Method for determining level of bacterial wilt resistance in solanum melongena, method for producing solanum melongena of bacterial wilt-resistant, molecular marker for bacterial wilt resistance in solanum melongena, and determination kit for determining solanum melongena of bacterial wilt-resistant

Abstract

To provide a method for determining the level of bacterial wilt resistance in Solanum melongena.SOLUTION: One aspect of the present invention is a method for determining the level of bacterial wilt resistance in Solanum melongena. The method includes a step of determining, in Solanum melongena, whether at least one of the nucleotide polymorphisms listed in Table 1, or a contiguous polynucleotide sequence containing such a nucleotide polymorphism, is a molecular marker for the bacterial wilt resistance in the Solanum melongena.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for determining the degree of bacterial wilt resistance in eggplant plants, a method for producing bacterial wilt-resistant eggplant plants, a molecular marker for bacterial wilt resistance in eggplant plants, and a determination kit for determining whether an eggplant plant is resistant to bacterial wilt. [Background technology]

[0002] Bacterial wilt, the most important soil disease of solanaceous plants, affects 90% of tomato producing areas and 60% of eggplant producing areas worldwide, and it is reported that in Japan, countermeasures cost 30 billion yen per year.

[0003] Regarding bacterial wilt resistance, approaches based on QTL analysis have also been used. For example, Non-Patent Document 1 describes that QTL analysis was performed on eggplant plants, and that a QTL (quantitative trait locus) showing resistance to eggplant bacterial wilt is located on chromosome 9, that QTL detected on chromosomes 2 and 5 is associated with partial resistance, and that markers closely linked to these three QTLs are important for breeding eggplant plants with a wide range of resistance to eggplant bacterial wilt. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Salgon S, Jourda C, Sauvage C, Daunay MC, Reynaud B, Wicker E, Dintinger J. Eggplant Resistance to the Ralstonia solanacearumSpecies Complex Involves Both Broad-Spectrum and Strain-Specific Quantitative Trait Loci. Front Plant Sci. 2017 May 19;8:828. doi: 10.3389 / fpls.2017.00828. PMID: 28580001; PMCID: PMC5437220. Summary of the Invention [Problem to be solved by the invention]

[0005] However, it cannot be said that sufficient progress has been made in organizing information on genetic resources related to bacterial wilt resistance or in developing resistance selection markers, and efficient breeding of resistant varieties has not been realized. Therefore, the current method involves cultivating plants in a field artificially inoculated with the bacterial wilt pathogen and visually assessing the severity of the disease. However, this method requires a lot of time and effort for the artificial inoculation of the pathogen, the cultivation of the plants, and the visual assessment of the severity of the disease.

[0006] One aspect of the present invention has been made to solve the above-mentioned problems, and aims to realize a technique for selecting eggplant plants resistant to bacterial wilt disease. [Means for solving the problem]

[0007] One aspect of the present invention includes, for example, the following. 1) A method for determining the level of bacterial wilt resistance in an eggplant plant, the method comprising the step of determining, in the eggplant plant, at least one of the nucleotide polymorphisms shown in Table 1, or a contiguous polynucleotide containing said nucleotide polymorphism, as a molecular marker for bacterial wilt resistance in the eggplant plant. 2) A method for producing a bacterial wilt-resistant eggplant plant, comprising a selection step of selecting a bacterial wilt-resistant eggplant plant by the method described in 1). 3) A molecular marker for bacterial wilt resistance in eggplant plants, comprising at least one of the nucleotide polymorphisms shown in Table 1, or a continuous polynucleotide containing the nucleotide polymorphism. 4) A kit for determining whether an eggplant plant is resistant to bacterial wilt, the kit comprising primers for amplifying a region in the eggplant plant that contains a continuous polynucleotide sequence containing at least one of the nucleotide polymorphisms shown in Table 1. [Effects of the Invention]

[0008] According to one aspect of the present invention, a technique for selecting eggplant plants resistant to bacterial wilt can be realized. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 shows the results of a field resistance test conducted on 100 Nascore collections in the Examples. [Figure 2] FIG. 1 shows resistance regions detected by genome-wide association analysis using genomic polymorphism information of the Nascore collection in the Examples. [Figure 3] In the examples, this figure shows the effect of resistance regions (2022) detected by classifying the disease index of the Nascore collection by the genotype at the polymorphic position of interest. [Figure 4] In the examples, this figure shows the effect of resistance regions (2023) detected by classifying the disease index of the Nascore collection by the genotype at the polymorphic position of interest. [Figure 5] FIG. 1 is a dot plot diagram of markers (KASP) for discriminating genotypes in the chromosome 6 region obtained in the Examples. [Figure 6]FIG. 1 shows the results of agarose gel electrophoresis using markers (CAPS) for discriminating genotypes in the chromosome 6 region, obtained in the Examples. [Figure 7] FIG. 1 shows a dot plot of markers (KAPS) for discriminating genotypes in the chromosome 1 region, obtained in the Examples, and the results of agarose gel electrophoresis using markers (CAPS). [Figure 8] FIG. 1 shows the results of agarose gel electrophoresis using markers (CAPS) for discriminating genotypes in the chromosome 9 region, obtained in the Examples. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention will be described in detail below. All of the documents described in this specification are incorporated herein by reference. Furthermore, unless otherwise specified in this specification, "A to B" representing a numerical range means "A or more (including A and greater than A) and B or less (including B and less than B)."

