshirazu' recombinant inbred line.

[0056] (Field inoculation test of soybean stem blight fungus) For the "Fukuyutaka" / "Himeshirazu" recombinant inbred lines and the "Fukuyutaka" / "Enrei" recombinant inbred lines, 12 plants per line × 2 replicates were used for testing. Seeds were sown in cell trays at the end of June and transplanted to the field (6a) in early July, 10 days after seedling rearing. In mid- and late August, 150L of stem blight fungus solution (used strains: N1, HR1, Th1) (zoospore concentration (10)) was used. 3 The entire area was sprayed with (1 / ml), and immediately flooded (for 2 days) was carried out to promote subsequent disease development. Stem blight disease was monitored at 14-day intervals from late August to mid-September to evaluate the degree of resistance. In addition, QTL analysis of chromosome 15 was performed in the same manner as above.

[0057] The results of the QTL analysis are shown in Figure 3 ("Fukuyutaka" / "Himeshirazu" recombinant inpollinated line) and Figure 4 ("Fukuyutaka" / "Enrei" recombinant inpollinated line). From Figures 3 and 4, resistance QTLs were detected on chromosome 15 in the field inoculation test of soybean stem blight fungus, as in the laboratory inoculation test, in both the "Fukuyutaka" / "Enrei" recombinant inpollinated line and the "Fukuyutaka" / "Enrei" recombinant inpollinated line. Moreover, the presence of resistance QTLs was observed in close regions of chromosome 15 in both the laboratory and field studies. Therefore, it was suggested that resistance in "Fukuyutaka" can be expected regardless of the background of the variety.

[0058] <Experimental Example 3: Genotype Analysis of Resistance Gene Candidate Regions in Fine Mapping> The genotypes of "Fukuyutaka," "Himeshirazu," "Fukuminori," and each recombinant inbred line were determined by typing arrays using markers described in Songet al. (2010) (Non-Patent Literature 4) and Sayama et al. (2011) (Non-Patent Literature 5). The markers examined are shown in Table 1, of which indel-1, indel-4, indel-5, indel-7, and indel-8 were markers discovered by the present inventors. More detailed genotyping analysis of candidate resistance gene regions was performed by determining the amplification length of PCR amplification fragments using DNA extracted from each line as a template, using agarose gel electrophoresis or DNA sequencing. The primers used are shown in Table 2. [Table 1] [Table 2]

[0059] Whole-genome sequencing and RNA-seq analysis were performed on QTLs on chromosome 15 of the 'Fukuyutaka' and 'Himeshirazu' varieties, and the above 10 high-precision variety identification markers (BARCSOYSSR_15_1228, BARCSOYSSR_15_1233, BARCSOYSSR_15_1235, indel-1, indel-4, indel-5, indel-7, indel-8, BARCSOYSSR_15_1254, BARCSOYSSR_15_1262) were selected. Using these markers, the candidate resistance gene region was further narrowed down to approximately 260kb (nucleotides 45401325 to 45662116 of the genomic DNA on chromosome 15; SEQ ID NO: 2) as shown in Figure 5. From RNA-seq analysis, it was estimated that 10 genes (genes encoding SEQ ID NOs: 13 to 22) reside in this approximately 260kb region. This region is different from previously reported QTL regions (e.g., Patent Documents 1 and 2). Furthermore, it is not included in the stem blight resistance genes or gene regions reviewed in Non-Patent Document 2, suggesting that the 10 genes located in the approximately 260kb region of chromosome 15 may be novel field resistance genes.

[0060] Of the 10 candidate resistance genes located around 45 Mb of chromosome 15 (the RpsF arrow region in Figure 5), approximately 260 kb, the genes indicated by the black arrows possess resistance gene-like amino acid sequences and their expression was confirmed. The genes indicated by the gray arrows are expressed genes with resistance gene-like sequences but lack an LRR (Leucine-Rich Repeats) domain and were therefore expected to be functionally incomplete. The genes indicated by the white arrows are expressed genes that do not possess resistance gene-like sequences.

[0061] Experimental examples 1-3 showed resistance to at least 10 pathogenic types of stem blight fungi, suggesting that 'Fukuyutaka' may exhibit field resistance to a wider range of fungal strains than conventionally known resistant varieties. Furthermore, it is expected that plant disease-resistant soybean plants can be easily created by introducing at least a portion of the candidate resistance gene region (SEQ ID NO: 2) on chromosome 15, which has the highest contribution rate, through breeding. In addition, it is expected that plant disease-resistant soybean plants can be easily selected by utilizing the high-precision variety identification markers mentioned above.

Claims

1. A method for selecting soybean plants resistant to plant diseases, comprising detecting the presence of a marker region containing 50 or more consecutive base sequences in the base sequence shown in Sequence ID No. 1, and comprising 50 or more consecutive base sequences in a marker selected from Sequence ID No. 3 to 12, from the genomic DNA of a soybean plant to be tested.

2. A method for selecting soybean plants resistant to plant diseases, comprising detecting the presence of a DNA region consisting of a base sequence encoding a protein that has 90% or more sequence identity with at least one amino acid sequence selected from Sequence ID No. 13 to 22, and that can confer plant disease resistance to the soybean plant, from the genomic DNA of a soybean plant under test.

3. The selection method according to claim 1 or 2, wherein the plant disease includes soybean stem blight.

4. The selection method according to claim 1 or 2, wherein the detection is performed by PCR using the genomic DNA of a soybean plant under test as a template and a primer capable of detecting the marker region or the DNA region.

5. A method for producing plant disease-resistant soybean plants, comprising introducing DNA into soybean plants consisting of a base sequence that has 90% or more sequence identity with at least one amino acid sequence selected from Sequence IDs 13 to 22, and that encodes a protein capable of conferring plant disease resistance to soybean plants.

6. The method for producing the plant disease according to claim 5, wherein the plant disease includes soybean stem blight.

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