[0011] As used herein, the term "polynucleotide" is used interchangeably with "nucleic acid" or "nucleic acid molecule," and refers to a polymer of nucleotides. Here, nucleic acids can exist in the form of DNA (e.g., cDNA or genomic DNA) or RNA (e.g., mRNA). DNA or RNA may be double-stranded or single-stranded. Single-stranded DNA or RNA may be a coding strand (sense strand) or a non-coding strand (antisense strand). As used herein, bases are represented by the single-letter symbols defined by IUPAC and IUB, as appropriate.

[0012] In one embodiment of the present invention, eggplant plants broadly include plants classified as eggplant (Solanum melongena L.). Eggplant plants may be, for example, plants that serve as rootstocks for grafting, or plants that serve as seedlings or scions.

[0013] Eggplant plants may be candidate plants for breeding material or plants obtained through a breeding process. Candidate plants for breeding material include, for example, parent plants used in crossbreeding and plants used in molecular breeding using genetic engineering techniques. Furthermore, plants obtained through a breeding process include, for example, plants obtained by intraspecific hybridization of eggplant plants and their progeny lines. Eggplant plants may be, for example, plants obtained by intraspecific hybridization of individuals resistant to bacterial wilt, and their progeny lines. Eggplant plants may be, for example, plants obtained by intraspecific hybridization of individuals resistant to bacterial wilt with individuals not resistant to bacterial wilt, and their progeny lines. Eggplant plants may be, for example, plants obtained by intraspecific hybridization of individuals whose bacterial wilt resistance is unknown, and their progeny lines. Eggplant plants may be, for example, plants obtained by intraspecific hybridization of individuals whose bacterial wilt resistance is unknown with individuals whose bacterial wilt resistance is known, and their progeny lines. The eggplant plant may be, for example, a plant obtained by intraspecific hybridization between individuals known to have resistance to bacterial wilt, and its progeny lineage.

[0014] As used herein, a plant may refer to a part or the whole of a plant. Examples of parts of a plant include rootstocks, propagation materials (e.g., pollen, seeds, embryos), flowers, and roots. As used herein, "an eggplant plant having resistance to bacterial wilt" means, for example, that the plant has better resistance to bacterial wilt compared to a reference eggplant plant. As used herein, "an eggplant plant not having resistance to bacterial wilt" means, for example, that the plant has equal or less resistance to bacterial wilt compared to a reference eggplant plant. Reference eggplant plants are appropriately selected from eggplant plants recognized as having no or low resistance to bacterial wilt. Representative examples of reference eggplant plants (eggplant plants recognized as having no or low resistance to bacterial wilt) include, but are not limited to, the varieties "Senryo No. 2," "Chikuyo," and "Aminori No. 2." The presence or absence of resistance to bacterial wilt can be determined, for example, by defining a disease index according to the degree of disease symptom appearance on leaves, as described in the Examples, and classifying the presence or absence of resistance of eggplant plants to bacterial wilt based on whether the disease index is above or below a predetermined value. In this specification, bacterial wilt refers to a pathological condition caused in eggplant plants by infection with Ralstonia solanacearum in a broad sense, and in particular refers to a pathological condition caused in eggplant plants by infection with Ralstonia pseudosolanacearum in the classification system described in SAFNI, I. et al. (2014): Int. J. Syst. Evol. Microbiol. 64: 3087-3103 and subsequent publications.

[0015] [Molecular markers for bacterial wilt resistance in eggplant plants] A molecular marker according to one embodiment of the present invention is a molecular marker for bacterial wilt resistance in eggplant plants. The molecular marker is a molecular marker that can be used to select eggplant plants resistant to bacterial wilt, and one example of such a molecular marker is a molecular marker for detecting alleles of single nucleotide polymorphisms (SNPs) that can be used to select eggplant plants resistant to bacterial wilt.

[0016] The molecular marker in an eggplant plant comprises at least one of the nucleotide polymorphisms shown in Table 1 (see Examples), or a contiguous polynucleotide containing the nucleotide polymorphism. The molecular marker in an eggplant plant comprises two or more of the nucleotide polymorphisms shown in Table 1, or a contiguous polynucleotide containing the nucleotide polymorphism. The contiguous polynucleotide containing a nucleotide polymorphism may contain only one of the nucleotide polymorphisms shown in Table 1 within its molecule, or may contain two or more of the nucleotide polymorphisms shown in Table 1 within its molecule.

[0017] A molecular marker according to one embodiment of the present invention is located on chromosome 1, 3, 6, 7, 9, or 12 of an eggplant plant. A molecular marker according to one embodiment of the present invention is preferably located on chromosome 1, 6, or 9 of an eggplant plant, and more preferably on chromosome 6 of an eggplant plant. Furthermore, a molecular marker according to one embodiment of the present invention can be used to identify loci associated with bacterial wilt resistance on these chromosomes of an eggplant plant. A locus associated with bacterial wilt resistance refers to a quantitative trait locus or gene region that affects bacterial wilt resistance. A quantitative trait locus (QTL) generally refers to a chromosomal region involved in the expression of a quantitative trait. A QTL can be defined using a molecular marker that indicates a specific locus on a chromosome.

[0018] Examples of molecular markers according to one embodiment of the present invention include SNP markers, AFLP (amplified fragment length polymorphism) markers, RFLP markers, microsatellite markers, SCAR markers, and CAPS markers.

[0019] In one embodiment of the present invention, the molecular marker may be a nucleotide polymorphism that is in linkage disequilibrium with any of the nucleotide polymorphisms shown in Table 1. "Linkage disequilibrium" refers to two alleles that are inherited in a linked relationship with each other at a frequency greater than when each allele is inherited independently. In one embodiment of the present invention, the molecular marker is a nucleotide polymorphism that is related to any of the nucleotide polymorphisms shown in Table 1 by a linkage disequilibrium coefficient r2 ≧ 0.9, preferably a nucleotide polymorphism that is related to a linkage disequilibrium coefficient r2 ≧ 0.95, and more preferably a nucleotide that is related to a linkage disequilibrium coefficient r2 ≧ 1. By typing a nucleotide polymorphism that is in linkage disequilibrium with a nucleotide polymorphism shown in Table 1, the nucleotide species of the corresponding nucleotide polymorphism shown in Table 1 can be indirectly determined, and bacterial wilt resistance of eggplant plants can be determined.

[0020] (SNP markers) An SNP marker can be (i) the base corresponding to the SNP itself, (ii) a continuous polynucleotide containing the SNP, or (iii) a continuous polynucleotide containing two or more SNPs. Here, SNP refers to a DNA polymorphism in which a single base mutation is observed within a specific region of the DNA base sequence. The SNP is a single nucleotide polymorphism based on the genomic sequence of an eggplant plant (for example, the publicly available sequence of the entire genome or sequence information obtained by analyzing a specific variety).

[0021] The above-mentioned (ii) contiguous polynucleotide containing an SNP refers to (a1) a contiguous polynucleotide containing an SNP (identical to the reference base sequence except for the SNP); (a2) a polynucleotide represented by a base sequence in which one or more bases have been substituted, deleted, added, or inserted relative to the region other than the SNP in the base sequence of the polynucleotide (a1), and which functions to determine bacterial wilt resistance in eggplant plants; or (a3) ​​a polynucleotide represented by a base sequence having 90% or more identity relative to the region other than the SNP in the base sequence of the polynucleotide (a1), and which functions to determine bacterial wilt resistance in eggplant plants. In (a3), the identity of the base sequence relative to the region other than the SNP can be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Such base sequence identity can be determined by aligning two base sequences using analytical software such as BLAST or FASTA.

[0022] The sequences around 200 bp before and after the polymorphism in Table 1 and their partial sequences (which contain SNPs) are examples of the above-mentioned (ii) contiguous polynucleotides containing SNPs. Longer sequences including the sequences around 200 bp before and after the polymorphism are also examples of contiguous polynucleotides containing SNPs.

[0023] [Eggplant plants resistant to bacterial wilt] An eggplant plant according to one embodiment of the present invention is a plant obtained by the production method described below. The eggplant plant having resistance to bacterial wilt disease is a plant that has the above-mentioned SNP identified by the above-mentioned molecular marker and has resistance to bacterial wilt disease.

[0024] According to one embodiment of the present invention, a bacterial wilt-resistant eggplant plant can be obtained by using the molecular markers described above to identify bacterial wilt-resistant eggplant plants from a plant obtained by crossbreeding eggplant plants with another eggplant plant and its progeny, or from a self-pollinated progeny of the eggplant plant, as described in the production method described below. The scope of the present invention also includes bacterial wilt-resistant eggplant plants that have been genetically engineered to contain the SNPs described above. An example of a genetically engineered eggplant plant that is resistant to bacterial wilt is an eggplant plant into which a genomic fragment containing a bacterial wilt resistance allele (SNP) and a QTL for bacterial wilt resistance has been genetically engineered.

[0025] [Method for determining the level of bacterial wilt resistance in eggplant plants] A method for determining the degree of bacterial wilt resistance in an eggplant plant (determination method) according to one embodiment of the present invention includes a step of determining, in the eggplant plant, at least one of the nucleotide polymorphisms shown in Table 1, or a contiguous polynucleotide containing the nucleotide polymorphism, as a molecular marker for bacterial wilt resistance in the eggplant plant.

[0026] A determination method according to one embodiment of the present invention may include a step of determining two or more of any of the nucleotide polymorphisms shown in Table 1, or a contiguous polynucleotide containing such nucleotide polymorphisms, as molecular markers for bacterial wilt resistance in eggplant plants.

[0027] The molecular markers used in the determination method according to one embodiment of the present invention are located, for example, on chromosome 1, 3, 6, 7, 9, or 12 of eggplant plants, and preferably on chromosome 1, 6, or 9 of eggplant plants. When two or more molecular markers are used, they may be located on the same chromosome, but it may be preferable for them to be located on different chromosomes. For example, bacterial wilt resistance may be determined using a molecular marker located on chromosome 6 and a molecular marker located on chromosome 1 or 9. For details of the molecular markers used, please refer to the description in the above section "Molecular markers related to bacterial wilt resistance in eggplant plants."

[0028] A determination method according to one embodiment of the present invention determines the level of bacterial wilt resistance in a test eggplant plant using the molecular marker according to one embodiment of the present invention described above. The determination method according to one embodiment of the present invention determines whether the eggplant plant has bacterial wilt resistance by identifying the base species of any of the nucleotide polymorphisms shown in Table 1 in the genome of the test eggplant plant.

[0029] In one embodiment of the determination method of the present invention, the test eggplant plant may be, for example, a candidate plant for breeding material or a plant obtained through a breeding process. The test eggplant plant may be, for example, a hybrid between a bacterial wilt-resistant eggplant plant and a bacterial wilt-resistant eggplant plant, a hybrid between a bacterial wilt-resistant eggplant plant and a non-resistant eggplant plant, or a hybrid between a non-resistant eggplant plant and a non-resistant eggplant plant, as well as their progeny. The test eggplant plant may also be a hybrid between an eggplant plant with known bacterial wilt resistance and an eggplant plant with unknown bacterial wilt resistance, or a hybrid plant obtained by crossing eggplant plants with unknown bacterial wilt resistance, as well as their progeny. Furthermore, the test eggplant plant may be a self-pollinated progeny of a bacterial wilt-resistant eggplant plant. The test eggplant plant may be a leaf, stem, root, or the like.

[0030] A determination method according to one embodiment of the present invention determines the level of bacterial wilt resistance of an eggplant plant based on whether the SNP shown in Table 1 is a resistant allele or a susceptible allele. For example, if the plant is homozygous for the resistant allele, it is determined to be resistant to bacterial wilt, and if the plant is homozygous for the susceptible allele, it is determined to not be resistant to bacterial wilt. In the case of a heterozygous plant between a resistant allele and a susceptible allele, it is determined, depending on the SNP in question, as 1) resistant to bacterial wilt, 2) not resistant to bacterial wilt, or 3) intermediate bacterial wilt resistance.

[0031] In the determination method according to one embodiment of the present invention, the method for testing the level of bacterial wilt resistance in eggplant plants using molecular markers is not particularly limited, and known SNP analysis methods can be used, such as SNP analysis methods that involve detecting SNPs in PCR-amplified fragments of a test eggplant plant. The determination method according to one embodiment of the present invention may amplify a region containing the molecular marker in the DNA of the eggplant plant using primers that amplify a region containing the molecular marker. One example of such primers is a primer that amplifies a region containing any of the SNPs listed in Table 1. In other words, primers that amplify a region containing any of the SNPs listed in Table 1 can be used in a determination kit for determining whether an eggplant plant is bacterial wilt-resistant according to one embodiment of the present invention.

[0032] Amplification of a region containing a molecular marker in the DNA of a test eggplant plant can be performed, for example, by polymerase chain reaction (PCR) using DNA (genomic DNA) extracted from the test eggplant plant as a template and a primer set that amplifies a region containing the SNP. The base (genotype) of the SNP in the resulting amplified fragment is then determined, and the level of bacterial wilt resistance in the eggplant plant is assessed based on data showing the relationship between the determined base (genotype) and the level of bacterial wilt resistance in the eggplant plant. Methods for determining the genotype of the amplified fragment can be conventionally known, such as DNA sequence analysis, agarose gel electrophoresis (e.g., CAPS markers), and TaqMan analysis using real-time PCR.

[0033] The primer set used in PCR is not particularly limited as long as it can amplify a DNA fragment containing the target SNP. The primer set may be designed to shorten the length of the amplified fragment. For example, the primer set is designed so that the length of the primer-amplified fragment is preferably 400 base pairs (bp) or less, 350 bp or less, 320 bp or 310 bp or less, 300 bp or less, 250 bp or less, 220 bp or 210 bp or less, 200 bp or less, 150 bp or less, or 100 bp or less. For example, in the case of KASP markers, the amplified fragment can also be designed to be shorter (e.g., 200 bp or less, 150 bp or less, or 100 bp or less, but not less than 40 bp). The primer set includes a forward primer and a reverse primer. The length of these primers may be, for example, 15 bp or more, 16 bp or more, 17 bp or more, 18 bp or more, 19 bp or more, 20 bp or more, or 25 bp or more, or 50 bp or less, 40 bp or less, or 30 bp or less. Such a primer set may constitute a determination kit for determining the level of bacterial wilt resistance according to one embodiment of the present invention. The nucleotide sequences of specific examples of primer sets are shown in the tables in the Examples (Tables 2, 4, 7, 8, and 9). A primer set may be configured to contain at least a portion or all of this nucleotide sequence (for example, based on the above-mentioned primer lengths, the lower limit is set to 15 bp or more, 16 bp or more, 17 bp or more, 18 bp or more, 19 bp or more, 20 bp or more, or 25 bp or more consecutively).

[0034] PCR in SNP analysis may be either singleplex PCR, which amplifies DNA fragments in a reaction system containing a single primer set, or multiplex PCR, which amplifies genes in a reaction system containing multiple primer sets. In the case of multiplex PCR, primer sets labeled with fluorescent substances having different wavelengths (e.g., NED, 6-FAM, VIC, PET, HEX) may be mixed.

[0035] PCR reaction conditions can be appropriately set depending on the type of DNA polymerase and PCR device used, the length of the amplified fragment, etc. Cycling conditions include a three-step PCR method, in which one cycle consists of three steps: a denaturation step, an annealing step, and an extension step, and a two-step PCR method, in which one cycle consists of two steps: a denaturation step, an annealing step, and an extension step. An example of reaction conditions for three-step PCR is 10 to 40 cycles (e.g., 35 cycles) of a denaturation step at 90 to 100°C for 20 to 60 seconds (e.g., 94°C for 30 seconds), an annealing step for 10 to 60 seconds (e.g., 30 seconds), and an extension step at 65 to 80°C for 10 to 90 seconds (e.g., 72°C for 60 seconds). An example of the annealing temperature is a condition in which the temperature is gradually decreased in each predetermined cycle (e.g., decreased by 1°C per cycle) from an initial annealing temperature of 55 to 70°C (e.g., 60°C) to a final annealing temperature of 40 to 60°C (e.g., 45°C). However, the annealing temperature may be constant for each cycle. PCR reaction conditions may be adjusted depending on the state of the template DNA to stably detect SNPs. An example of reaction conditions for two-step PCR is 10 to 50 cycles of a denaturation step at 90 to 100°C for 15 to 60 seconds (e.g., 94°C for 20 seconds) and an annealing and extension step at 45 to 70°C for 10 to 90 seconds. For example, the annealing and extension steps may be performed under conditions in which the temperature is gradually decreased for each predetermined cycle from an initial reaction temperature of 55 to 70°C to a final reaction temperature of 45 to 60°C, but the annealing temperature may be constant for each cycle, or may be a combination of a condition in which the temperature is gradually decreased for each predetermined cycle and a condition in which the temperature is constant for each cycle. PCR reaction conditions may be adjusted depending on the state of the template DNA in order to stably detect SNPs.

[0036] As the PCR in SNP analysis, real-time PCR such as TaqMan®-PCR, which performs PCR amplification and identifies SNP markers, may be used. That is, a TaqMan® probe may be further used to detect SNPs contained in the amplified fragments amplified using a primer set. The use of real-time PCR can provide a high-throughput discrimination method. Furthermore, SNPs in the amplified fragments amplified by PCR may be identified by analyzing the nucleotide sequence of the amplified fragments using an automated DNA sequencer or the like.

[0037] The method for extracting DNA (genomic DNA) to be amplified by PCR from eggplant plant specimens is not particularly limited, and known DNA extraction methods can be used. Alternatively, DNA may be extracted using a commercially available DNA extraction kit. Depending on the type of specimen and the amount of contaminants, appropriate pretreatment may be performed before the DNA extraction step. Furthermore, the DNA extracted from the specimen may be washed or purified as necessary for use as a template in the PCR reaction. Furthermore, the DNA extracted from the specimen may be digested with two restriction enzymes, and the resulting restriction enzyme fragments may be amplified by PCR.

[0038] Furthermore, in the determination method according to one embodiment of the present invention, genetic polymorphisms that are in linkage disequilibrium with the SNPs shown in Table 1 may be analyzed to identify the alleles of the SNPs shown in Table 1. As explained in the section on molecular markers, the above-mentioned linkage disequilibrium state is, for example, a linkage disequilibrium state in which the linkage disequilibrium coefficient r2 is 0.9 or more.

[0039] According to a method for determining whether an eggplant plant is resistant to bacterial wilt using molecular markers, bacterial wilt-resistant eggplant plants and their progeny lines can be selected based on the results of the determination.

[0040] According to one aspect of the present invention, the method of determining bacterial wilt resistance in eggplant plants can be selected at the seedling stage, significantly shortening the time required for breeding selection. Furthermore, by planting and growing only the seedlings of eggplant plants with bacterial wilt resistance selected at the seedling stage, the efficiency of farmland utilization can be improved.

[0041] [Method for producing eggplant plants resistant to bacterial wilt] A production method according to one aspect of the present invention is a method for producing a bacterial wilt disease-resistant eggplant plant, and includes a selection step of selecting a bacterial wilt disease-resistant eggplant plant using the evaluation method according to one aspect of the present invention. The production method may also include repeating the selection step one or more times on progeny of the bacterial wilt disease-resistant eggplant plant selected in the selection step.

[0042] The production method may include a crossbreeding step in which eggplant plants are crossbreeded with each other prior to the selection step, and in the selection step, eggplant plants resistant to bacterial wilt may be selected from the eggplant plants obtained by the crossbreeding step or other progeny eggplant plants using the determination method according to one embodiment of the present invention.

[0043] Furthermore, the production method may include a step of obtaining a self-pollinated progeny of a bacterial wilt-resistant eggplant plant before the selection step, and in the selection step, a bacterial wilt-resistant eggplant plant may be selected from the obtained self-pollinated progeny of the bacterial wilt-resistant eggplant plant using the determination method according to one embodiment of the present invention.

[0044] Therefore, the explanations regarding the molecular marker, the bacterial wilt resistant eggplant plant, and the determination method according to one embodiment of the present invention are also used to explain the method for producing a bacterial wilt resistant eggplant plant.

[0045] In the crossbreeding step, eggplant plants used as parent plants may be, for example, eggplant plants resistant to bacterial wilt, eggplant plants not resistant to bacterial wilt, eggplant plants whose level of bacterial wilt resistance is unknown, and their progeny lines.

[0046] Furthermore, the eggplant plant used as the parent plant in the crossing step may be a bacterial wilt-resistant eggplant plant according to one embodiment of the present invention. Furthermore, the eggplant plant used in the crossing step may be a bacterial wilt-resistant eggplant plant selected by the determination method according to one embodiment of the present invention. That is, the production method according to one embodiment of the present invention may further include, prior to the crossing step, a discrimination step of discriminating a bacterial wilt-resistant eggplant plant from test eggplant plants by the determination method according to one embodiment of the present invention.

[0047] In the discrimination step, a bacterial wilt-resistant eggplant plant is discriminated from the eggplant plants obtained in the crossing step or from eggplant plants of progeny lines by a discrimination method according to one aspect of the present invention.

[0048] According to a production method of one aspect of the present invention, it is possible to determine whether an eggplant plant is resistant to bacterial wilt using a molecular marker, and to produce a bacterial wilt-resistant eggplant plant selected based on the determination results.

[0049] 〔summary〕 As described above, the scope of the present invention includes, for example, the following: 1) A method for determining the level of bacterial wilt resistance in an eggplant plant, the method comprising the step of determining, in the eggplant plant, at least one of the nucleotide polymorphisms shown in Table 1, or a contiguous polynucleotide containing said nucleotide polymorphism, as a molecular marker for bacterial wilt resistance in the eggplant plant. 2) The method according to 1), comprising a step of determining two or more of any of the nucleotide polymorphisms shown in Table 1, or a continuous polynucleotide containing such nucleotide polymorphisms, as a molecular marker for bacterial wilt resistance in an eggplant plant. 3) The method according to 1) or 2), wherein the molecular marker is located on chromosome 1, chromosome 6, or chromosome 9 of the eggplant plant. 4) The method according to 3), wherein the molecular marker is located on chromosome 6 of the eggplant plant. 5) The method according to any one of 1) to 4), wherein in the determining step, the region in the DNA of the eggplant plant is amplified using primers that amplify the region containing the molecular marker. 6) A method for producing a bacterial wilt-resistant eggplant plant, comprising a selection step of selecting a bacterial wilt-resistant eggplant plant by the method described in any one of 1) to 5). 7) The production method according to 6), wherein the selection step is repeated one or more times on the progeny of the bacterial wilt-resistant eggplant plant selected in the selection step. 8) A molecular marker for bacterial wilt resistance in eggplant plants, comprising at least one of the nucleotide polymorphisms shown in Table 1, or a continuous polynucleotide containing the nucleotide polymorphism. 9) A kit for determining whether an eggplant plant is resistant to bacterial wilt, the kit comprising primers for amplifying a region in the eggplant plant that contains a continuous polynucleotide sequence containing at least one of the nucleotide polymorphisms shown in Table 1.

[0050] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention. [Example]

[0051] Example 1: Field resistance testing of 100 Nascore collections Four individuals from each of the 100 strains of the Nascore collection described in Reference 1 were cultivated in the field, and a bacterial wilt inoculation test was conducted to evaluate the severity of the disease symptoms. In the bacterial wilt inoculation test, a strain of Ralstonia pseudosolanacearum (R. pseudosolanacearum), the causative bacterium of bacterial wilt, isolated from the field, was used, and the grown plants were inoculated by root drench. Specifically, the roots of eggplant plants one month after sowing were cut with a sickle with a blade length of approximately 15 cm, and 1.0 × 10 8The plants were inoculated by pouring in a solution of R. pseudosolananaceum bacteria prepared at a concentration of 10 ... 0: No symptoms 1: Symptoms present on 25% or less of the leaves 2: Symptoms present on 25-50% of leaves 3: Symptoms present on 50-75% of leaves 4: Symptoms present throughout the plant (including withering) Each individual was evaluated according to the above criteria, and the average value of the four individuals was calculated as the disease index for each line.

[0052] (Reference 1) Miyatake K et al. (2019) Construction of a core collection of eggplant (Solanum melongena L.) based on genome-wide SNP and SSR genotypes. Breeding Science 69(3):498-502.

[0053] Example 2: Resistance region detected using genomic polymorphism information from the Nascore collection For each lineage in the Nascore collection used in Example 1, large-scale base sequence information was obtained using short-read analysis by Illumina, and this information was mapped to the genome reference sequence to obtain polymorphism information. The genome reference sequence was constructed by integrating sequence data obtained from long-read sequencing analysis by Oxford Nanopore Technologies and short-read analysis by Illumina for one lineage in the core collection. The "BWA" package was used for mapping, and the "GATK" package group was used for polymorphism information analysis.

[0054] Genome-wide association analysis was performed using the polymorphism information and disease index (Example 1) obtained for each line. The R package "gaston" was used for the analysis. The significance level of the polymorphism position was calculated as the false discovery rate (FDR) using the Benjamini-Hochberg method, and a -log satisfying FDR < 0.01 was used. 10 The -log(p) value was used. 10 The p-values ​​are indicated by black dots in Figure 2. Table 1 lists all of the significant polymorphic positions (168 positions) found. In Table 1, No. is the reference number assigned to each marker and corresponds to the sequence number in the sequence listing. For example, to explain only marker No. 1, the marker name is Chr01_3719262, and since the chromosome number is Chr01, it is located on chromosome 1. The polymorphic position on chromosome 1 is 3719262. The p-values ​​for 2022 and 2023 are as shown in the table, with the bacterial wilt resistance allele being C and the susceptibility allele being T. The SNP is indicated in parentheses as [C / T] in the sequence 200 bp around the polymorphism. In the sequence listing, this SNP is represented by w, r, m, k, y, or s, and the resistance allele and susceptibility allele can be identified by referring to Table 1. Typically, the smaller the p-value in Table 1, the more significant it is.

[0055] [Table 1] JPEG2025152365000002.jpg235170JPEG2025152365000003.jpg235170JPEG2025152365000004.jpg235170JPEG2025152365000005.jpg235170JPEG2025152365000006.jpg235170JPEG2025152365000007.jpg235170JPEG2025152365000008.jpg235170 Example 3: Effect of detected resistance regions In Example 2 (results in Figure 2), for seven areas that were commonly detected in 2022 and 2023, the highest or top -log 10 Polymorphic positions with (p) values ​​were selected, and the disease index of the Nascore collection (2022 and 2023) was classified by the genotype of each polymorphic position. Figure 3 shows the effect of the detected resistance region (for 2022), and Figure 4 shows the effect of the detected resistance region (for 2023). In each figure, the plot shows the average value, and the error bars show the standard deviation.

[0056] Example 4: Creation of a marker (KASP) for discriminating genotypes in the chromosome 6 region For the region of chromosome 6 in Figure 2 detected in Example 2, -log 10 Polymorphic positions with the highest p values ​​were selected to create markers capable of distinguishing the genotype at each polymorphic position. The sequences of the designed PCR primers, the sequences of the amplified products generated by the PCR reaction, and the positions and base types of the target alleles (resistant / susceptible alleles) are shown in Table 2. The PCR primers were a combination of a resistance allele-specific primer and a common primer, and a susceptible allele-specific primer and a common primer. One of the allele-specific primers was labeled with FAM and the other with HEX. The PCR reaction conditions are summarized in Table 3. Item 4) Measurement and analysis of fluorescence intensity in Table 3 was performed by measuring the fluorescence intensity of FAM and HEX using a real-time PCR device and determining the genotype from a dot plot. The results are shown in Figure 5. [Table 2]

[0057] [Table 3] Example 5: Creation of markers (CAPS) for discriminating genotypes in the chromosome 6 region Markers (CAPS) capable of discriminating the genotype at each polymorphic position by agarose gel electrophoresis were created at the same polymorphic positions as in Example 4 (shown in Figure 5). The sequences of the PCR primers used, the sequences of the amplified products generated by the PCR reaction and their restriction enzyme sites, the positions and base types of the target alleles (resistant / susceptible alleles), and the restriction enzymes used are shown in Table 4. The conditions for the PCR reaction, restriction enzyme treatment, and agarose gel electrophoresis are summarized in Tables 5 and 6. The results are shown in Figure 6. [Table 4] [Table 5]

[0058] [Table 6] Example 6: Creation of a marker (KASP) for discriminating the genotype of the chromosome 1 region For the region of chromosome 1 in Figure 2 detected in Example 2, -log 10 Polymorphic positions with the highest (p) values ​​were selected, and markers capable of distinguishing their genotypes were created. The sequences of the designed PCR primers, the sequences of the amplification products generated by the PCR reaction, and the positions and base types of the target alleles (resistant / susceptible alleles) are shown in Table 7. The PCR primers were a combination of a resistance allele-specific primer and a common primer, and a combination of a susceptible allele-specific primer and a common primer, with one allele-specific primer labeled with FAM and the other with HEX. The PCR reaction conditions were the same as those summarized in Table 3 of Example 4. The results are shown on the left side of Figure 7.

[0059] [Table 7] Example 7: Creation of markers (CAPS) for discriminating genotypes in the chromosome 1 region A marker (CAPS) capable of discriminating the genotype at the polymorphic position by agarose gel electrophoresis was created at a nearby polymorphic position in the region of chromosome 1 in Figure 2 detected in Example 2, different from that in Example 6. The sequences of the PCR primers used, the sequences of the amplified products generated by the PCR reaction and their restriction enzyme sites, the positions and base types of the target alleles (resistant / susceptible alleles), and the restriction enzymes used are shown in Table 8. The conditions for the PCR reaction, restriction enzyme treatment, and agarose gel electrophoresis were the same as those summarized in Tables 5 and 6 of Example 5. The results are shown on the right side of Figure 7.

[0060] [Table 8] Example 8: Creation of markers (CAPS) for discriminating genotypes in the chromosome 9 region For the region of chromosome 9 in Figure 2 detected in Example 2, -log 10 Polymorphic positions with the highest (p) values ​​were selected, and markers (CAPS) capable of distinguishing their genotypes were created. The sequences of the PCR primers used, the sequences of the amplified products generated by the PCR reaction and their restriction enzyme sites, the positions and base types of the target alleles (resistant / susceptible alleles), and the restriction enzymes used are shown in Table 9. The conditions for the PCR reaction, restriction enzyme treatment, and agarose gel electrophoresis were the same as those summarized in Tables 5 and 6 of Example 5. The results are shown in Figure 8. [Table 9] [Industrial Applicability]

[0061] The present invention can be used in the fields of agriculture, plant breeding, etc.

Claims

1. A method for determining the level of bacterial wilt resistance in an eggplant plant, comprising: A method comprising the step of determining, in an eggplant plant, at least one of the nucleotide polymorphisms shown in Table 1, or a contiguous polynucleotide containing said nucleotide polymorphism, as a molecular marker for bacterial wilt resistance in the eggplant plant. 【Table 1】

2. The method according to claim 1, comprising a step of determining two or more of any of the nucleotide polymorphisms shown in Table 1, or a continuous polynucleotide containing the nucleotide polymorphisms, as a molecular marker for bacterial wilt resistance in an eggplant plant.

3. The method of claim 1 or 2, wherein the molecular marker is located on chromosome 1, chromosome 6, or chromosome 9 of the eggplant plant.

4. The method of claim 3 , wherein the molecular marker is located on chromosome 6 of the eggplant plant.

5. The method according to claim 1 or 2, wherein in the determining step, the region in the DNA of the eggplant plant is amplified using primers that amplify the region containing the molecular marker.

6. A method for producing a bacterial wilt resistant eggplant plant, comprising: A step of selecting eggplant plants resistant to bacterial wilt by the method according to claim 1 or 2. A manufacturing method comprising:

7. The method according to claim 6 , wherein the selection step is repeated one or more times on progeny of the bacterial wilt-resistant eggplant plant selected in the selection step.

8. A molecular marker for bacterial wilt resistance in eggplant plants, comprising: A molecular marker comprising at least one of the nucleotide polymorphisms shown in Table 1, or a continuous polynucleotide containing the nucleotide polymorphism.

9. A kit for determining whether an eggplant plant is resistant to bacterial wilt, comprising: A determination kit comprising primers for amplifying a region containing a continuous polynucleotide containing at least one of the nucleotide polymorphisms shown in Table 1 in an eggplant plant.

Images